Body-Fluid Diagnostics in Microliter Samples

Home , Hydrodynamic voltammetry, Xanthurenic acid

BODY-FLUID DIAGNOSTICS IN MICROLITER SAMPLES

GAUTAM N. SHETTY

Submitted in partial fulfillment of requirements

for the degree of Doctor of Philosophy

Thesis Advisor: Dr. Miklós Gratzl

Co-advisor: Dr. Koji Tohda

Department of Biomedical Engineering

CASE WESTERN RESERVE UNIVERSITY

May, 2006 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Gautam N. Shetty .

candidate for the Ph. D. degree *.

(signed) Miklós Gratzl (chair of the committee)

Koji Tohda

Barry Miller

Clive. R. Hamlin

Mark D. Pagel

(date) 01/26/06

*We also certify that written approval has been obtained for any proprietary material contained within.

I grant to Case Western Reserve University the right to use this work, irrespective of any

students, agents and employees. I further agree that the University may reproduce and

provide single copies of the work, in any format other than in or from microforms, to the public for the cost of reproduction.

Gautam N. Shetty . (sign)

To my hardworking parents

iv TABLE OF CONTENTS

List of figures…………………………………………………………………………….vii

List of tables………………………………….………………………………………...... ix

Acknowledgements……………………………………………………………………...... x

List of Abbreviations……………………………………………………………………..xi

Abstract…………………………………………………………………………………..xii

Introduction: Significance, hypotheses and specific aims………………………………...1

Part I Optimization of RSS system parameters

Chapter 1 Hydrodynamic Electrochemistry in 20 μL drops in the Rotating

Sample System……………………………………………………………………4

Part II Investigation of RSS performance in biological samples

Chapter 2 Rotating Sample System: Hydrodynamic Electrochemistry in

Biological Matrices………………………………………………………………28

Chapter 3 Rotating Sample System: A Simple Tool for Rheological Examination

of the Air-Solution Interface……………………………………………………..50

Part III Investigation of electrode ‘fouling’ in biological samples

Chapter 4 Protein Adsorption on the electrode of the Rotating Sample System…66

Chapter 5 Electrochemical Desorption of Proteins……………………………...82

Part IV Trace Pb analyses

Chapter 6 Rotating Sample System: Trace Pb(II) Analyses in Serum and Blood

Samples…………………………………………………………………………..96

v Part V

Chapter 7 Summary and Future Work………………………………………….114

Appendix A……………………………………………………………………………..122

Appendix B……………………………………………………………………………..129

Appendix C……………………………………………………………………………..137

Bibliography……………………………………………………………………………147

vi List of Figures

Figure 1-1 Schematic diagram of home-made Rotating Sample System

Figure 1-2 Schematic diagram of microfabricated Rotating Sample System

Figure 1-3 Cyclic voltammograms for electrode position close to the axis of rotation at

different rotation rates of the sample

Figure 1-4 Images of dye injection to visualize bulk flow patterns

Figure 1-5 Trace Pb analyses in aqueous (non-biological) samples using the RSS

Figure 2-1 Schematic diagram of Rotating Sample System (Top and Front view)

Figure 2-2 Cyclic voltammetry in rotated and stationary samples containing different

dilutions of fetal bovine serum

Figure 2-3 Lipid-protein interplay illustrated by cyclic voltammetry

Figure 2-4 Affect of electrode protein adsorption on mass transport properties

Figure 3-1 Cyclic voltammetry in rotated sample of different BSA concentration

Figure 3-2 Cyclic voltammograms depicting lipid-protein interfacial interactions

Figure 3-3 Calculating CMC from plateau currents in RSS

Figure 4-1 Using Hydrogen UPD to get electrode active surface area

Figure 4-2 Adsorption kinetics

Figure 4-3 Adsorption kinetics with and without Nafion coating

Figure 4-4 Comparison in voltammograms with and without Polyurethane coating

Figure 5-1 Adsorption and Desorption kinetics

Figure 5-2 Desorption in fetal bovine serum matrix

Figure 6-1 Anodic stripping voltammetry of 2.5 ppm Pb for optimization of CAP

vii membrane

Figure 6-2 Repeatability of detection of Pb in 10 μL serum samples

Figure 6-3 Convection properties in hemoglobin samples

Figure 6-4 Pb analysis in human blood

Figure 6-5 Trace Pb detection in human blood

viii List of Tables

Table 1-1 Diffusion layer thickness as a function of the position and the inner diameter

of nozzle for a single air jet

Table 1-2 Plateau and edge currents at various air flow rates for different positions of the

Pt mini-disc electrode, using two anti-parallel air jets for sample rotation

Table 4-1 Comparison of different membrane for coating electrode

Table A-1 Comparison of electrode area obtained using different techniques

Table C-1 Summary of Standards and Regulations for Pb

ix Acknowledgements

I would like to thank my advisor Prof. Gratzl; I am fortunate to have had the opportunity

to learn under him. I am indebted for the education which I am certain will hold me in

good stead for the future. I would also like to thank my co-advisor Dr. Tohda, who has

always been a great resource (all things except whitewater rafting!). I am grateful to Dr.

Barry Miller, Dr. Clive Hamlin and Dr. Marty Pagel for serving on my committee; I

would also like to thank for their constant guidance and encouragement. I would like to

thank my colleagues at the Laboratory for Biomedical Sensing; my research experience

would be incomplete without you all.

I would like to sincerely thank for first employers at the Center for Health Promotion

Research, Department of Epidemiology and Biostatistics for their support in my initial

period here; I would never have made it this far if not for my job there. I would like to thank my friends at Case, Cleveland chapter of Asha for Education, the Cleveland

Cricket Club and numerous others in the Cleveland community for enriching my

Cleveland experience.

I would like to thank my family and friends for their constant support. Coming from a country where one in three children do not have access to primary schooling, I would like

to thank all my teachers; I am indebted to them for the gift of education.

x List of Abbreviations

BSA : Bovine serum albumin

CAP : Cellulose acetate hydrogen phthalate

CCD : Charge coupled device

CV : Cyclic voltammetry

CMC : Critical micelle concentration

HSA : Human serum albumin ppm : parts-per-million ppb : parts-per-billion ppt : parts-per trillion

RDE: Rotating disc electrode

RSS : Rotating sample system

WE : Working electrode

UPD : Under-potential deposition

xi Body-fluid Diagnostics in Microliter Samples

Abstract

Gautam N. Shetty

The Rotating Sample System (RSS) has been conceived in our laboratory for diagnostics of microliter samples. The design of the RSS enables effective convection generation in microliter sized samples. Convection aids in mass-transport and is essential in improving sensitivity in applications such as trace metal diagnostics. In diagnostics applications such as titration and enzyme activity measurements, convection helps in homogenization of the sample. Capability to investigate microliter sized samples is essential to extend diagnostic capability for neonates and small children without having to draw body-fluids

(e.g blood) in the order of milliliters for analyses. Also, smaller size of the system would make it portable and attractive for use in point-of-care applications eliminating need for storage and transportation of samples. Small samples also ensure that storage and disposal issues are minimal. Natural physical properties such as surface tension, which are usually ignored for larger sample volumes, become prominent with microliter sample sizes; these can be engineered to develop simple yet robust tools for body fluid diagnostics. Optimization of system parameters for optimal system performance has been undertaken as part of this work. Study of the hydrodynamic performance of the RSS in

xii biological matrix was conducted and revealed interplay between proteins and lipids at the liquid-air interface. The RSS is unique in the sense that it imparts convection to the sample via its surface. Hence, utility of the RSS as a tool to probe the interfacial properties of samples containing surface-active molecules has been investigated. The

RSS by providing information about both bulk and surface properties of a sample fosters better diagnostics of biological samples. Challenges are posed to electrochemical analysis by non-specific adsorption of proteins onto electrode surfaces; hence methods to protect the electrode by coating with a suitable spacer polymer membrane have been developed.

For the first time, a technique to electrochemically effect desorption of proteins is demonstrated. Using the RSS’ favorable convective properties, trace Lead (Pb) analyses in model aqueous samples and detection capability in serum and blood matrices has been demonstrated. A detection limit of 260 ppt for Pb was achieved in aqueous samples.

xiii INTRODUCTION: Significance, hypothesis and specific aims

Conventional analytical systems and techniques are limited in their ability to address small samples. These systems designed for larger samples cannot be scaled down in size to work with microliter sized samples - a pre-requisite for biomedical applications.

Natural properties of liquids such as surface tension, which tend to be ignored for larger volumes, become significant in smaller volumes. This has been engineered to enable us to build a platform for analysis of microliter sized samples. The Rotating Sample System

(RSS) was thus conceived in our laboratory.

The RSS consists of a microliter sized drop placed atop a hydrophilic substrate such as glass and kept in position by a hydrophobic ring. The internal diameter of the ring is calculated such that the sample forms a hemisphere. Strong surface tension forces ensure that the sample is indeed hemispherical. Tangential air-jets are applied to the sample drop.

The linear gas jet velocity couples onto the sample surface and translates into rotation of the drop thereby generating convection in it. The sensing elements which include a working electrode (WE) are embedded in the substrate.

Various convection-based systems are available for analysis; e.g. rotating wires, streaming mercury electrodes, vibrating electrodes, and systems where there is forced flow past a stationary electrode such as conical, tubular, screen, packed-bed electrodes in fluid streams, channel electrodes, bubbling electrodes, and most prominently, the

Rotating Disc Electrode (RDE) system. These systems are limited in their ability to address microliter sized samples- a pre-requisite in the biomedical context.

The aim of my PhD study is to test the hypothesis that the Rotating Sample System can

be employed for diagnostics of microliter sized biomedical samples. To this end, the

specific aims are:

Specific Aim 1: To investigate hydrodynamic variations at the base of the rotating drop

and optimize system parameters

The RSS affords convection to a microliter sized drop by coupling air jet velocity to the

surface of the sample. Due to the non-rigid nature of an aqueous sample, it is plausible

that variations in the hydrodynamics may manifest along the radius of the sample. This

variation will be investigated by varying the electrode position in the substrate and

evaluating the electrochemical performance at each position for a given rotation of the

sample. Results of this study will lead to optimal design and performance for specific

RSS applications. This aim is addressed in Part I, chapter 1, appendix A.

Specific Aim 2: To investigate the hydrodynamic performance of the RSS in a biological matrix

Biological matrices manifest different rheological properties as compared to non-

biological samples. This may modulate the performance of the RSS since it affords

convection to the biological sample through its surface. This will be investigated by studying the hydrodynamics in different dilutions of fetal bovine serum solutions and model protein solutions. This aim is addressed in Part II, chapter 2.

2 Specific Aim 3: To evaluate electrode ‘fouling’ caused by non-specific adsorption of

proteins in biological matrices

Proteins are known to block access to the electrode by non-specific adsorption. This

limits the lifetime of the electrode in a biological environment. This problem is more

severe when investigating microliter sized samples since the electrode sizes involved are

smaller, loss of electrode area would undermine its ability to analyze the sample.

Electrochemical methods to investigate protein adsorption kinetics will be devised and

methods to protect the electrode from fouling will be explored. This aim is addressed in

Part III, chapter 4.

Specific Aim 4: To analyze trace Lead (Pb) in microliter samples

Pb analysis using current state-of-art employs bulky instrumentation requiring vials of

blood (order of milliliters) to be drawn from patients. This is more serious an issue with

small children who are more prone to the toxic effects due to Pb exposure. Model samples containing Pb will be analyzed for detection using RSS to check for sensitivity;

Pb would then be analyzed in serum and blood matrices. This aim is addressed in Part IV, chapter 6.

3 Chapter 1

Hydrodynamic Electrochemistry in 20 μL Drops in the Rotating Sample System

Gautam N. Shetty, Nilofar Syed, Koji Tohda, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

This work was published in Analytical Science, 2005, 21(10), 1155-1160 (reproduced with kind permission)

4 1.1 Abstract

The Rotating Sample System (RSS) has been conceived in the authors’ laboratory as a

convection platform for microliter-sized solution volumes. Convection is achieved by

rotating a small drop of sample on a stationary substrate by humidified gas jets directed

tangentially at the drop base with the working electrode and a liquid junction embedded

in it. Simplicity and portability of the device, and substrates complete with

microfabricated electrode and junction made potentially disposable, are further

competitive advantages with respect to competing, conventional analytical systems. In

this work the RSS’ performance with variation of system parameters such as the position

and size of gas jets used for sample rotation, and position of the working electrode in the

substrate are studied. Trace levels of Pb could be detected with this system and is

reported here.

1.2 Introduction

Conventional electrochemistry typically involves planar macro-electrodes and is

diffusion limited. One dimensional diffusion in these systems leads to progressive depletion at the working electrode (WE), making the more desirable stationary techniques out of reach, and sensitivities often insufficient. Forced convection can be used to limit diffusive transport to within the immediate vicinity of the WE, resulting in higher sensitivities as well as steady state mass transport and thus, stationary voltammetry. Besides the obvious advantages in analytical applications, this renders also mechanistic studies of electrode processes as well as related homogeneous reactions,

5 more efficient. To achieve the requisite conditions, different methodologies have been

proposed such as rotating wires, streaming mercury electrodes, vibrating electrodes, and

systems where there is forced flow past a stationary electrode such as conical, tubular, screen, packed-bed electrodes in fluid streams, channel electrodes, bubbling electrodes, and most prominently, the Rotating Disc Electrode (RDE) system [Bard, 2001].

The Rotating Sample System (RSS, Figure 1) which has been conceived and developed

in the authors’ laboratory [Cserey et al., 1997; Cserey, 2001] derives its inspiration from

the RDE approach. The RSS cell generates convection by rotating a liquid microsample

atop a stationary hydrophilic substrate with the working electrode and liquid junction

embedded flush with it (Figure 1) and surrounded by a hydrophobic ring to keep the

sample in position. This in effect provides a very simple equivalent to the costly,

complex, and much larger RDE system. Yet, the RSS approach does not involve any

moving mechanical parts since rotation is achieved by employing a humidified gas (e.g.,

air) jet or jets. The fact that the sample volume can be very small (in the order of ten to

twenty microliters), and that the substrate can be microfabricated and disposable, renders

the RSS system attractive for use in biomedical and environmental applications. Its

utility can be extended to monitoring and measuring contaminants in industrial samples,

and for basic electrochemical studies.

The RSS approach has been shown to achieve diffusion layer thicknesses in the order of

10 μm and less, at comfortable gas flow rates (around 100 mL/min) [Cserey et al., 1997].

This performance is equivalent to a disc electrode rotated at about 3,000 rpm, yet no

signs of deformation of the sample drop are apparent to the eye: it remains semi-

spherical which indicates the remarkable strength of surface tension at the boundaries of

6 a miniature aqueous drop and air. Earlier findings [Cserey et al., 1997] corroborated the

expected trend that the diffusion layer thickness decreases with increasing sample

rotation rate. Also, considering that the results are very reproducible suggest that laminar

flow conditions exist in the rotating drop.

Using the Rotating Sample platform with a microfabricated platinum ring electrode

(Figure 2), determination of Mercury [Cserey et al., 1997] in aqueous samples has been

shown to be feasible using anodic stripping voltammetry. Copper detection capability in

an acid pre-treated and filtered (to remove serum proteins) serum matrix has been

demonstrated in our laboratory [Cserey, 2001]. In addition, the RSS’ utility in detection

of other metal ions has also been explored [Gratzl et al., 2001-2003]. Given the potential

for useful applications, a better understanding of the RSS’ performance vis-à-vis its system parameters is warranted.

While similarities can be drawn between the RSS and the RDE [Levich, 1952],

differences exist in specific details. The unique aspect of the RSS is that sample drop

integrity is maintained during rotation due to surface tension. Also, since a gas jet (or

jets) is used to achieve sample rotation instead of directly rotating a “rigidly” coupled

electrode as in the RDE approach, a “soft” mechanical coupling of the gas jet and the

surface of the liquid sample is to be considered. In this work these aspects, unique to the

Rotating Sample System, are explored experimentally. Dependence on the number, and placement of gas jet(s) relative to the sample to examine coupling of the air jet(s) with the sample, and the effects of gas flow rate and nozzle diameter on the ultimate electrochemical properties of the RSS, are also investigated.

7 Trace analysis of Pb in 20 microliter samples is also reported here. Pb in blood is

considered to have several deleterious effects on human health [Needleman, 2004]. The

RSS here could provide with an alternative to the expensive, bulky and labor intensive current state-of-the-art AAS [Baralkiewicz et al., 1996] and ICP-MS [Hansen et al.,

2002] techniques. Several electrochemical approaches to Pb detection such as the RDE

[Brihaye et al., 1983], sonoelectroanalysis [Banks et al., 2004], flow injection [Jaenicke

et al., 1998], and stirred systems [Bartlett et al., 2000] have also been explored. The RSS

operates with much smaller sample volume than is required in the aforementioned

methodologies. Moreover, the RSS can be modified to employ different electrode

materials such as carbon [Brihaye et al., 1983; Bartlett et al., 2000], boron doped

diamond electrodes [Banks et al., 2004; Maeda et al., 2003] and mercury [Jaenicke et al.,

1998; , Tekutskaya et al., 1999; Duinker et al., 1977] for Pb detection purposes.

1.3 Experimental

1.3.1 Materials

All chemicals were from Sigma (St Louis, MO, USA); solutions were made with Milli-Q

water (18.2 MΩ cm Milli-QUV plus from Millipore, Billerica, MA, USA). For trace Pb

analysis the water was distilled using a quartz distiller to obtain ultra-pure water. Also,

polypropylene flasks (Nalge Nunc International, Rochester, NY, USA) were used for

storing Pb solutions to preempt any contamination due to storage in glass containers

[Prichard, 1996]. The two RSS setups used in this work (Figures 1 and 2) were

characterized using 1.0 mM K3[Fe(CN)6]with 0.1 M KNO3 as supporting electrolyte.

8 Capillary tubes (0.025 mm wall thickness; A.H. Thomas Co., Philadelphia, PA, USA) of

0.5 mm inner diameter (i.d.) were used to rotate the drops in experiments with flexible cell geometry (Figure 1). Capillaries of different i.d. values were used in the experiments

involving the microfabricated RSS cell (Figure 2). In order to visualize flow patterns within a rotating drop, Evans blue dye was inserted into a rotating drop using a pulled glass capillary.

1.3.2 Apparatus

To evaluate system performance by changing the position of the air jet and the diameter

of the air-nozzle used for sample rotation, a microfabricated RSS cell was used with a Pt

ring WE, deposited symmetrically around the junction hole (Figure 2). Fabrication of

this device has been described earlier [Cserey et al., 1997; Cserey, 2001]. The nozzle of

the humidified air jet was mounted on a three axis manipulator (WPI, Sarasota, FL,

USA). The nozzle positions were recorded by taking a top view image of the RSS setup

using a CCD camera (CV-S3200 from JAI, Copenhagen, Denmark).

To evaluate system performance for different positions of the WE in the substrate with

respect to the sample drop, another system with a Pt mini-disc electrode and flexible

geometry was adopted. The corresponding electrochemical cell (Figure 1) was fabricated

using a part of a microscope glass slide (7.5 cm × 2.5 cm and 0.1 cm thick, from Fisher

Scientific, Pittsburgh, PA, USA) as substrate. The WE was made from 150 μm diameter

platinum wire (Alfa Aesar, Ward Hill, MA, USA). Silicone elastomer (DOW Corning,

Midland, MI, USA) was applied to form the hydrophobic ring that confines the sample

9 drop into a semi-sphere. Epoxy resin (ITW Performance Polymers, Riviera Beach, FL,

USA) was used for gluing purposes. A similar setup was also used for trace Pb analyses.

The Ag|AgCl reference (BAS, West Lafayette, IN, USA) and stainless steel counter electrodes (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma). Humidified air jets driven by an aquarium pump (Wollinger Bros., Oakland, NJ, USA) were directed toward the sample from 0.5 mm i.d. glass capillaries. Air flow rates were measured with volumetric flow meters (Cole-Parmer Instrument Co., Niles, IL, USA). For trace Pb analysis, a humidified nitrogen gas jet was used instead of air jet.

Electrochemical measurements for testing effects of the position of gas jets were performed using BAS 100W potentiostat (BAS, West Lafayette, IN, USA) with microfabricated sensors [Cserey et al., 1997] (Figure 2). For all other studies a CH100 electrochemical workstation (CH Instruments, Austin, TX, USA) was used. Images of the rotated sample drop were acquired using a CCD camera (see above).

All calculations were done using Matlab 6.0 (Math Works, Natick, MA, USA).

1.3.3 Procedures

1.3.3.1 Fabrication of the electrochemical cell with flexible geometry (Figure 1-1)

Two holes 1 mm in diameter were drilled through the glass slide. Glass of the same material was heat melted around the tip of the platinum wire with 150μm diameter so as to cover the tip completely with a glass bead, a little larger than the drilled hole. This end of the wire was inserted in one of the holes so that it fits in snugly. The bead was then

10 glued onto the substrate using epoxy resin. The substrate surface was then polished to

leave a flush platinum mini-disc exposed to form the WE (Figure 1-1). The other hole is

for the liquid junction. Silicone elastomer was printed to form a hydrophobic ring to

keep the sample in position; the internal diameter of this ring is calculated as 4.2 mm for a 20 μL drop to ideally form a perfect hemisphere. Different positions of the electrode were realized by simply re-applying the elastomer in different positions by shifting the relative position of the circular hole in the elastomer. This way the axis of rotation of the sample vis-à-vis the electrode position could be shifted while ensuring similar electrode surface conditions; therefore an exact comparison in the responses is possible. This arrangement also allowed the investigation of system characteristics with the working electrode positioned at the center of the sample drop.

Junction hole 20μL sample drop Stationary disc electrode Hydrophobic ring

Air jet

Glass substrate Air jet

Figure 1-1 Schematic diagram of the Rotating Sample System as used in this work to explore flexible cell design. The position of the 150μm Pt mini-disk working electrode (WE) is variable with respect to the axis of rotation by moving the hydrophobic silicone elastomer ring accordingly. Liquid junction is made of a hole, filled with agar gel, to connect the sample drop to an underlying reference compartment where an Ag|AgCl reference and a stainless steel counter electrode are housed, bathed in 0.1 M KNO3 solution (not shown).

It is noted that the presence of an electrical contact line between the electrode and the

contact pad of the substrate may have an averaging effect along the radius of the drop.

This was minimized by fabricating a contact line very thin with respect to the ring electrode in the cell shown in Figure 2. In the setup shown in Figure 1, electrical contact is made on the other side of the glass slide which eliminates this problem altogether.

Also, mechanical polishing of the WE is possible in contrast to earlier microfabricated electrodes (Figure 1-2) since this is in comparison a mechanically more robust electrode.

y x o (e) (f) (d)

(b) (c) (a)

Figure 1-2 Schematic diagram (top view) of a microfabricated RSS cell, as used in exploring the effects of position of the air jet, its nozzle diameter, and gas flow velocity. The thick solid line (a) indicates the microfabricated Pt ring electrode and its connection to the contact pad (b). The connecting wire (c) is buried under the silicone elastomer (d) to ensure that a contiguous hydrophobic barrier surrounds the sample drop. The nozzle (e) was placed on top of the substrate; its horizontal position with respect to the vertical axis of rotation of the sample drop is defined by the x and y coordinates as shown in the top view. The liquid junction (f) and reference / counter electrode compartment are similar to the RSS cell with flexible geometry shown in Figure 1-1.

12 Two anti-parallel humidified air jets were employed in this device for more axis-

symmetric rotation of the sample. Humidified air-jets ensure that loss of sample volume

due to evaporation is minimal [Cserey et al., 1997]. The position of the electrode vis-à-

vis these jets was kept such that the electrode – junction hole line was parallel to the axes

of the jets in all experiments (Figure 1-1).

1.3.3.2 Determination of the effective surface area at, and edge current of, the working

electrode and the diffusion layer thickness

Electrode area was determined by chronoamperometry where the transient current

response for a microdisk electrode is given by Cottrell’s equation, i.e., the first term in

Equation 1 [Bard, 2001]:

⎛ A ⎞ )( nFDCti 4r (1) = ⎜ 2/1 + e ⎟ ⎝ πDt)( ⎠

2 3 where re is WE disc radius, A (cm ) the active surface, 1mM concentration C (mol/cm ) of potassium ferricyanide with diffusion coefficient D (D = 7.3×10-6 cm2/s [Cserey et al.,

1997]). Thus, the slope of a linear fit to the current versus inverse square root of time

plot also has information regarding active electrode surface area. The obtained electrode

area was also verified by using cyclic voltammetry at different scan rates [Appendix A].

The bias of the linear fit from the chronoamperometry experiment gives the edge current

(the second, “microelectrode”, term in Equation 1, iedge). Edge currents are negligible in

the case of macro-electrodes; however it is no longer negligible for a mini-disc electrode

such as the one used here (150 μm diameter).

13 Plateau currents of stationary CV plots, obtained when the sample was efficiently rotated

in the RS system, have been used earlier [Cserey et al., 1997] to estimate the diffusion layer thickness (δ), correcting here also for edge currents:

nFADC δ corrected = (2) plateau − ii edge

1.3.3.3 Trace Pb analysis

Pb measurement with the RSS in the 160-1600 parts-per-billion (ppb) range was first performed using differential pulse stripping voltammetry. Mercury was pre-deposited onto the electrode from a 1.5 mM HgCl2 solution containing 5% HCl for 250 seconds.

This is done to prevent hydrogen evolution, which would mask the Pb stripping current on a bare platinum electrode [Bartlett et al., 2000; Yekutskaya et al., 1999; Duinker et al.,

1977]. After pre-concentration of Pb on the WE during rotation of the sample for 30 s, a

30 s quiet time was applied with the air jets were switched off. The potential at the WE was clamped at deposition potential during all this time to avoid any oxidation of Pb that might otherwise occur due to corrosion processes. Blank runs (with samples containing no Pb), simulating identical experimental conditions were interspersed between subsequent Pb tests so that the system self-cleans itself and ensures no residual Pb contributed to the stripping current.

Two 0.5 mm i.d. glass capillaries were glued to the glass substrate to preempt any

variation in diffusion layer thickness which may have been caused due to slight variation

in nozzle positions. Thus for each individual sample, RSS system parameters such as

electrode position, nozzle position and gas flow rate (180 mL/min) were fixed.

14 Pb solutions were prepared by serial dilutions of a Pb standard solution for atomic

absorption spectrometry. A solution with 0.1 M KNO3 was used as background at pH 2.3,

acidified with trace-select HNO3. Three sets of calibrations (increasing followed by

decreasing followed by increasing concentration of Pb: ‘up-down-up’) were performed.

A background solution with no Pb was also used as a blank. A 300 μm diameter Pt mini-

disc electrode with its center positioned 1.8 mm from the axis of rotation was used.

To test for reproducibility and to confine enhanced sensitivity by using RSS parameters

tuned for lower Pb concentrations, measurements were also performed for 16 ppb

samples (n=5). Hg pre-deposited, Pt mini-disc electrode with 100 μm diameter, whose center was positioned 1.8 mm from the axis of rotation was used in this case for a 750 second pre-concentration time for Pb.

1.4 Results and Discussion

1.4.1 Air-nozzle study

To optimize the air jet – drop mechanical coupling, the relative jet – drop position as

well as the nozzle inner diameter for air jet were systematically varied with the same volumetric gas flow rate. The experiments were conducted with a single gas jet and using the microfabricated version of the RSS cell incorporating a Pt ring electrode

(Figure 1-2). Plateau current of cyclic voltammetry (CV) was used to determine the

15 diffusion layer thickness (Equation 3); the active electrode area was obtained from

chronoamperometry.

The narrower the nozzle the thinner the diffusion layer, i.e., the better is the

electrochemical performance of the system (Table 1-1). Thus, the same volumetric gas

flow rate that translates to higher linear gas velocities in narrower tubing apparently induces higher rotation rate of the drop. This observation indicates that deterioration of the soft mechanical coupling between jet and drop due to decreasing contact area with reduced nozzle inner diameter is less pronounced than the simultaneous positive effect of increasing air jet velocity.

It is noted that average air jet velocity is inversely proportional to the square of nozzle

inner diameter while the contact area is proportional to the diameter. Thus, their mutual

effects could, in principle, compensate each other for the same volumetric gas flow. The

fact that this is not the case suggests that divergence of the air jet upon exit from the

nozzle is quite significant, even over the short distance from nozzle to drop. The contact

area therefore decreases to a lesser extent than the cross sectional area of the nozzle. Air

jet velocity therefore increases faster than the contact area decreases, which explains the

findings.

16 Table 1-1. Diffusion layer thickness as a function of the position and the inner diameter of nozzle for a single air jet.

Nozzle position is defined by the horizontal coordinates of the capillary outlet (nozzle) at the axis (x and y, in mm, see Figure 2); δ is diffusion layer thickness as obtained from the plateau current of cyclic voltammetry of 1mM K3[Fe(CN)6] at the microfabricated Pt ring electrode (Figure 2). The volumetric gas flow rate was kept 140 mL/min throughout for all air jets. With the nozzle placed on top of the substrate, the height of the axis of the air jet is at half its inner diameter plus 0.025mm (due to tubing wall) above the base.

The effect of variation of air jet – drop relative position is more sensitive in the direction

perpendicular to the air jet (y direction) than in the direction tangential to the drop (x direction). The best hydrodynamic effect, i.e., the thinnest diffusion layer, for the same nozzle inner diameter is ensured when the axis of the air jet is nearly tangential to the

17 drop (lowest value of y in Figure 1-2, see Table 1-1) and closest to it (highest x value).

This position with the narrowest nozzle used can ensure a diffusion layer in the order of

8 microns thick with this simple system and using just a single gas jet for sample rotation.

1.4.2 Variation in diffusion layer thickness with WE position along the drop radius

The RSS with flexible geometry (Figure 1-1) was used to compare system performance

for various positions of the working electrode. Two anti-parallel, tangential air jets were

employed.

The active surface area of the Pt mini-disc electrode of this cell was determined using a chronoamperometry experiment. The values for electrode area were also validated using

cyclic voltammetry experiments using different scan rates. The area thus obtained

compared well with each other as well as with the nominal area (wire cross-section), the

latter being always somewhat larger (Appendix A).

Relative contribution of the edge current to total current becomes more significant at

lower gas flow rates and / or shorter electrode distances from the axis of rotation, (Table

1-2). This results in a decrease in total current, and hence an increase in diffusion layer thickness. The edge current is about 26 % of the total current at a gas flow of 39 mL/min and in the closest position of the WE to the center, 0.82 mm. It makes up, however, only about 7 % total current in more hydrodynamically efficient settings, i.e., at 140 mL/min

18 flow rate and r = 1.89 mm. The diffusion layer thickness was evaluated according to

Equation 2 at different positions of the electrode at different air flow rates (Table 1-2).

Table 1-2. Plateau and edge currents at various air flow rates for different positions of the Pt mini-disc electrode, using two anti-parallel air jets for sample rotation.

Electrode position is measured from axis of rotation of the sample drop. Edge current in each case was calculated using chronoamperometry. The system in Figure 1-1 was used in these experiments.

19 For the working electrode positioned at the axis of rotation of the drop, as the rotation rate is increased, the current slightly increases indicating increasing hydrodynamic flow at the surface of the electrode, but peaks in the cyclic voltammograms are still discernable (Figure 1-3a-f) as opposed to the plateau current which was observed for electrode positions away from the axis of rotating drop (not shown). This indicates that while some convection effects are noticeable with slightly increased peak heights, quiescent conditions exist close to the axis of rotation. Diffusion here is in between semi-infinite and diffusion layer limited regimes.

Figure 1-3 Cyclic voltammograms (CV) in a 20μL sample drop containing 1 mM K3[Fe(CN)6] with 0.1M KNO3 as background electrolyte for the Pt mini-disc electrode positioned at the axis of rotation on the substrate with various air jet flow rates (a=0ml/min, b=20ml/min, c=39ml/min, d=63ml/min, e=98ml/min, f=140ml/min, g=140ml/min). Initial potential 350 mV, scan rate = 100 mV/s. Two anti-parallel air jets were used, except for the highest air flow rate where CV was also obtained with one air jet.

To explain this effect, a very small amount of Evans blue dye solution was introduced at the apex of a rotating drop from a pulled capillary tip for the observation of hydrodynamic flow pattern. It was found that the trajectory pattern visualized by the applied dye solution indicates a spiral path down towards the base of the drop along the surface; the trace then re-appeared at the axis of rotation beginning to move up before dilution of the dye (Figure 1-4). This observation indicates that in addition to the primary motion of rotation, secondary flow patterns exist in the bulk of the drop. Close to the axis of rotation of the drop the dye trace indicates an upward flow exists. This leads to quiescent conditions there as evidenced from the cyclic voltammetry experiments.

t=0 t=0.5

t=1.5 t=2.0

Figure 1-4 Images showing flow patterns in a rotating drop by injection of Evans dye at the apex of the drop. Pictures of the drop rotated by 39 ml/min gas jet were taken.

21 When one of the air jets was switched off in this experiment, however, the peak in the

center position disappeared and a plateau current became apparent with a significantly

increased current level (Figure 1-3g). The reason for this anomalous behavior is that the

system in this case becomes asymmetric and the axis of rotation is therefore shifted

away from the geometrical axis where the electrode is located; dye-insertion provided

with a visual proof of the same. Thus, a system having two air jets provides for more

axis-symmetric, i.e., more ideal rotation patterns since the jets themselves are in this case

axis-symmetric.

An analysis of data obtained at the WE positioned away from the center when only one air jet is used for rotation results in somewhat thicker diffusion layers (not shown) than those obtained with double jets at the respective positions. The increase in thickness of

the diffusion layer (and the corresponding decrease in current) is, however, no greater

than 10% at the studied WE positions. This corroborates the earlier observation that the

actual jet linear flow velocity is more important in hydrodynamic efficiency than the

contact area between drop and air jet(s).

1.4.3Trace Pb analysis

Three sets of “up-down-up” calibration experiments were carried out with samples

containing various concentrations of Pb (Figure 1-5) using differential pulse stripping

voltammetry. Each set of calibration was performed with increasing Pb concentrations,

followed by decreasing concentrations and then followed by the same concentrations in

increasing order. Excellent linearity of calibration was obtained in each set with a

22 unified regression coefficient of 0.9927 (Figure 1-5). Using 3σ [Banks et al., 2004;

Analytical Methods Committee, 1987], these settings yielded a detection limit of 14 ppb

Pb level.

The amount of Pb, N, deposited during time t, is the difference between the initial

amount in the sample and the decreased amount at the end of the deposition step.

Diffusion limited depletion being proportional to concentration in the sample, deposition

flux decreases exponentially with time [Cserey et al., 1997]:

AD ⎛ − t ⎞ ⎜1−= eVCN Vδ ⎟ (3) 0 ⎜ ⎟ ⎝ ⎠

where V is the volume of the sample (20 microliters), C0 is the initial concentration of Pb

in the drop, A is the area of the electrode, D is diffusion coefficient of Pb2+ in aqueous

solution, and δ is the diffusion layer thickness. The amount deposited can be obtained experimentally by integration of the stripping current. Equation 3 suggests that exhaustive deposition of the analyte requires long deposition times and/or a large electrode area. Since the electrode size used here is small and the deposition times are moderate, the deposited amount can be estimated with a linear approximation of

Equation 3 that is valid for short time period. The diffusion layer thickness (δ) for Pb stripping analysis is therefore estimated using

ADtC δ = 0 (4) N

23 The estimated diffusion layer thickness was 10.2 + 1.6 μm obtained for set of experiment of decreasing Pb concentrations.

Figure 1-5 Up-down-up calibrations for anodic stripping analysis of trace Pb in 20 μl aqueous drops. Linear regression is shown for peak differential currents of samples containing 160, 400, 800, 1200 and 1600 ppb Pb. Pre- concentration potential of -750 mV was used as measured against Ag|AgCl reference electrode with junction (3M KCl) with a pre-concentration time of 30 s. In stripping step differential pulse voltammetry (DPV), the following parameters were used: 25 mV of amplitude, 4 mV of voltage step, 0.05 s of pulse width, 0.012 s of sampling period, 0.1 s of pulse period. Nitrogen gas jets for the sample rotation were switched off 30 s before the stripping step; the potential was clamped at -750 mV during this period (quiet time). A Hg pre-deposited, Pt mini-disc with 300 μm diameter was used as WE. Inset: Stripping Voltammograms for samples containing 16 ppb Pb (n=5). Pre- concentration time used here was 750 s for a 100 μm Hg layer pre-deposited onto the Pt mini-disc electrode. DPV parameters were the same as those used for up-down-up calibrations.

24 Repeatability and sensitivity for trace Pb detection using the RSS in the lower tens of ppb range was then tested. A Hg pre-deposited, platinum mini-disc electrode with 100

μm diameter was used for detection of 16 ppb Pb. Improved signal to noise ratio was achieved in this case by increasing the deposition time to 750 seconds (inset, Figure 1-5).

The measurement was repeated five times to test for reproducibility. The coefficient of

variation for stripping peak heights and peak areas were 1.8% and 1.4% respectively.

The diffusion layer thickness estimated from the five measurements was 9.2 + 0.1 μm.

With improved signal-to-noise ratio, this setup and protocol yielded a detection limit of

240 ppt (parts-per-trillion) Pb level based on 3σ [Banks et al., 2004; Analytical Methods

Committee, 1987].

1.5 Conclusions

The Rotating Sample System is based on the idea that for very small aqueous samples, effective hydrodynamic electrochemistry can be realized by turning the Rotating

Electrode System upside down and keeping the electrode steady while rotating the sample drop on top via soft mechanical coupling with tangential air jets. At such small volume of the sample, surface tension ensures that drop integrity is maintained during rotation.

25 Increasing the number of air jets with the same air velocity each does not necessarily

increase rotation very much, but provides for more axis-symmetric rotation of the drop.

At least two air jets would be required for axis-symmetric rotation of the sample.

Secondary bulk flow patterns exist in addition to the primary rotation of the sample. This

secondary flow within the drop bulk modulates the diffusion layer thickness. These

secondary flow patterns also contribute to the mixing effect in the sample. Minor

variations of diffusion layer thickness are hence manifest with varying the distance of

the working electrode from the axis of rotation. At the center of the sample, quiescent

condition exists and offers an ideal location for the reference junction to be positioned.

Currents can be increased, and in stripping detection, deposition times shortened, however, by positioning the electrode as close to the drop edge as possible since the diffusion layer is the thinnest here. This position is empirically the most optimal position for trace metal analysis. At the expense of increased pre-concentration time, improved signal-to-noise ratio can be obtained by decreasing the electrode size. Larger electrodes reduce the deposition time, but their detection level is limited by the background that forms the baseline. Therefore, for lower concentration ranges, as the results show, using a smaller electrode will enhance sensitivity significantly.

26 1.6 Acknowledgements

The authors gratefully acknowledge Prof. C.-C. Liu’s assistance in providing access to fine drilling equipment. The Pb analysis work was partially supported by Vision Sensors

LLC. We would like to thank the Case School of Engineering for a Case Prime

Fellowship to GS.

27 Chapter 2

Rotating Sample System: Hydrodynamic Electrochemistry in

Biological Matrices

Gautam N. Shetty, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

This work was submitted to Analytical Chemistry, October 2005 (reproduced with kind permission)

2.1 Abstract

The rotating sample system (RSS) has been conceived in the authors’ laboratory as a

convection platform for analysis in microliter sized samples. The sample is placed atop a

stationary substrate such as glass and kept in position by a hydrophobic ring with

electrode(s) for electrochemical analyses embedded flush with the substrate. Tangential

air jets rotate the sample about its axis, generating vigorous convection in it. This

enhances mass transport and thus increases sensitivity and/or reduces detection time in

electrochemical stripping analysis. In other applications the mixing effect of convection

helps homogenize the sample. Due to its ability to address miniature samples, the RSS is

ideal for analyses of biological fluids. Convection properties of the RSS in aqueous, non-

biological samples have been established in earlier works. We report here how

biomolecular components such as proteins and lipids affect the rotation of acid pre-

treated fetal bovine serum solutions. Effects of protein adsorption at the air-sample and

adsorption at sample-electrode interfaces on the convective properties in the sample are

also discussed. The utility of lipids in effecting better convection properties in protein-

containing samples is also reported.

2.2 Introduction

A number of techniques have been employed to generate convection in aqueous samples to aid electrochemical analysis, such as rotating wires, streaming mercury electrodes,

29 vibrating electrodes, and systems where there is forced flow past a stationary electrode such as conical, tubular, screen, packed-bed electrodes in fluid streams, channel electrodes, bubbling electrodes, and most prominently, the rotating disc electrode (RDE) system [Bard, 2001]. The rotating sample system (RSS) in comparison to the above operates with a sample size in the order of microliters. Surface tension, which is negligible in comparison to body forces in larger samples, becomes a significant factor in small volumes that are analyzed using the RSS. The resultant near semi-spherical shape of the sample drop is a result of a trade-off between surface tension, gravity and adhesion between the drop and the substrate [Neumann, 1996]. Surface tension is so dominant a factor that drop integrity is maintained even in the presence of strong centrifugal forces at high rotation speeds. Titration [Xie et al., 1996] and trace metal analyses [Chapter 1;

Cserey et al., 1997] in microliter samples have been shown to be feasible in model aqueous samples using the RSS. This makes the RSS an attractive platform for the analysis of biological samples.

Proteins constitute a major component of biological fluids such as serum and blood [Voet,

1998]. Presence of proteins alters the physicochemical properties of aqueous samples. In addition to inducing changes in viscosity, proteins have a tendency to migrate to the air- liquid interface and alter surface properties as well [Aschaffenburg et al., 1946]. The latter is of particular interest vis-à-vis the RSS since it is a convective system where convection is imparted to the target sample drop via coupling of a tangential gas jet(s) with the sample surface. This is in contrast to all other systems where convection is afforded to the sample bulk. Surface tension of serum (50 dyne/cm [Geigy

30 Pharmaceuticals, 1962]) is much lower than that of water (72 dyne/cm [Ross, 1988]).

This reduction in surface tension affects the ability of the gas jets (e.g. nitrogen jets) in

rotating the sample drop effectively and is one of the motivations of this study.

We have shown that pre-treatment by filtering out proteins improves the convection properties of serum [Cserey, 2001]. However, filtering is not a practical approach in case

of microliter sized samples. The only practically feasible pre-treatment that is also

sometimes essential, is the dilution of the biological sample to reduce viscosity. For trace

metal analysis in biological matrices, dilution with a strong acid is essential for extraction

of the target metal ion from its protein complex [Cserey, 2001; Banks et al., 2004;

Kruusma et al., 2004]. Dilution of the biological samples also ensures that the problem of electrode fouling is reduced, since the amount of protein adsorbed is proportional to its concentration [Jaenicke et al., 1998].

Convection assists in mass transport since diffusion in a convected system is limited

across a thin layer at the electrode called the ‘diffusion layer’. The RSS has been shown

to achieve a diffusion layer thickness of less than 10 μm when rotated by air-jets with

flow rates of about 100 mL/min [Chapter 1; Cserey et al., 1997]. Protein adsorption onto the electrode surface also causes reduction in active surface area available for electrochemical analysis [Rai et al., 2003; Guo et al., 1996] called electrode ‘fouling’. It is believed that the layer of adsorbed proteins is a monolayer thick [Young et al., 1988].

In the case of insulin however, there is aggregation of insulin monomers leading to a thicker (greater than monolayer) coverage by the adsorbed protein molecules [Nylander

31 et al., 1994]. It is not clear in what way this adsorbed layer impedes access to the

electrode and affects the hydrodynamic performance in convection-based electrochemical

systems. Increased diffusion layer thickness due to the adsorbed protein layer would

results in reduced sensitivities and increased detection times.

In light of all of the above a better understanding of the convective properties of

biological matrices is warranted.

2.3 Experimental

2.3.1 Materials

All chemicals were from Sigma (St Louis, MO); solutions were made with 18.2 MΩ

Milli-Q water (Milli-QUV plus from Millipore, Billerica, MA). Regular fetal bovine

serum (Equitech-Bio Inc., Kerrville, TX) and dialyzed (triple 0.1 micron filtered) fetal

bovine serum (Gibco, Invitrogen Corp., Carlsbad, CA) were used to study the

hydrodynamic performance of RSS in a biological matrix. Serum samples were diluted

with pH 1 sulphuric acid (Sigma). These serum samples were characterized

electrochemically using analytical grade potassium ferrocyanide (Sigma) such that its

final concentration in a 20 microliter sample is 2.5mM. Lyophilized bovine serum

albumin (BSA) (Sigma) and lyophilized human serum albumin (HSA) (Sigma) were used

to simulate the presence of protein component in serum samples; lyophilized human hemoglobin (Sigma) was used to simulate the same in a blood matrix. Intralipid 20%

(Baxter Healthcare Corp., Deerfield, IL), a 20% lipid fat emulsion was used to simulate

32 the presence of lipids in blood and serum. Capillary tubes (A.H. Thomas Co.,

Philadelphia, PA) having 0.025 mm wall thickness and 0.5 mm i.d. were used to direct

the nitrogen jets to rotate the samples. Nitrogen flow rate was measured with a

volumetric flow meter (Cole-Parmer Instrument Co., Niles, IL) and was maintained at

140 mL/min when the samples were rotated.

2.3.2 Apparatus

Electrochemical measurements were made using CH100 electrochemical workstation

(CH Instruments, Austin, TX). Homemade substrates with platinum mini-disc electrodes

were employed. The electrochemical cell (Figure 1) was fabricated using Corning glass

slide (7.5 cm × 2.5 cm and 0.1 cm thick, from Fisher Scientific, Pittsburgh, PA) as

substrate. The WE was made from 150 μm and 250 μm diameter platinum wires (Alfa

Aesar, Ward Hill, MA). Silicone elastomer (DOW Corning, Midland, MI) was applied to

form the hydrophobic ring that confines the sample drop into a semi-sphere (Figure 1).

Electrodes were polished on Microcloth polishing pad (Buehler, Lake Bluff, IL) mounted

on a Delta 31-120 disk sander (Delta, Jackson, TN). Alumina polishing paste (Buehler) of

1 and 3 micron sizes were used for polishing.

The Ag|AgCl (3N KCl)reference electrode (BAS, West Lafayette, IN) and gold wire

spiral counter electrode (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma)

(Figure 1).

33 YM-10 centrifuge filter units (Millipore) were used for filtration of serum with a

Labofuge 400 (Heraeus Instruments, Hanau, Germany) centrifuge. Rotating disc electrode (RDE) system (Pine Instruments, Grove City, PA) was used for comparative studies.

2.3.3 Procedures

2.3.3.1 Fabrication of the electrochemical cell

Silicone elastomer was deposited on a glass micro slide substrate to form a hydrophobic

ring to keep the sample in position; internal diameter of this ring was calculated as 4.2

mm for a 20 μL drop to ideally form a hemisphere (Figure 2-1). Two holes 1mm in

diameter were drilled through the glass micro slide. Glass of the same material was

melted around the tip of a 250 μm diameter platinum wire so as to cover the tip

completely with a glass bead that was a little larger than the drilled hole. This end of the wire was inserted in one of the holes so that it fits in snugly. The bead was then glued onto the substrate using an epoxy resin. The substrate surface was then polished to leave a platinum 250 μm diameter mini-disc electrode (working electrode - WE) flush with the

glass substrate. The WE position was offset 1.8 mm from the center of the hydrophobic

ring. The other hole, drilled at the center is for the liquid junction and consists of a 1 wt% agar liquid junction that connects the sample to a chamber containing an Ag|AgCl

reference and gold counter electrodes (Figure 2-1).

Anti-parallel gas jets TOP VIEW

Glass slide substrate

Silicone elastomer ring

20 μL sample SIDE VIEW

1 wt% agar gel junction 250 μm dia. Pt electrode

Figure 2-1 Schematic diagram of Rotating Sample System. Nitrogen is humidified before directing it to the sample for rotation. The agar gel junction connects the sample to an underlying compartment housing the reference and counter electrodes immersed in 0.1 M KNO3 solution (not shown).

2.3.3.2 Electrode Polishing

The electrode on exposure to a biological matrix is fouled [Rai et al., 2003; Guo et al.,

1996] by non-specific adsorption of proteins. Hence, after each experiment involving electrode contact with a biological sample, the electrode was polished before it could be re-used. The electrode was polished with 1 and 3 micron alumina powder on a polishing disk mounted on a disk sander. After every polishing routine, electrode responses in non- biological solutions were used for comparison and ensure uniformity of electrode surface conditions.

35 2.3.3.3 Assessing RSS’ convective properties in various fetal bovine serum dilutions and comparison with RDE

Cyclic voltammetry of potassium ferrocyanide has been used to characterize convection in stationary and rotated model aqueous samples [Chapter 1, Cserey et al., 1997].

However, due to the reducing properties of serum proteins [Sakoguchi et al., 1984;

Bryant et al., 1929], freshly made potassium ferrocyanide was employed to investigate the convective properties of samples with different fetal bovine serum concentrations of

1%, 5%, 10% and 50%, all diluted in pH 1 sulphuric acid. Cyclic voltammograms were conducted in both stationary and rotated samples. The flow rate in the gas-jets for the rotated samples was maintained at 140 mL/min in all cases. In order to compare the results obtained with the RSS with a conventional convection platform, the above set of experiments was repeated using an RDE with the electrode rotated at 3000 rpm.

2.3.3.4 Studying convection in the RSS with filtered serum samples

As a control to understand the contribution of proteins to the convective properties in biological matrices using the RSS, experiments to study convection were conducted in samples where the proteins were filtered out. Dialyzed fetal bovine serum, which consists of only high molecular weight proteins was used. This dialyzed serum was then filtered using a Centricon Millipore filter (10 kDa nominal molecular weight limit); centrifugation was done for 15 minutes at 3500 rpm. The filtrate thus obtained was the non-protein component of serum. The convection properties of samples containing different concentrations of this filtrate were analyzed using cyclic voltammetry.

2.3.3.5 Studying convection using model proteins simulating serum samples

36 In order to understand the mechanisms underlying the convective properties in serum samples, convection studies were done in samples of model protein solutions. The protein contribution to the convective properties in serum samples was simulated with samples containing BSA. Albumin is the most abundant protein present in serum; it constitutes about 60% of the total protein content [Geigy Pharmaceuticals, 1962]. Hence, the properties of BSA would dominate the convective properties of serum. Diluting a stock solution containing 3.9 g/dL BSA (native concentration of BSA in serum) using the same dilution protocols used for fetal bovine serum used before, convection studies were done using cyclic voltammetry.

Dilution of biological samples is an established method to reduce their viscosity and also alleviate the problem of protein adsorption onto electrodes by reducing its concentration.

Samples of solutions containing 3.9 mg/dL (0.1% of native concentration) BSA and 0.39 mg/dL (0.01% of native concentration) BSA were investigated for their convective properties. This was done to find out the lowest concentration level of BSA that its presence would affect convection properties in the RSS. The convective properties of

BSA samples were also tested at pH 7.4.

Although HSA and BSA have different molecular weights, their structures share analogous regions [Sakata et al., 1980] and the two can be considered as homologous to each other [Bradshaw et al., 1969]. Its convective properties were also tested to ensure that the findings of the rotation studies of samples containing BSA would extend to human serum samples. In blood hemoglobin is the most significant protein component.

Therefore, for blood analyses, it is important that the convective properties of hemoglobin solutions be understood. From a stock solution of 13 g/dL of hemoglobin

37 (native hemoglobin concentration in blood [Geigy Pharmaceuticals, 1962]), different dilutions were tested for their convective properties.

2.3.3.6 Convection study in protein solutions with lipid addition

In addition to proteins, lipids constitute an important component of biological fluids.

Samples of model protein solutions simulate the protein contribution to the convective

properties of serum. In order to better characterize the convection properties of serum,

lipid addition to model protein samples is necessary. Intralipid (a widely used intravenous

lipid emulsion) was used as the model lipid component, and is known to contain all the

essential fatty acids [Wretlind, 1981]. Intralipid was diluted using pH 1 sulphuric acid

and added to the protein sample such that 1 wt% Intralipid was present in the sample,

also containing 2.5mM potassium ferrocyanide.

2.3.3.7 Effect of protein adsorption on mass-transport to electrode surface

Non-specific adsorption of proteins causes reduction in active surface area of the

electrode; this is electrode fouling. If this adsorbed protein layer forms a diffusion

limiting barrier by increasing the diffusion layer thickness, it would result in longer

detection times and/or reduced sensitivities, especially in applications such as trace metal

analyses involving hydrodynamic electrochemistry. In order to understand the effect of

protein adsorption on mass-transport to the electrode, we conducted cyclic voltammetry

experiments first in samples containing no protein but 2.5 mM potassium ferrocyanide

acidified by pH 1 sulphuric acid. The peak current corresponding to the oxidation of

potassium ferrocyanide in a stationary sample is given by [Bard, 2001]:

3 1 1 5 2 2 2 i peak ×= 1069.2 CAFDn ν (1)

38 where n is no of electrons exchanged, A (cm2) is the electrode area, F is the Faraday’s

constant (96500 C/mol), D (cm2/s) is the diffusion constant of potassium ferrocyanide, C

(mol/cm3) is the concentration of potassium ferrocyanide and v is the scan rate (V/s). In a rotated sample, the potassium ferrocyanide oxidation current is limited by a diffusion layer δ (cm) and an increased plateau current becomes visible given by [Chapter 1]:

nFADC i = (2) plateau δ

A stationary sample containing 0.39 g/dL BSA (10% of native BSA concentration),

acidified by pH 1 sulphuric acid was placed for 10 seconds to allow for the BSA to

adsorb onto the electrode. The sample was then replaced by one containing only 2.5 mM potassium ferrocyanide acidified at pH 1. The peak current in a stationary sample measured using cyclic voltammetry is given by equation 1. The ratio of the peak currents before and after adsorption of BSA provides the fraction of accessible electrode surface area, considering all other parameters remain the same. In rotated samples, the peak current is replaced by a plateau current given by equation 2. The diffusion layer thicknesses being identical, then the ratio of plateau currents before and after BSA

adsorption also characterizes the fraction of accessible electrode surface area. The above

two ratios would be identical if all other parameters remain unchanged. Hence, a

comparison of the above ratios was done to identify the effect of protein adsorption on

mass-transport to the electrode. The same set of experiments was also repeated for BSA

adsorption times of 5, 25, 35, 50 and 75 seconds. Comparisons were made for currents

measured in BSA free samples to ensure that changes in diffusivity of potassium

ferrocyanide in protein matrices is not a factor [Chang et al., 2005].

39 2.4 Results and Discussion

2.4.1 Convective properties of fetal bovine serum dilutions assessed with the RSS and

comparison with RDE

Convective properties in samples containing different concentrations of acidified fetal

bovine serum were studied using cyclic voltammetry in both stationary and rotated samples. Oxidation current of potassium ferrocyanide in the sample was monitored in

each case. In stationary samples, a reduction in the ferrocyanide oxidation peak was

observed with increasing serum concentration (Figure 2-2A). This implies increased

protein adsorption onto the electrode surface and reduced diffusivity, both of which result

in reduced currents with increasing serum concentration. However, results with rotated

samples yielded a contrasting trend (Figure 2-2B); increasing serum concentrations

elicited increased currents (Figure 2-2B). Efficient convection properties were expected

at lower concentrations of serum due to lower viscosity. The increased current densities

at higher serum concentration are indicative of significantly better convective properties

with increasing concentrations of serum. Improvement in convection properties with

increasing serum concentrations was observed despite reduced diffusivity, increased

viscosity and reduced electrode area.

0.9 A B 0.9 No serum 0.8 0.8 0.7 0.7 0.6 0.6

0.5 No serum 0.5

A A μ 50% μ 0.4 0.4 1% 10% 5% 0.3 0.3

Current / Current 5% / Current 0.2 0.2 1% 0.1 0.1

0 10% 0 -0.1 50% -0.1 100 200 300 400 500 600 100 200 300 400 500 600 PotentialPotential (vs (Vs Ag|AgCl) Ag|AgCl) / mV / mV PotentialPotential (Vs (vs Ag|AgCl)Ag|AgCl) / mV

Figure 2-2 Cyclic voltammetry in 20 μL samples of different fetal bovine serum dilutions acidified with pH 1 H2SO4 and also containing 2.5 mM K4[FeCN6] at a 250 μm Pt disc electrode. Initial potential 100 mV, 100 mV/s scan-rate. (A) Cyclic Voltammetry in non-rotated samples showing decreasing peak size of ferrocyanide oxidation with increasing serum concentration. (B) Cyclic Voltammetry in samples rotated by humidified, anti-parallel nitrogen jets having 140 mL/min flow rate. Serum samples show increasing currents observed with increasing serum concentrations.

In order to compare the response obtained in the RSS with another convection-based

analytical system, the same experiments were repeated using a RDE. With the RDE it

was observed that the current decreased with increasing serum concentration, when the

electrode was rotated at 3000 rpm similar to the trend observed in quiescent conditions

when the electrode was not rotated (not shown).

The RDE is very different than the RSS in the manner in which it imparts convection to a

solution. The RDE is immersed in the solution and hence affords convection to its bulk.

The result with the RDE reflects the increase in viscosity with increasing serum

concentration causing a corresponding decrease in currents.

41 The RSS imparts convection to a solution sample via its surface; a better understanding

of the surface properties of biological solutions provides the basis for understanding the

results obtained with the RSS.

In order to identify the reason for the surprising results obtained in rotated serum samples

using the RSS, another control was run by separating the protein and non-protein

components of dialyzed serum by filtration through centrifugation. The filtrate, which

consists of the non-proteinaceous fraction of serum showed results consistent with the

RDE (not shown). This implies that proteins were responsible for the poor convection

properties in a sample even when present at low concentrations levels in the sample.

2.4.2 Effect of presence of proteins on the convective properties of RSS

Bovine serum albumin (BSA), the largest protein component of bovine serum was used to simulate the effect of the presence of proteins on the convective properties of RSS in serum. Cyclic voltammetry experiments were conducted in samples containing different dilutions of a 3.9 g/dL BSA solution (native concentration of BSA in serum) and 2.5 mM potassium ferrocyanide. Even at 39 mg/dL BSA content (equivalent to 1% native concentration of BSA in serum), the rotated samples did not show an increase in current

(not shown) in comparison to the currents observed in a stationary sample. This was similar to the result obtained at low serum concentrations. However, unlike the trend with bovine serum samples, there was no increase in currents with increasing BSA concentration (not shown). This implies that although the presence of proteins in the sample causes poor convective properties in a sample, for higher concentrations of serum, the presence of proteins alone does not characterize its convective properties. This is

42 because better convection in the sample was observed with increasing concentrations of

serum.

Studies of BSA in solution [Poole et al.., 1984, 1989] have indicated its proclivity to migrate to the air-water interface and adsorb onto it [Langmuir et al.., 1940; Gonzalez et al.., 1970]. The adsorbed proteins form a contiguous film, which is elastic [Poole, 1989].

This film is known to be resistant to shear and contributes to surface viscosity [Prins et al.,

1998]. The presence of this film explains the fact that no convection effects are manifest in samples containing only BSA. The RSS affords convection to the sample via coupling of the air-jet onto the sample surface. Alteration in the surface properties of the sample affects the ability of the RSS to translate linear air jet velocities into rotation of the sample drop. Hence, the poor convective properties exist in samples containing only BSA, unless the concentration of BSA is too low to form a contiguous film at the surface of the sample drop.

The above set of experiments was repeated with human serum albumin, and similar

results were observed (not shown). Convective properties in solutions of hemoglobin, the

most significant protein component in blood, showed similar results to those obtained

with BSA. Hence, the results obtained with BSA can be considered representative of

convective properties of human serum and blood matrices.

2.4.3 Convection in BSA solutions with added lipid

Lipids besides proteins form another important constituent of biological fluids. Therefore,

the effect of the presence of lipids on the convective properties in protein-containing samples was investigated. Cyclic voltammetry of 2.5 mM potassium ferrocyanide in the

43 samples was used to investigate its convection properties. Cyclic voltammograms in a sample containing 0.39 g/dL BSA (10% of native BSA concentration in serum) showed a marginal increase in the oxidation current (Figure 2-3b) even when it was rotated. The current measured was almost similar to currents measured in quiescent conditions (Figure

2-3a). In a rotated drop with no proteins or lipids, a substantial increase in the oxidation current was observed, indicative of efficient convection in it (Figure 2-3e). On addition of lipid (1 wt% Intralipid) to a 0.39 g/dL BSA sample, when rotated produced an increased current indicating better convection in it than in the presence of BSA alone (Figure 2-3c).

1.2

(e) 1 (d)

0.8

μ (c)

/ 0.6

0.4

Current

0.2 (b) (a)

-0.2 50 100 150 200 250 300 350 400 450 500 550 600

PotentialPotential (Vs(vs Ag|AgCl) Ag|AgCl) / mV / mV Figure 2-3 Interaction between proteins and lipids illustrated by cyclic voltammetry at a 250 μm Pt disc electrode in 20 μL samples acidified by pH 1 H2SO4 containing (a) 2.5 mM K4[Fe(CN)6] only in stationary state, (b) 2.5 mM K4[Fe(CN)6] and 0.39 g/dL BSA in rotated state, (c) 2.5 mM K4[Fe(CN)6], 0.39 g/dL BSA and lipid in rotated state, (d) 2.5 mM K4[Fe(CN)6] and lipid in rotated state and (e) 2.5 mM K4[Fe(CN)6] in rotated state. Convection in rotated samples was effected by humidified, anti-parallel nitrogen jets with 140 mL/min flow rate. Initial potential 100 mV, scan rate = 100 mV/s.

44 Cyclic voltammetry in a rotated sample containing only lipid showed a substantial

increase in the oxidation current that was more than that observed in the presence of BSA,

but slightly less than in the case with neither BSA nor lipid in the sample (Figure 2-3d).

Lipids have been known to displace proteins from the air-water interface [Langmuir et al.,

1940; Poole et al., 1986; Poole, 1989], and disrupt protein films. In case of the RSS too,

the presence of lipids disrupt the contiguity of the protein film resulting in a fractured sample surface. This fractured surface provides for better convection properties than when a contiguous protein film exists at the interface. In the absence of lipids, the gas jet velocity is expended in merely stretching of the elastic protein film without generating any convection in the sample (Figure 2-3b).

In light of the above results, we can infer that at lower serum concentration trace amount

of lipid did not disrupt the protein film sufficiently enough to improve convection in the sample. However, at higher serum concentration the lipid concentration was sufficiently

high to effectively fragment the protein film and hence effect better convection in it.

2.4.4 Effect of protein fouling on mass-transport to the electrode surface

Cyclic voltammetry experiments were used to assess the effect of protein adsorption on

mass-transport to the working electrode surface (Figure 2-4). Cyclic voltammetry of potassium ferrocyanide in stationary BSA-free samples serve as a reference for electrode surface area before protein adsorption. The same in rotated samples provides information about the mass-transport properties in the absence of protein adsorption onto the

electrode surface. Adsorption of BSA onto the electrode surface was allowed from a

stationary sample containing 0.39 g/dL BSA for 10 seconds. This BSA sample was then

45 replaced by a sample containing only 2.5 mM potassium ferrocyanide. A reduction in peak and plateau currents were observed in cyclic voltammograms in stationary and rotated states respectively, corresponding to the reduction in electrode surface area due to protein adsorption.

1.2 Before BSA addition After BSA addition rotated 1

0.8

A μ 0.6 /

0.4

Current stationary 0.2

-0.2 100 200 300 400 500 600 PotentialPotential (Vs (vs Ag|AgCl) Ag|AgCl) / mV / mV

Figure 2-4 Effect of protein adsorption on mass-transport to the electrode studied by cyclic voltammetry on a 250 μm Pt disc electrode in pH 1 H2SO4 acidified 20 μL samples containing 2.5 mM K4[FeCN6] before and after adsorption of 0.39 g/dL BSA for 10 s. Initial potential 100 mV and scan rate = 100 mV/s. The ratio of peak oxidation currents in stationary samples is 1.17 and was found to be equal to the ratio of convective plateau currents found to be 1.18. Initial potential 100 mV, scan rate = 100 mV/s.

Comparing voltammograms in rotated samples before and after BSA adsorption provides information on the convective properties in the sample before and after protein adsorption and also loss of electrode area. Comparing voltammograms in stationary samples before

46 and after BSA adsorption however, provides information only about the loss of electrode

area due to protein adsorption. Results from both can be combined to identify whether

adsorbed proteins act as a mass-transport barrier. Comparison of the ratios of currents before and after protein adsorption in stationary and rotated samples showed that the ratios were identical indicating that there was no increase in the diffusion layer thickness even after protein adsorption..

It was found that for adsorption times 5, 25, 35, 50, 75 seconds from a 0.39 g/dL BSA

sample, the aforementioned ratios were identical (variance in difference between ratios

for each adsorption time was 1.5%), even though the absolute measured current reduced

with increasing adsorption times. This implies that there was no appreciable increase in

diffusion layer thickness due to BSA adsorption. The adsorbed protein layer does not

impede mass-transport to the electrode; but, it blocks parts of the electrode surface

reducing the active surface area available for analysis. It has been shown that the

adsorbed protein layer by itself therefore does not contribute to increasing the diffusion

layer thickness.

2.5 Conclusions

The RSS’ ability to address microliter sized samples makes the system well-suited for

analyses of biological samples. The RSS by coupling linear, tangential air-jets with the

surface of a liquid micro-sample is unique in the way it affords convection to the sample.

Changes in the surface properties of the sample modulate the convection generated in the

sample. This is true also for biological matrices since bio-molecules have an affinity to

47 migrate to the liquid-air interface, thereby affecting the properties of the sample surface.

Usual convection based systems are oblivious to surface modifications since they afford convection directly to the sample bulk. A better understanding of the interaction that exists between lipids and proteins at the surface of biological samples can be leveraged to ensure that the RSS generates effective convection in the sample.

Proteins such as BSA have an affinity to migrate to and adsorb at the air-water interface forming an elastic film. When tangential gas-jets are applied, only stretching of this film occurs; linear gas-jet velocity does not translate into rotation of the sample drop. Lipids however displace proteins from the liquid-air interface and disrupt the contiguity of the protein film. If the concentration of lipid is high enough, then it negates the effect of protein film formation on rotation of the sample. This finding can be leveraged to ensure

efficient convection in a sample containing proteins such as BSA. This effect was

observed in protein samples at both acidic and neutral pH and is true of protein samples present in human serum and blood matrices. This interaction between lipids and proteins at the air-water interface can be exploited to generate efficient convection in the sample for applications such as trace metal analyses, titration, electrochemical reaction kinetics etc.

With increasing serum concentration, the increased amounts of lipids present in it provide

for better convection properties in the sample. Hence, in the case of the RSS dilute

samples with trace amounts of lipid in it have poorer convection properties. However,

high serum concentration samples with higher protein content contribute to greater

48 fouling of the electrode by non-specific adsorption onto it and also have a higher viscosity. The above two can be reconciled by addition of lipid additives such as

Intralipid to disrupt the protein films in diluted biological samples.

The protein layer adsorbed onto the electrode is a monolayer thick. This work

experimentally proves that in a convected system the proteins adsorbed on the electrode

surface do not impede mass-transport to the electrode surface by way of an increase in

the diffusion layer thickness. The adsorbed proteins do however reduce the active surface

area available for analysis by blocking access to the electrode surface.

Modification of surface properties of the sample modulates the response of the RSS. The

RSS is hence is a unique tool to probe both sample bulk and surface properties.

2.6 Acknowledgements

This work was partly supported by NSF grant 0352443. We would like to thank Dr. Clive

Hamlin, Pathology- CASE, for his invaluable inputs and providing access to centrifuge

facilities at University Hospitals. Advice from Drs. Koji Tohda, Barry Miller and Mark

Pagel are also acknowledged. We would also like to thank Case School of Engineering

for partial Case Prime Fellowship to GS.

49 Chapter 3

Rotating Sample System: A Simple Tool for Rheological Examination of the Air-Solution Interface

Gautam N. Shetty, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

This work is to be submitted to Journal of Colloid and Interface Science

50 3.1 Abstract

The Rotating Sample System (RSS) has been conceived in our laboratory as a convection platform for analyses of microliter sized samples. Convection is afforded to the sample via its surface using tangential air-jets that rotate it. The RSS is hence unique in the way that it generates convection in the sample, i.e. via its surface. Changes in the surface properties of aqueous samples therefore modulate the hydrodynamic performance of the

RSS. Therefore, the RSS provides a simple tool to study the surface properties of the sample. We report in this work how protein-protein and protein-lipid interactions at the air-liquid interface can be analyzed by investigating bulk hydrodynamics using the RSS.

We also report the use of the RSS as a tool for calibration-free determination of the

Critical Micelle Concentration (CMC) of surfactants using the example of Triton X-100.

3.2 Introduction

The RSS consists of an aqueous microliter sample placed atop a stationary substrate and kept in position by a hydrophobic film ring with inner diameter such that the sample forms a near-hemisphere [Chapter 1, Cserey et al., 1997]. An electrode for electrochemical analysis is embedded flush with the substrate. This sample is rotated about its axis by tangential air jets that generate convection in it. Convective properties of the RSS have been documented with trace Pb [Chapter 1] and Hg [Cserey et al., 1997] analyses. The RSS is the only known convective system that affords convection to a sample through its surface. Hence, it is in a unique position to probe not only the sample

51 bulk (e.g. trace metal analysis [Chapter 1, 6]) but also the sample surface, both of which

provide very useful information about the sample.

The study of interfaces is of great interest to researchers across different areas of science.

Examination of a surface containing surface-active molecules such as proteins, lipids, surfactants and the like provide an insight into their physicochemical properties. Several

techniques that have been employed include FRAP (fluorescence recovery after

photobleaching) [O’Connell et al., 2005, Kragel et al., 1999], radioactivity [Small et al.,

1992] and UV spectroscopy [Suzuki et al., 1970].

Adsorption of protein molecules at the air-water interface has been investigated by

researchers to study their conformation [Burgess et al., 1992] and the effects of pressure

on adsorption of proteins at air-water interfaces [Gonzalez et al., 1970; Langmuir et al.,

1940; Graham et al., 1979]. Protein adsorption at interfaces plays an important role in

many biological processes [Burgess et al., 1992]. Surface of solution containing proteins

and lipids has been investigated to study the interactions that exist between them [Sprong

et al., 2001; Verger et al., 1982; Nylander, 1998]. The breakdown of lipids by the enzyme

lipase occurs at the air-liquid interface [Pieroni et al., 1990]; monitoring change at this

changing interface provides information of the enzyme activity. Surfaces of solutions

containing emulsifying and foaming agents used in food, pharmaceuticals, paper,

petrochemical products etc. have been studied; protein-lipid interactions at these

interfaces play a crucial role in the stability of these systems [Nylander, 1998; Poole et al.,

1984, 1986, 1989]. Pulmonary surfactants used to prevent collapsing of lung alveoli in

52 case of patients suffering from asthma and also deep-sea divers, are a mixture of specific phospholipids and apoproteins [Ruiz et al., 1989]; another example of the importance of interfacial interaction of lipids and proteins. Pulmonary surfactants reduce alveolar surface tension, and this allows reduction in the work needed for breathing and helps stabilize the alveoli. Measuring the surface tension of amniotic fluid is used as a measure of fetal lung maturity [Kashiwabara et al., 1986].

Surface tension is hence an important parameter to be monitored for studying the properties of a given solution containing surface-active components such as proteins and lipids. Various techniques are in use to provide with information on surface tension viz.,

Wilhelmy plate [Gonzalez et al., 1970; Kashiwabara et al., 1986], Lecomte du Nouy ring tensiometer [Lunkenheimer et al., 1981], pulsating bubble surfactometer [Enhorning,

2001] etc. Surface tension is also used as a measure of CMC (critical micelle concentration) of surfactants [Tsujii, 1998]. The CMC value is very important in order to ensure optimal use of surfactants [Tsujii, 1998].

In this work, the RSS platform is introduced as a simple tool for examination of surface properties by examination of BSA, BSA-lipid and Triton X-100 samples. The RSS platform has also been used to evaluate the CMC (critical micelle concentration) of

Triton X-100.

3.3 Experimental

53 3.3.1 Materials

All chemicals were from Sigma (St Louis, MO); solutions were made with 18.2 MΩ

Milli-Q water (Milli-QUV plus from Millipore, Billerica, MA). Lyophilized bovine serum albumin (BSA) (Sigma) and Intralipid (Baxter Healthcare Corp., Deerfield, IL), a

20% lipid fat emulsion was used in the interfacial study of proteins and lipids. The samples were characterized electrochemically using freshly prepared analytical grade potassium ferrocyanide (Sigma) such that its final concentration in a 20 microliter sample is 2.5mM. Capillary tubes (A.H. Thomas Co., Philadelphia, PA) having 0.025 mm wall thickness and 0.5 mm i.d. were used to direct the nitrogen jets to rotate the samples. The air flow rates were fixed at 160 mL/min for all measurements. A 1000 ppm stock solution of surfactant Triton X-100 (Sigma) in distilled water was used to prepare different dilutions of the surfactant.

3.3.2 Apparatus

Electrochemical measurements were made using CH100 electrochemical workstation

(CH Instruments, Austin, TX). Homemade substrates with platinum mini-disc electrodes were employed. The electrochemical cell was fabricated using Corning glass slide (7.5cm

× 2.5cm and 0.1cm thick, from Fisher Scientific, Pittsburgh, PA) as substrate. The WE was made from 150μm, 250μm diameter Platinum wire (Alfa Aesar, Ward Hill, MA).

Silicone elastomer (DOW Corning, Midland, MI) was applied to form the hydrophobic ring that confines the sample drop into a semi-sphere (fabrication procedure is described earlier [21]). Electrodes were polished on Microcloth polishing pad (Buehler, Lake Bluff,

54 IL) mounted on a Delta 31-120 disk sander (Delta, Jackson, TN). Alumina polishing

paste (Buehler) of 1 and 3 micron sizes were used for polishing.

The Ag|AgCl (3N KCl) reference electrode (BAS, West Lafayette, IN) and gold wire

spiral counter electrode (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma).

3.3.3 Procedures

3.3.3.1 Using the RSS to investigate protein-protein interaction by rheological

examination of samples containing different concentrations of BSA

It is known that proteins have a proclivity to migrate towards the air-liquid interface and

adsorb there [Gonzalez et al., 1970; Langmuir et al., 1940; Poole et al., 1984]. Once at

the surface, they undergo a change in conformation and reside in their lowest energy state

[Prins et al., 1998]; lateral interactions occur between adjacent proteins resulting in the formation of a contiguous protein film. We investigated these properties by conducting cyclic voltammetry experiments in 20 μl rotated samples containing 0.039, 0.0039 and

0.00039 g/dL BSA. These samples were prepared in pH 1 sulphuric acid; this implies that the BSA was in a completely unfolded state [Carter et al., 1994]. Potassium ferrocyanide was also added to the sample such that its final concentration was 2.5 mM. Potassium ferrocyanide was used to characterize the electrode response. One nitrogen jet was used

and same position of the jet vis-à-vis the sample was maintained to ensure reproducible

coupling of the sample surface and gas-jet in each case. Nitrogen flow rate was

maintained at 160 mL/min.

55 3.3.3.2 Assessing lipid-protein interaction at the surface

Investigation of lipid-protein interaction at the surface shows the competing tendencies of

proteins and lipids to occupy the air-liquid interface. This interaction has been the

continued object of investigation by researchers [Sprong et al., 2001, Verger et al., 1982;

Nylander, 1998, Pieroni et al., 1990; Poole et al., 1984, 1986, 1989]. Intralipid, a 20%

intravenous fat emulsion was used as a model lipid solution [Wretlind, 1981]. For a given

concentration of BSA (0.39 g/dL) the lipid content was varied and cyclic voltammetry of experiments were conducted in pH 1 acidified samples rotated by same gas-jet flow rate.

Here too, one nitrogen jet was used and same position of the jet vis-à-vis the sample was maintained to ensure uniform coupling of the sample surface and gas-jet for accurate comparisons. As a control, cyclic voltammetry for samples containing lipid only (no protein) were conducted using the same experimental parameters. For a constant lipid concentration (10:4000 dilution of Intralipid), the BSA concentration was varied and rotation properties of these samples were investigated using cyclic voltammetry. Nitrogen flow rate was maintained constant at 160 mL/min in all cases and the air-jet position was kept same for repeatable results .

3.3.3.3 Evaluating Critical Micelle Concentration (CMC) using RSS

Surfactants at concentrations lower than CMC are loosely integrated into the water

structure. However in the region of CMC, the surfactants form a monolayer at the surface

and micelles in the interior. Surfactants are known to reduce the surface tension of water

at the air-water interface. With increasing surfactant concentration, the surface tension

decreases until it reaches a value close to the CMC. Beyond this value, the surface

56 tension remains constant [Tsujii, 1998]. Tensiometers have been used to evaluate CMC

[26].The use of surface tension to evaluate CMC was attempted using the RSS. Surfaces with lower surface tension are by nature elastic. The elasticity of the surface alters the rotation of the sample and this has been probed by conducting cyclic voltammetry experiments in rotated samples with incremental concentrations of Triton X-100 surfactant (100 ppb to 250 ppm). In this case too, one nitrogen jet was used and same position of the jet vis-à-vis the sample was maintained to ensure uniformity. Also, using the same RSS setup 10 μL samples were used since they were better able to withstand the shear caused by the gas-jet for low values of surface tension at higher concentrations of the surfactant. Nitrogen flow rate was maintained constant at 160 mL/min in all cases.

3.4 Results and Discussion

3.4.1 Assessing rheological properties of samples containing different BSA concentrations

For concentration of BSA at 0.01 wt%, cyclic voltammetry in a rotated sample showed the redox peaks of potassium ferrocyanide (figure 3-1); this response is similar to when the sample is not rotated and is unlike the plateau currents observed in rotated (non- protein) aqueous samples [Chapter 1, 2]. The presence of these peaks is indicative of a stationary sample, despite the air-jet used to rotate it. Rotated samples containing higher

BSA concentrations (greater than 0.01 wt%) also show similar results [Chapter 2]. When the concentration of BSA is reduced further at 0.001 wt%, the current slightly increases in the rotated sample (Figure 3-1), and this is indicative of convection in the sample.

57 When the BSA amount is diluted to 0.0001 wt%, the current observed in the voltammograms becomes comparable to a sample with no BSA in the sample (Figure 3-

1).

At 0.01 wt% of BSA and above, the there is complete coverage of the sample surface by

BSA. There exists here lateral hydrophobic interaction with adjacent BSA molecules at the surface and a contiguous film is formed [Poole et al., 1984; Prins et al., 1998]; this shows protein-protein interaction at the air-water interface. This film is central to the stability of foams [Poole et al., 1984, 1986; Prins et al., 1998] and is known to be resistive to shear [Prins et al., 1998]; this manifests in the no increase in current even when rotation of the sample is attempted. Due to this shear-resistive film the sample surface, the gas jet velocity does not into rotation of the sample. At 0.001 wt % BSA and lower, the concentration of BSA is no longer sufficient to ensure complete coverage of the sample surface. This manifests in the slightly increased current in comparison to the case where a contiguous protein film exists. The current observed is however much lower compared to case when there is no protein in the sample. Presence of proteins at the interface reduces the surface tension; lower surface tension surfaces are inherently more elastic in comparison to a high surface tension surface. Hence part of the air-jet velocity that rotates the sample is expended in stretching of this elastic surface which results in less efficient rotation of the sample than in a sample with higher surface tension. This effect of low surface tension due to the presence of proteins progressively decreases with decreasing protein concentration. At 0.0001 wt%, the current observed is almost similar to the sample with no protein. Hence, results from the study of solutions with different

58 concentrations of BSA provide information on their properties and concentration at the air-water interface.

Figure 3-1 Cyclic voltammetry of 2.5 mM K4[FeCN6] at a 125 μm Pt disc electrode in rotated 20 μL samples containing 0.01 wt% BSA, 0.001 wt% BSA, 0.0001 wt% BSA solutions in pH 1 H2SO4. Control cyclic voltammograms of rotated samples also containing 2.5 mM K4[FeCN6] in pH 1 H2SO4 solution. Initial potential 100 mV, scan-rate = 100 mV/s. Samples were rotated by humidified nitrogen jets at 160 mL/min in each case.

3.4.2 Assessing lipid-protein surface interaction by rheological examination

In 0.01 wt% BSA solution, the cyclic voltammogram showed no current increase even in rotated samples indicating convection in the sample (Figure 3-1) due to the formation of a contiguous protein film at the air-water interface. With increasing amounts of lipid added

59 the BSA sample, the redox peaks of potassium ferrocyanide in rotated samples containing

BSA only was now replaced by a plateau current (Figure 3-2). This plateau current increased with increasing lipid concentration. On the other hand, plateau currents in a control with only lipid in the sample resulted in a decreasing current trend with increasing lipid concentration. This is due to the effect of reduced surface tension with increasing lipid concentrations at the air-water interface (Figure 3-2). Reduced surface tension imparts greater elasticity to the surface. Hence, when rotated the gas jet velocity does not completely translate into rotation of the sample, part of it is expended in stretching of the elastic surface. This variable elasticity of the surface is manifest in variation in plateau currents obtained.

Lipids are known to disrupt protein films [Poole et al., 1986]. Hence, in a sample with a given protein concentration i.e. 0.01 wt% BSA, with increasing lipid concentration, the plateau currents obtained increases with increasing disruption of the protein film. This fragmented surface provides for better rotation of the sample by the gas-jet than contiguous protein film at the surface (Figure 3-2). From the lipid control, we can infer that the increasing current trend is due to the protein film gets increasingly fragmented due to disruption caused by the increasing lipid content, despite evidence that there is a net reduction in the surface tension.

The above trend was verified by repeating the same experiment with 0.1 wt% BSA concentration. Plateau currents however were lower in the latter case due to increased viscosity with higher BSA concentration (not shown).

Figure 2 Plateau currents obtained from cyclic voltammetry of 2.5 mM K4[FeCN6] on a 125 μm Pt disc electrode in rotated 20 μL samples containing 0.01 wt% BSA and different concentrations of Intralipid in pH 1 H2SO4 demonstrating protein-lipid interactions at the air-liquid interface. Plateau currents from cyclic voltammograms 2.5 mM K4[FeCN6] in samples containing only Intralipid in pH 1 H2SO4 are shown as a control. Initial potential 100 mV, scan-rate = 100 mV/s. Samples were rotated by humidified nitrogen jets at 160 mL/min.

Using a fixed concentration of the lipid in the sample (1 wt% Intralipid in pH 1 sulphuric

acid) the BSA concentration was varied from 0.01 wt% to 0.05 wt% resulted in the

plateau currents of cyclic voltammograms showing a decreasing trend with increasing

BSA concentration (not shown). This is due to increasing viscosity of the sample with increasing protein concentration. The surface area of the hemispherical sample is fixed.

Lipids and proteins compete to occupy the air-water interface, the composition of which is dictated by their individual affinities for the interface. We have showed that at 0.01

61 wt% BSA concentration a protein film encapsulates the sample. On addition of a fixed

amount of lipid, the increased affinity of the lipid displaces some of the protein molecules

from the interface. Increasing the protein concentration does not alter the surface

composition; the increased protein concentration however does contribute to an increase

in viscosity of the sample. This higher viscosity results in lower currents. Hence, once the

surface of the sample is standardized, changes in the bulk properties (e.g. viscosity) of the

sample can be investigated.

This rheological study of lipid-protein solutions articulates how their surface interactions

can be investigated using the RSS. By standardizing the properties of the surface, it is

possible to investigate the bulk properties of such solutions. Hence, the RSS can be used

as a simple tool to study sample surface as well as bulk.

3.4.3 Evaluating Critical Micelle Concentration (CMC) using RSS

Since the RSS affords convection to the liquid micro-sample through its surface, changes

in surface tension are reflected in the cyclic voltammograms of rotated samples. At low concentrations of surfactants in water, surfactant molecules are loosely integrated in the solvent (i.e. water) and some present at the air-water interface with the hydrophobic part preferring to reside at the air-water interface and reducing the surface tension. At CMC, the surfactant molecules in the bulk of the solution form micelles and there is monolayer coverage of surfactant molecules at the air-water interface. For concentrations of surfactant greater than CMC, there are more micelles formed in the bulk, but no change in the constitution of the monolayer at the air-water interface. Therefore, the surface tension becomes constant at concentrations higher than CMC.

62 Cyclic voltammograms in rotated samples containing 2.5 mM potassium ferrocyanide

and different concentrations of surfactant Triton X-100 revealed decreasing plateau currents with increasing surfactant concentration. However for surfactant concentration

of 125 ppm and greater there was no further decrease in plateau current. It is known that

surfactants reduce the surface tension of the air-water interface. However beyond a

certain concentration called the Critical Micelle Concentration (CMC), there is no further

decrease in surface tension. In this context, the point where the plateau current does not

reduce further indicates the Critical Micelle Concentration of Triton X-100 at 125 ppm

(Figure 3- 3). The value obtained here is in good agreement with that observed in the

literature [Courtney et al., 1986; Mandal et al., 1988; Dow Corning].

0.9

0.8

0.7 CMC

0.6

0.5 Normalized Plateau Current

0.4

0.3 -1 0 1 2 10 10 10 10 Surfactant Concentration / ppm Figure 3 Plateau currents obtained from cyclic voltammetry of 2.5 mM K4[FeCN6] on a 250 μm Pt disc electrode in rotated 10 μL samples containing different concentrations of surfactant Triton X-100. CMC value obtained at concentration at which plateau current stops changing . Initial potential 100 mV, scan-rate = 100 mV/s. Samples were rotated by humidified nitrogen jets at 160 mL/min.

63 From the electrochemical investigations of surfactant solutions, we have demonstrated

the utility of the RSS as a simple tool to calculate the CMC.

3.5 Conclusions

The presence of surface-active molecules such as proteins, lipids and surfactants (e.g.

Triton X-100) at the air-liquid interface of rotated samples in the RSS modulates the

hydrodynamic electrochemistry of potassium ferrocyanide. Each of the aforementioned

samples modulates rotation of the sample differently; this modulation has been used to

interpret the interfacial properties of the above samples.

Samples containing protein BSA were used as an example to demonstrate ability of the

RSS platform to study protein-protein interactions at the air-water interface. At high enough concentration of the protein, this interaction leads to the formation of a contiguous film that encapsulates the aqueous protein sample; this film is known to oppose shear stresses and stabilize foam surfaces. The presence of this film was determined electrochemically using the RSS, where currents in air-jet rotated samples are equivalent to those obtained with stationary samples. For concentrations of protein not sufficient enough to form a film, electrochemical response from rotated samples are reflective of the change in surface tension associated with different concentrations of protein in the sample.

The RSS’ ability to study lipid-protein interaction at the air-liquid interface demonstrated.

Lipids are known to disrupt protein films; this was illustrated by the fact that currents

64 increased during rotation of the sample with increasing lipid concentrations for a fixed

protein concentration. This was not the case when no lipid (and also at low lipid

concentration) was present in the sample. Lipids and proteins compete for the air-liquid

interface and it was demonstrated that by rotation of the sample, quantifiable

measurements are possible to study lipid-protein solutions and the interaction that exists

between them.

The RSS is sensitive to changes in surface tension. Changes in sample surface tension are

reflected in changes in currents measured; lower currents correspond to lower surface

tension. The advantage of the RSS here is that since convection is afforded through the

surface by air/gas jets, there is no change in the interface as opposed to conventional

methodologies where contact is made with the surface in order to measure surface tension.

This was used to evaluating the CMC of surfactant Triton X-100.

Another advantage of using rotation of a liquid sample by air-jets to study its surface properties would mean that the surface would reach equilibrium faster. The favorable mass transport properties of the sample would enable the surface-active components to reach the surface faster; the surface is analyzed in steady-state conditions. The ability to investigate surface properties of small samples is a significant advantage in the analyses of biological and biomedical applications where sample sizes available are smaller.

65 Chapter 4

Electrochemical Determination of Protein Adsorption onto the

Electrode in Rotating Sample System

Gautam N. Shetty, Koji Tohda, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

66 4.1 Abstract

When exposed to biological matrices, electrodes are confronted with the problem of non-

specific adsorption of proteins onto their surface. This results in reduction of the active

surface area available for electrochemical analyses. In order to obtain a measure of the

extent of this inhibition of the electrode surface several techniques have been investigated

by several research groups. In this work, we seek to employ a method to estimate

adsorption of proteins onto the electrode surface in the Rotating Sample System (RSS).

By monitoring the current due to the under-potential deposition (UPD) of hydrogen and

its subsequent oxidation in a cyclic voltammogram on a platinum disc electrode, we were

able to study the extent of fouling of the electrode surface by proteins. Coating electrodes

with polymer spacer membranes have been employed to help alleviate the problem of

protein adsorption. Suitable choices of membrane to coat the electrode to be used in

biological matrices have been explored in this work.

4.2 Introduction

The surface activity of proteins is a fundamental property of these complex

macromolecules. Substrates of almost any type that come into contact with proteins tend

to become quickly occupied by proteins, leading to profound alterations in the physicochemical and biological properties of the substrate. Electrochemical analyses in biological matrices are hampered by the adsorption of proteins onto the electrode surface reducing the active surface area available for analyses [Brabec et al, 1981]. This limits

67 the lifetime of the electrode and hence its efficacy. The process of protein adsorption onto electrode surfaces is thought to be irreversible [Clark, 2002, Fang, 2001], and is known as electrode ‘fouling’.

Protein adsorption is a major concern in the electrochemical analyses of microliter sized samples, since the electrode size involved is already small. Any further reduction in the electrode active area due to non-specific adsorption of proteins would severely impair its capability for analyses in a biological matrix. Also, in the case of analyses of microliter sized samples, conventional filtering protocols are inaccessible.

Several techniques have been employed to study adsorption of proteins onto surfaces viz. ellipsometry [Logothetidis et al., 2005], surface enhanced resonance Raman scattering

(SERRS) [Rospendowski et al., 1991], attenuated total reflection infrared spectrometry

(ATR) [Ishida et al., 1991], total internal reflection fluorescence (TIFR) spectroscopy

[Fisher 1996], photon correlation spectroscopy (PCS) [Gun'ko et al., 2003], small angle x-ray scattering (SAXS) [Rosenfeldt et al., 2004], radioactive labeling [Rosenbloom et al.,

2004], solution depletion [Cornelius et al., 1992], scanning tunneling microscopy (STM)

[Friis et al., 1997], surface plasmon resonance [Silin et al., 2003], atomic force microscopy (AFM) [Friis et al., 1997] and cantilevers [Moulin et al., 1999].

Electrochemical techniques such as impedance spectroscopy [Xie et al., 2003] and voltammetry of potassium ferrocyanide [Guo et al., 1996] have also been used to study adsorption of proteins onto electrode surfaces.

68 A commonly used electrochemical technique that provides information on the free electrode area is explored in this work for the purpose of investigating protein adsorption for the first time. Electrochemical analysis of potassium ferrocyanide for studying protein adsorption is not possible in acidic pH due to its poor stability at low pH [Potassium ferrocyanide MSDS sheets]. However, assessing protein adsorption at low pH is especially important in case of trace metal diagnostics in biological samples, since the pre-treatment protocol involves acid dilution. We have explored in this work the use of under potential deposition of hydrogen as a technique to measure the electrode active area [Angerstein-Kozlowska, 1984]. This technique has been used to study the crystal structure of electrode materials [Yeager, 1978] and hence is a sensitive technique.

Coating of the electrode with a membrane has been used as an approach to alleviate the problem of fouling by proteins. To that end, we have explored in this work suitability of

Nafion, polyurathene and cellulose acetate hydrogen phthalate as possible choices. Their adhesion to the sensor substrate (including the electrode) and permeability were investigated.

4.3 Experimental

4.3.1 Materials

All chemicals were from Sigma (St Louis, MO); solutions were made with 18.2 MΩ

Milli-Q water (Milli-QUV plus from Millipore, Billerica, MA). Lyophilized bovine

serum albumin (Sigma) solutions were used to test repeatability of adsorption kinetics

69 obtained. Fetal bovine serum (Equitech-Bio Inc., Kerrville, TX) was used as model

matrix to simulate a biological matrix. Dilutions of the fetal bovine serum were made in

pH 1 sulphuric acid (Sigma). For studying adsorption of proteins in rotated samples,

capillary tubes (A.H. Thomas Co., Philadelphia, PA) having 0.025 mm wall thickness

and 0.5 mm i.d. were used to direct the nitrogen jets to rotate the samples. The air flow rates were fixed at 160 mL/min. The efficacy of Nafion (Sigma), polyurathene (Fluka) and cellulose acetate hydrogen phthalate (Sigma) was tested to provide protection to the electrode from adsorption of proteins.

4.3.2 Apparatus

Electrochemical measurements were made using CH100 electrochemical workstation

(CH Instruments, Austin, TX). The RSS platform was employed as the electrochemical

cell to study adsorption onto the electrode surface. Homemade substrates with platinum

mini-disc electrodes were employed [Chapter 1, 2]. The electrochemical cell was

fabricated using Corning glass slide (7.5cm × 2.5cm and 0.1cm thick, from Fisher

Scientific, Pittsburgh, PA) as substrate. The WE was made from 150μm diameter

Platinum wire (Alfa Aesar, Ward Hill, MA). Silicone elastomer (DOW Corning, Midland,

MI) was applied to form the hydrophobic ring that confines the sample drop into a semi- sphere (fabrication procedure is described earlier [Chapter 1]). Electrodes were polished on Microcloth polishing pad (Buehler, Lake Bluff, IL) mounted on a Delta 31-120 disk

sander (Delta, Jackson, TN). Alumina polishing paste (Buehler) of 1 and 3 micron sizes

were used for polishing.

70 The Ag|AgCl (3N KCl) reference electrode (BAS, West Lafayette, IN) and gold wire

spiral counter electrode (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma).

Humidified nitrogen gas jets were directed toward the sample from 0.5 mm i.d. glass capillaries.

4.3.3 Procedures

4.3.3.1 Underpotential deposition of hydrogen as a measure of electrode active (free)

area

Cyclic voltammetry in pH 1 sulphuric acid was used to study how the electrode active

area changes with protein adsorption. Underpotential deposition of hydrogen occurs just

before the evolution of hydrogen gas. This deposition corresponds to monolayer coverage

of hydrogen [Angerstein-Kozlowska, 1984]; this region of the cyclic voltammogram has

been used to calculate the electrode area. We use in this work the section of the

voltammogram corresponding to the oxidation of the underpotential deposited hydrogen

monolayer (hydrogen desorption) (Figure 4-1); this too provides information on the

electrode free area.

Cyclic voltammetry is repeated several times until voltammograms become reproducible

[Yeager, 1978]. This protocol is repeated before each experiment to study adsorption on

the electrode as a standardization procedure. Voltammograms measured during

adsorption are compared to this voltammogram conducted in a solution containing pH 1

sulphuric acid only.

0.3

0.2

0.1

-0.1 Current /Current uA -0.2

-0.3

-0.4

-0.5 1000 800 600 400 200 0 -200 -400 Potential (vs Ag|AgCl) / mV

Figure 4-1 Underpotential deposition of hydrogen was used to estimate free electrode surface area. Shaded region was monitored for loss of electrode area due to protein adsorption; this region corresponds to the desorption of hydrogen monolayer (hydrogen desorption). Cyclic voltammogram in pH 1 sulphuric acid at 0.5 V/s scan rate.

4.3.3.2 Investigating adsorption from different concentrations of fetal bovine serum

Adsorption kinetics from a sample containing 0.39 g/dl BSA acidified by pH 1 sulphuric acid were obtained. This was repeated 4 times and the kinetics obtained in each case were compared. Cyclic voltammograms were then conducted in pH 1 sulphuric acid solutions containing different concentrations of fetal bovine serum; 1%, 10% and 25% serum solutions were investigated. Back-to-back cyclic voltammograms were conducted and the areas corresponding to the hydrogen monolayer oxidation were compared to the same in the voltammogram without serum. This comparison yielded the relative free surface area

72 [Guo et al., 1996]. Comparison of each of the back-to-back conducted voltammograms

gave the adsorption kinetics.

4.3.3.3 Comparing protein adsorption in rotated and stationary samples

It is imperative to know how adsorption of proteins occurs in convected systems. Since,

the RSS application for body-fluid diagnostics would entail rotation of the sample,

adsorption from samples containing 10% fetal bovine serum was compared in stationary

and rotated samples. The relative free areas were evaluated in each case.

4.3.3.4 Protecting electrode by coating with Nafion

Nafion has been employed as a membrane coated on the electrode to protect it from

protein adsorption [Jaenicke et al., 1998; Kruusma et al., 2004]. Efficacy of the Nafion coated electrode was tested by cyclic voltammograms in a 10% fetal bovine serum solution and this was compared to the same with no serum. This comparison was also made for different thicknesses of the Nafion membrane viz. 1μm, 3 μm and 5 μm

(thicknesses were calculated from the values of densities of Nafion and its concentration provided by the manufacturer). This is done to provide with an optimal thickness of the

Nafion membrane. The choice of membrane thickness would be such that it affords maximum protection to the electrode but at the same time contributing significantly to an increase in the diffusion layer thickness. It is also essential that the membrane demonstrate good adhesion to the sensing substrate especially when the sample is rotated.

This was also tested by rotating a 10% fetal bovine serum sample with a humidified nitrogen jet at 160 mL/min.

4.3.3.5 Testing for efficacy of polyurethane and cellulose acetate hydrogen phthalate

(CAP) as an electrode coating membrane

73 Polyurethane has been employed for coating of biosensors [Shin et al., 2004] and hence

was investigated if it can be use as an electrode coating material in the RSS setup. Cyclic

voltammograms were conducted in pH 1 sulphuric acid solutions and a comparison was

made between voltammograms with and without the membrane. Cellulose acetate

hydrogen phthalate [CAP] was also investigated as an electrode coating membrane. It has

been employed as an enteric coating for capsules or tablets [Maharaj et al., 1984] and is

insoluble at low pH [Spitael et al., 1980]. Same experiments as those conducted for

polyurathene were conducted to test for efficacy of CAP.

4.4. Results and Discussion

Protein adsorption onto a platinum disc electrode was characterized by investigating the

reduction in free surface area by UPD of hydrogen. Cyclic voltammograms in a pH 1

sulphuric acid sample containing protein were compared with a voltammogram in a sample with no protein to monitor protein adsorption.

4.4.1 Adsorption from different concentrations of fetal bovine serum

In order to test repeatability of the results obatined, back-to-back voltammograms were

conducted in 0.4 wt% BSA solutions. Each voltammogram when compared to the

corresponding voltammogram in a sample containing pH 1 sulphuric acid only (figure 4-

2A) provides information about the relative decrease in free electrode area at that instant.

A plot of relative free electrode area obtained from each voltammogram provides the

adsorption kinetics (Figure 4-2).

1 0.3 A 0.9 B 0.2 0.8 0.1 0.7

0 0.6

-0.1 0.5

Current / uA 0.4

-0.2 area free Relative 0.3 -0.3 0.2 -0.4 0.1

-0.5 0 1000 800 600 400 200 0 -200 -400 0 50 100 150 200 250 300 Potential (vs Ag|AgCl) / mV Time / s

Figure 4-2 (A) Monitoring the region of hydrogen desorption in the CV provides information on protein adsorption; (B) comparison with region without protein gives relative free area and on repeating the same gives information on protein adsorption kinetics. Cyclic voltammogram in pH 1 sulphuric acid and 10 % fetal bovine serum at 0.5 V/s scan rate.

Adsorption kinetics was obtained in samples containing 1%, 10% and 25% serum in pH 1

sulphuric acid for 300 s. With increasing serum concentration the relative free area

decreased indicating increasing protein adsorption onto the electrode. This is consistent

with the fact that there will be increased adsorption with increased concentration of the

protein. Hence, it is possible to employ the method of using UPD of hydrogen to study

adsorption of proteins onto the electrode.

4.4.2 Adsorption in stationary and rotated samples

75 Adsorption studies in a 10% serum sample in stationary and rotated samples revealed that

there is increased amount of adsorption of the protein in a rotated sample than a

stationary sample. Favorable mass-transport properties exist in rotated samples.

Adsorption of proteins is inhibited however by steric hindrances caused by already adsorbed proteins [Yang et al., 2003], and this limits adsorption of proteins despite

favorable mass-transport properties in a rotated sample. The enhanced mass-transport however, provides for denser packing of the adsorbed protein molecules and hence the slightly increased amount of protein adsorption. Enhanced mass-transport also provides for the protein molecule with the most affinity for adsorption onto the electrode [Slack,

1995] faster despite its lower concentration in some cases.

4.4.3 Nafion as an electrode coating material for protection from fouling

In the comparison of a bare electrode, it is observed that more relative free area is exists

when the electrode is coated with a Nafion membrane (Figure 4-3). To understand how

membrane thickness can affect the relative extent of protein adsorption, adsorption from

10% fetal bovine serum solution onto an electrode coated with Nafion membrane of

different thicknesses was studied. It was observed that there was an improvement in the

relative free area when the membrane thickness was increased from 1μm to 3μm.

Comparison of electrode areas measured with 1μm to 3μm membranes in the absence of any proteins showed a reduction in the absolute area with an increase in membrane thickness. When the membrane thickness was increased to 5μm the relative free area did not increase relative to the electrode coated with a 3μm membrane. This shows that the there is improvement in protection from adsorption only up to a certain thickness of

76 Nafion (empirically estimated as 3 μm for Nafion); beyond this an increase in the membrane thickness would only contribute to an increase in the diffusion layer thickness.

However, when adhesion of Nafion was tested in a rotated serum matrix, it was observed

that the membrane flaked off from the substrate and consequently from the electrode.

Poor adhesion of Nafion onto electrode surfaces is known [Cha et al, 1993]; however this

becomes a more severe problem when the sample is rotated. Nafion helps protect the

electrode from adsorption of proteins, but due to its poor adhesion properties it is not

suitable in the context of the hydrodynamic electrochemistry in biological matrices.

1 bare electrode 0.9 3 micron Nafion

0.8

0.7

0.6

0.5

0.4 Relative free area free Relative 0.3

0.2

0.1

0 0 50 100 150 200 250 300 Time (s) Figure 4-3 Protein adsorption kinetics with and without Nafion. A 3 μm Nafion membrane was coated on a 250 μm Pt disc electrode. Cyclic voltammograms were conducted in pH 1 sulphuric acid and 10 % fetal bovine serum at 0.5 V/s scan rate.

4.4.4 Exploring polyurethane and CAP as electrode coating materials

Polyurethane has been employed as a biosensor coating material. A comparison of cyclic

voltammograms in stationary sample containing only pH 1 sulphuric acid before and after

77 coating the electrode with polyurethane showed severe attenuation of the current in case of the membrane coated electrode (Figure 4-4). The poor response of a polyurethane coated electrode indicates that the membrane does not allow efficient partitioning of water and the electrolyte into it. Hence, despite its superior adhesion properties, polyurethane is not a suitable choice as an electrode coating membrane.

-6 x 10 6 bare electrode

4 Polyurethane covered electrode

-2 Current /A

-4

-6

-8 1000 800 600 400 200 0 -200 -400 Potential (vs Ag|AgCl) / mV

Figure 4-4 Cyclic voltammetry of pH 1 sulphuric acid on a bare 250 μm dia. Pt disc electrode compared with the same coated with a 2 μm thick polyurethane membrane. Cyclic voltammogram in pH 1 sulphuric acid at 0.5 V/s scan rate.

The above experiment when repeated for a CAP coated electrode also showed severe attenuation of the electrode response (not shown); this was in case of a contiguous CAP membrane. The contiguous CAP membrane was formed when the solvent acetone was allowed to evaporate in an atmosphere of acetone. However, when the solvent was

78 allowed to evaporate without confining it to an atmosphere of acetone, it resulted in the formation of pinholes in the CAP membrane. This CAP membrane is hence porous.

Permeability and convection properties of the porous CAP membrane were demonstrated by cyclic voltammetry of potassium ferrocyanide in stationary and rotated samples on a

CAP coated platinum electrode. (Protection from protein adsorption and membrane thickness optimization discussed in chapter 6). Also, due to the presence of the phthalate group, the polymer is able to form hydrogen bonds with the glass substrate resulting in better adhesion to the substrate and the electrode. Hence, among the polymers tested for protection of the electrode in biological matrices, a porous CAP membrane is the most suitable choice of membrane in the RSS setup (Table 4-1).

Table 4-1 Suitability of different polymer membranes for coating a platinum electrode

Requisites Membrane material Substrate adhesion Permeability

Nafion Poor Good Polyurethane Good Poor Cellulose Acetate Hydrogen Phthalate (CAP) Good Poor Porous CAP Good Good

The suitability of each was evaluated based on their adhesion properties to the electrode (and substrate in general) during rotation and their permeability for electrochemical analyses.

79 4.5 Conclusions

Adsorption of proteins reduces the active electrode area; this fact was used to study protein adsorption on electrodes. It was shown that by monitoring the region of a cyclic voltammogram corresponding to the underpotential deposition of hydrogen and its corresponding oxidation (hydrogen desorption), protein adsorption onto the electrode can be assessed. This process however also effects desorption of proteins (described in chapter 5) and hence cannot be employed as a quantitative tool for investigating protein adsorption on surfaces. However, it is an effective method to study the trends of adsorption and can also be employed as a simple tool to estimate adsorption for system design purposes.

Convection causes increased adsorption of proteins onto the surface. Adsorption on the surface is limited by steric hindrances caused by adsorbed proteins. Coating by protective polymer membranes helps prevent substantial reduction in active surface area due to protein adsorption. Coating by the membrane itself causes a reduction in electrode area since the polymer is in direct contact with the electrode. However, a larger electrode fabricated to off-set reduction of electrode area caused by the polymer coating can help overcome this problem.

Three polymer membrane materials viz. Nafion, Polyurethane and CAP were evaluated for suitability of coating of electrode and were tested for adhesion to the substrate and the electrode, and permeability. The porous form of CAP was found to be the most suitable

80 for coating of the electrode for analyses in biological matrices. Although a spacer

membrane helps protect the electrode from adsorption, it does not completely eliminate

adsorption of proteins. It does help however, in increasing the lifetime of the electrode in a biological matrix.

81 Chapter 5

Electrochemical Desorption of Proteins

Gautam N. Shetty, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

This work is to be submitted to Nature Methods

82 5.1 Abstract

Protein adsorption onto a surface is thought to be irreversible. Adsorption of proteins

hence severely impacts the workability and longevity of electrodes in biological environments. This phenomenon is also prevalent in food and dairy industries and is a matter of concern. A method to effect desorption of proteins electrochemically is reported in this work for the first time. In this work, using cyclic voltammetry desorption is demonstrated by effecting desorption of bovine serum albumin (BSA) and in a fetal bovine serum matrix. Controls to understand the underlying mechanisms are also reported.

5.2 Introduction

The fouling of metal surfaces by non-specific adsorption of proteins is a matter of

concern in biomedical sensing, in the food processing industry and in the dairy industry.

Protein adsorption during in vivo or in vitro electrochemical analyses affects the lifetime

of the electrode [Chapter 4]. Adsorptive adhesion of proteins enhances the adhesion of

bacteria, resulting in ‘biofouling’. In the dairy industry, deposition of thermally unstable

materials in heat-transfer surfaces poses a problem [Geesey et al., 2000]. The process of

protein adsorption onto these surfaces is until now known to be irreversible [Clark, 2002,

Fang, 2001]. There are several types of interactions that may cause a protein to adsorb onto a surface; Van der Waals forces, electrostatic interactions and the hydrophobic

83 effect [Nadarajah et al., 1995]. Researchers have demonstrated how varying the electrode potential affects adsorption onto it demonstrating electrostatic basis for protein adsorption

[Guo et al., 1996]. The hydrophobic effect is considered in most cases as the chief cause

of protein adsorption on surfaces [Tilton et al., 1991]. This also forms the basis for

irreversibility of protein adsorption.

Surfactants have been used to address the problem in the dairy industry [Biswas et al.,

2002]. Surfactants however interact with proteins and helps effect its desorption from the

surface [Arnebrant et al., 1995]. However, the use of surfactants in alleviating the

problem of ‘fouling’ by proteins has met with only limited success [Biswas et al., 2002].

Surfactants have not been applied for biosensor applications, and even less in the case of

in vivo analyses. Attempts to address the ‘fouling’ of electrodes in biosensors involved

coating the electrode with spacer membranes such as Nafion [Jaenicke et al., 1998],

cellulose acetate [Cserey et al., 2001] and the like. Although, this provides protection to

the electrode it attenuates the electrode response since it blocks off some part of the

electrode area itself [Chapter 4], and acts as a mass transport barrier. In addition,

reproducible fabrication of the membrane coating is a non-trivial task [Kruusma et al.,

2004]. Efforts have also been made to explore use of lipids to dissolve adsorbed proteins

[Wilson et al., 2004]. It is known that proteins with higher affinity for the substrate displace proteins previously adsorbed on the surface, which is known as the Vroman effect [Slack et al., 1995]; for obvious reasons this does not however alleviate the problem of fouling.

84 In this work for the first time a method by which desorption of proteins can effected

electrochemically. By successive cyclic voltammetry scans, the ability to desorb proteins

from the electrode surface is demonstrated.

5.3 Experimental

5.3.1 Materials

All chemicals were from Sigma (St Louis, MO); solutions were made with 18.2 MΩ

Milli-Q water (Milli-QUV plus from Millipore, Billerica, MA). Fetal bovine serum

(Equitech-Bio Inc., Kerrville, TX) was used as model matrix to demonstrate desorption

of protein in a heterogeneous protein matrix. Lyophilized bovine serum albumin (BSA)

(Sigma) to demonstrate and understand the mechanism of desorption. Solutions of BSA

and fetal bovine serum were made in pH 1 sulphuric acid (Sigma). For studying desorption of proteins in rotated samples, capillary tubes (A.H. Thomas Co., Philadelphia,

PA) having 0.025 mm wall thickness and 0.5 mm i.d. were used to direct the nitrogen jets

to rotate the samples. The air flow rates were fixed at 160 mL/min.

5.3.2 Apparatus

Electrochemical measurements were made using a CH100 electrochemical workstation

(CH Instruments, Austin, TX). The RSS platform was employed as the electrochemical

cell to study adsorption onto the electrode surface. Homemade substrates with platinum

mini-disc electrodes were employed [Chapter 1, 2]. The electrochemical cell was

85 fabricated using Corning glass slide (7.5cm × 2.5cm and 0.1cm thick, from Fisher

Scientific, Pittsburgh, PA) as substrate. The WE was made from 150μm diameter

Platinum wire (Alfa Aesar, Ward Hill, MA). Silicone elastomer (DOW Corning, Midland,

sander (Delta, Jackson, TN). Alumina polishing paste (Buehler) of 1 and 3 micron sizes

were used for polishing.

The Ag|AgCl (3N KCl) reference electrode (BAS, West Lafayette, IN) and gold wire

spiral counter electrode (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma).

5.3.3 Procedures

5.3.3.1 Standardizing electrode condition

Back-to-back cyclic voltammetry scans were conducted until reproducible cyclic

voltammograms were obtained [Yeager et al., 1978]. This ensures stable and

homogenous electrode surface conditions. This was undertaken before the sample was

investigated for adsorption and desorption of proteins. The number of repetitive runs varied from 60 to 300.

5.3.3.2 Demonstrating desorption

Back-to back cyclic voltammograms in a sample containing 0.39 mg/dL BSA in pH 1

sulphuric acid were conducted to obtain the adsorption kinetics (this protocol is identical

to the one discussed in Chapter 4). The serum sample was replaced by sample containing

86 pH 1 sulphuric acid. And back-to-back cyclic voltammograms were conducted and the

region of hydrogen desorption was monitored for duration of time equal to adsorption

which in this case was 300 s.

5.3.3.3 Controls for identifying factor causing desorption

In order to further understand the causative agents of desorption of proteins, we

conducted a control. From the experiment demonstrating desorption of proteins, we

identified two probable causes viz. desorption due to presence of acid and cyclic

voltammetry. We allowed adsorption of protein from a 5% fetal bovine serum solution

and obtained an adsorption kinetic profile for 300 s. Then, in order to decouple the effect of acid from the cyclic voltammograms, the serum solution was replaced by a pH 1 sulphuric acid solution and allowed to stand for 300 s after which a cyclic voltammogram was conducted to check for change in electrode area. This was then followed by back-to- back cyclic voltammograms in the pH 1 sulphuric acid sample to confirm the cause of desorption. Further controls to understand the mechanisms involved in desorption were conducted in samples containing BSA.

5.3.3.4 Demonstrating desorption of proteins in a protein matrix

In the above experiments desorption was demonstrated by replacing the serum/protein solution by sulphuric acid. In order for this to be applied in a practical scenario, we need to demonstrate the ability to desorb proteins in a biological matrix. For this purpose, we studied the adsorption kinetics in three scenarios using cyclic voltammetry. In the first case, back-to-back voltammograms and the adsorption kinetics was obtained. In the second case successive cyclic voltammograms were run with a 5 second delay between successive runs. In the third case, a 10 second delay was introduced between successive

87 runs. In all three scenarios voltammograms were conducted in a solution containing 5% fetal bovine serum solution in pH 1 sulphuric acid. The adsorption kinetics in each case was compared.

5.4 Results and Discussion

Desorption of proteins was first demonstrated in a solution containing a known protein viz. BSA. Desorption was then studied in a heterogeneous matrix such fetal bovine serum.

5.4.1 Demonstrating desorption of serum proteins

Scanning the electrode potential in the reduction direction (in this case +1.0V to -0.25V versus Ag|AgCl reference electrode), leads to the underpotential deposition (UPD) of a hydrogen monolayer; this is the hydrogen reduction step [Will et al., 1960]. On scanning back in the oxidation direction, this monolayer gets oxidized. Integrating either the oxidation or reduction current corresponding to this region of the cyclic voltammogram conducted in sulphuric acid solutions has been established as a method to obtain free electrode area [Angerstein-Kozlowska, 1984]. This technique has also been used to study the crystal structure of the electrode materials [Yeager et al., 1978]; hence, it is a technique sensitive in the atomic scale to estimate electrode area. Back-to-back cyclic voltammograms were measured in pH 1 sulphuric acid solution at 0.5 V/s scan rate, adsorption kinetics from 0.39 g/dL of BSA acidified to pH 1 by sulphuric acid was monitored for 300 seconds (60 back-to-back voltammograms). Successive cyclic voltammograms in this matrix showed decrease in current corresponding to oxidation and reduction of the hydrogen monolayer. This is attributed to the reduction in electrode

88 surface area due to protein adsorption [Jackson et al., 2000]. On replacing the BSA

sample with the one containing only pH 1 sulphuric acid solution and measuring

successive cyclic voltammograms for 300 seconds (60 voltammograms), successive

voltammograms showed an increase in the hydrogen monolayer reduction/oxidation

current; an indication of an increase in free electrode area. The first voltammogram in the

60 voltammogram series was identical to the last voltammogram obtained when allowing

for adsorption of BSA. Hence, this increase in current which corresponds to an increase

in free electrode surface area is indicative of desorption of BSA (Figure 5-1).

1 1 0.9 0.9

0.8 0.8

0.7 0.7

0.6 0.6 0.5 0.5 0.4 0.4 Relative free area free Relative Relative free area free Relative 0.3 0.3

0.2 0.2

0.1 A 0.1 B

0 0 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Time / s Time / s

Figure 5-1 (A) Adsorption kinetics of 0.39 g/dL BSA in pH 1 sulphuric acid monitored by cyclic voltammetry on a 250 μm Pt disc electrode. (B) Desorption kinetics by monitoring cyclic voltammetry in pH 1 sulphuric acid only. Cyclic Voltammetry: Initial potential 1.0V, low -0.25V; scan rate 0.5V/s. All potentials were measured against Ag|AgCl electrode. Cyclic voltammograms were conducted in stationary samples.

5.4.2 Controls to identify mechanism of desorption

The first control experiment was conducted to understand the mechanism of desorption

observed on measuring successive cyclic voltammograms in acidic solution. This control

was to test if desorption of proteins was due to any cleansing action of the acid or the cyclic scanning of the electrode potential.

In order to test if the acid cleansed the electrode of the protein adsorbed, proteins were

allowed to be adsorbed from a 5% fetal bovine serum for 300 s and this was monitored

using cyclic voltammetry. This serum sample was then was replaced by a pH 1 sulphuric

acid solution; a cyclic voltammogram was measured in this solution immediately and

compared with the same measured after waiting for 300 seconds. No difference in the

relative free area was observed, confirming that the strong acidic condition did not cause

the proteins to desorb. However with back-to-back cyclic voltammograms, the desorption

effect was visible again (not shown). This confirmed that successive cyclic

voltammograms are responsible for desorption of proteins.

Controls were then conducted to find which region within a cyclic voltammogram was

responsible for desorption of proteins. The potential in the voltammogram was scanned

between 1.0V and -0.25V (versus Ag|AgCl reference electrode). In the reduction scan,

the region between 1.0V and 0.3V corresponds to the reduction of Pt oxide [Will et al.,

1960]. The region between 0 and -0.25V corresponds to the underpotential deposition of

hydrogen during the reduction scan [Will et al., 1960]. In the oxidation scan, the potential

between -0.25V and 0V corresponds to desorption of hydrogen and 0.3V and 1.0V

corresponds to the formation of Pt oxide layer [Will et al., 1960].

90 Isoelectric point or ‘pI’ is the pH value at which the net charge on a protein is ‘zero’

[Voet, 1980]. For pH values below the pI, the protein exhibits a net positive charge. It is

known that by varying electrode potential, we can alter adsorption onto an electrode [Guo

et al., 1996]. Desorption experiments above were conducted in solutions at pH 1. The adsorbed proteins are hence in contact with a solution with pH much lower than their isoelectric point (pI). Although unknown before, it could be possible that electrode potential could effect desorption of proteins. To test this, two controls were run. In both

controls protein BSA was allowed to adsorb from a 0.39 g/dL solution of BSA in pH 1

sulphuric acid for 300 seconds. In the first control the electrode potential was clamped at

the most positive potential (i.e. +1.0V versus Ag|AgCl electrode) employed in the

voltammogram for 300 seconds in a pH 1 sulphuric acid solution. In the second control

the potential was cycled between +1.0V and +0.3V (positive potential region of the scan);

this was repeated for 300 seconds in all. In order to monitor the free active surface area

available and to test for desorption of the protein, after each control a cyclic

voltammogram was conducted by replacing the protein sample with a pH 1 sulphuric acid

solution and the potential was scanned between 1.0V and -0.25V. It was observed that in

each of the controls, no desorption of the protein could be observed. In the case of

clamping the potential at +1.0V, we did observe a large increase in the Pt oxide reduction

peak because of the clamping the electrode at that potential would have produced a large

amount of platinum oxide. This proves that desorption was not caused by electrostatic

repulsion and provides further evidence that the protein-electrode interaction is not

electrostatic. Hence, by the process of elimination, we can imply that the region of

91 hydrogen UPD is responsible for observed desorption of proteins and also that the

principal driving force of adsorption is the ‘hydrophobic effect’.

A third control that was conducted was repeating the above protocol for protein

desorption in a pH 4.8 sulphuric acid solution. BSA desorption was observed in this case

from cyclic voltammograms after allowing for adsorption of BSA from a 0.39 g/dL

solution of BSA. The pI of BSA is 4.9 [Lockwood, 2000, Teramoto, 1999]; desorption of

BSA at pH 4.8 further emphasizes the fact that desorption is not driven by electrostatic

repulsion.

Cyclic voltammograms at physiological pH (7.4) have been used to study the

conformation of proteins and this effect is not visible there [Jackson et al., 2000]. No

desorption of proteins was reported. Hence, in order to effect desorption increased

coverage of the hydrogen monolayer (i.e. higher density of hydrogen in the monolayer) is

required, which is available lower pH (acidic) solutions.

5.4.3 Desorption in the presence of proteins

All of the above experiments demonstrated desorption in acidic solutions using cyclic

voltammograms with no protein added in it (i.e. the original protein solution from which

adsorption had occurred was replaced by a solution containing only sulphuric acid). In

order to test for the efficacy of this technique in a protein-containing sample, in three

different scenarios, the electrode potential was scanned between 1.0 V and -0.25 V

(versus Ag|AgCl reference electrode) in a solution containing 10% fetal bovine serum diluted with pH 1 sulphuric acid and cyclic voltammograms were measured in each case.

Three sets of experiments involved one with successive cyclic voltammograms were run

92 back-to-back, second with 5 second delays between voltammograms and thirds with 10 second delay between voltammograms. The relative loss of free area due to protein adsorption was monitored in each case over a period of 300 seconds. It was observed that when back-to-back cyclic voltammograms were conducted more relative free area was observed and it was least when there were 10 second delays between successive voltammograms (Figure 5-3). This set of experiments is similar to that used to study adsorption of proteins onto the electrode [Chapter 4]. However, it demonstrates how successive cyclic voltammograms can be used to desorb proteins in acidic solutions even in the presence of proteins in the solution.

1 back-to-back CVs 0.9 5 second delay 10 second delay 0.8

0.7

0.6

0.5

0.4 Relative free area free Relative 0.3 0.2

0.1

0 0 50 100 150 200 250 300 Time (s)

Figure 5-2 Comparison between adsorption profiles from cyclic voltammograms conducted back-to-back, 5 second delay and 10 second delay in 5% fetal bovine serum samples acidified by pH 1 sulphuric acid. This is to demonstrate capability to effect desorption in protein matrices. Cyclic Voltammetry: Initial potential 1.0V, low -0.25V; scan rate 0.5V/s. All potentials measured against Ag|AgCl electrode. Cyclic voltammograms were conducted in stationary samples.

5.5 Conclusions

An understanding of hydrogen UPD reveals that the HUPD adatoms are intrinsically repulsive to each other [Zolfaghari et al., 1997]. In an aqueous solution, the hydrophobic regions of the proteins are first exposed to randomly oriented water molecules. However, an ordering of water molecules occurs around the hydrophobic regions. Close to the substrate an ordered layer of water molecules stays between the protein and the substrate, squeezing out randomly oriented water molecules. This results in adsorption of proteins onto a surface by way of the hydrophobic effect and is driven by a reduction of entropy around the hydrophobic region of the protein due to the presence of ordered water molecules [Tilton et al., 1991]. The protein molecule stays adsorbed onto the surface unless its vibrational energy makes adsorption untenable [Brash et al., 1995] due to an increase in its internal energy. There exists a layer of structured water molecules between the protein and the surface, therefore reduction of protein content in the solution does not produce desorption of the protein from the surface by way of concentration equilibrium.

This was demonstrated by the fact that a standing acid solution did not effect desorption.

It is possible however for a protein molecule to displace an already adsorbed protein molecule if it is able attain a lower entropic state than the previously adsorbed protein

[Slack et al., 1995]; this is the Vroman effect. The ability of surfactants to partially effect desorption of proteins is another example of this effect [Arnebrant et al., 1995]. The formation of repulsive UPD hydrogen adatoms is able to provide the protein molecule

94 with extra vibrational energy that causes to increase the internal energy of the protein

needed to overcome hydrophobic force causing adsorption of the protein.

This technique of being able to desorb proteins using cyclic voltammetry can be used in electrochemical sensing for biomedical applications that are hampered by protein adsorption onto electrode surfaces from biological samples. In case of dairy and food

manufacturing this method would eliminate the need to employ surfactants and eliminate

disposal problems.

We have exploited the use of hydrogen UPD to effect desorption of proteins. We have

shown in this work that hydrogen evolution is not necessary for protein desorption; the

electrode potential was scanned negative enough only to form a monolayer of hydrogen

and not electrochemically evolve hydrogen. Evolution of hydrogen showed increased

desorption rates, but would be impractical in protein containing solutions due to high

physical stability of any bubbles that may be formed. For applications requiring cleaning

of surfaces fouled by protein adsorption (e.g. dairy), evolving hydrogen by reduction of

water would be more effective method to desorb proteins.

95 Chapter 6

Rotating Sample System: Trace Pb(II) Analyses in Serum and

Blood Samples

Gautam N. Shetty, Koji Tohda, Miklós Gratzl Department of Biomedical Engineering CASE, Cleveland OH 44106

96 6.1 Abstract

The Rotating Sample System (RSS) has been conceived in the authors’ laboratory as a

convection platform for microliter sized samples. With favorable mass-transport

properties, we report detection capability of trace Lead (Pb) in microliter sized serum and

blood matrices on an Hg pre-deposited platinum electrode. Adsorption of proteins onto

electrodes is a deterrent to electrochemical analyses in biological matrices. We introduce

in this work the use of a Hg pre-deposited Pt electrode coated with porous cellulose

acetate hydrogen phthalate (CAP) membrane for protection from fouling by proteins. Due

to the miniature nature of the system, the RSS provides an enabling technology platform

for point-of-care screening for toxicity due to Pb exposure.

6.2 Introduction

Trace metals in body-fluids need to be monitored; some for their toxicity and some for

their necessity. With better understanding of the toxicity of certain metals even in trace

quantities, their monitoring in body fluids has gained increased importance [Wang, 1982].

Conventional methodologies for trace metal detection include Atomic Absorption

Spectroscopy (AAS) [Baralkiewicz et al, 1996], Inductively Coupled Plasma- Mass

Spectroscopy (ICP-MS) [Hansen et al, 2002] and electrochemical detection systems such

as rotating disc electrode (RDE) [Brihaye et al, 1983], sono-electroanalysis [Banks et al,

2004] and flow-injection [Jaenicke et al, 1998] techniques. AAS and ICP-MS techniques

involve bulky instrumentation and are labor intensive. They are also not amenable for

97 point-of-care applications and samples to be analyzed using these techniques would have to factor issues related to transportation and storage of these samples. Electrochemical techniques listed above need samples in the order of milliliters for analysis. This would entail drawing out of larger amounts of sample (e.g. blood) for analysis.

We consider the example of trace Pb analysis in this work. The deleterious effects of Pb

on human health include neurological [Goyer, 1996], renal [Batuman et al, 1981],

hematological [DeSilva, 1981], endocrine [ATDSR, 1999], cardiovascular [Victery et al,

1988], reproductive and developmental abnormalities and is considered as a probable

carcinogen [Cooper, 1976] (toxic effects on Pb are discussed in Appendix C). The toxic

effects of Pb are even more serious in case of children and neonates [Ernhart et al, 1986]

because of the severe impact of Pb on their development. Hence it is essential to provide

with a platform for analyses in microliter sized samples.

The RSS has the capability to generate efficient convection in microliter sized samples.

The aforementioned electrochemical systems employ different methods to generate

convection in the solutions they are employed to analyze. Convection in electrochemical

trace metal analysis is essential since it helps reduce the pre-concentration time needed for analysis due to improved mass transport in comparison to quiescent solutions

[Chapter 1]. The RSS generates convection by coupling linear gas jets’ velocities to the surface of a microliter sized sample drop placed on a stationary substrate and kept in position by a hydrophobic film in the shape of a ring. The inner diameter of the ring is calculated such that a 20 μL sample drop forms a near-hemisphere. The coupling of the

98 gas jets with the sample surface translates into rotation of the drop, thereby generating convection in it. The RDE system has been adapted to generate convection in samples as small as 500 μL [Miller, 1974]. The RSS however does not contain any moving mechanical parts and, also has an advantage that since the samples involved have minimal waste problems; also the sensing substrate can be microfabricated and made disposable. All of these make the RSS platform ideal for trace metal analyses in body- fluids. Efficient Convection properties of the RSS have been demonstrated in aqueous samples [Chapter 1, Cserey et al, 1997]. Trace Pb analysis has been demonstrated in aqueous samples using the RSS and a limit-of-detection of 260 ppt has been achieved

[Chapter 1].

Electrochemical analysis of blood for detection of Lead [Jaenicke et al, 1998; Kruusma et al, 2004], Cadmium [Kruusma et al, 2004], Zinc [Kruusma et al, 2004] has been demonstrated, although in much larger blood samples for large dilutions.

In this work, we will demonstrate detection capability of Pb in serum and blood matrices.

Although Pb is not present in serum since it is present primarily in blood [Appendix C], the example of Pb in serum is used to prove detection capability of other metals such as

Cu, Zn etc. that are analyzed in serum [Soylak et al, 2001]. Also in this work, for the first time the use of cellulose acetate hydrogen phthalate (CAP) as a suitable membrane to prevent fouling of the electrode in biological matrices is demonstrated.

6.3 Experimental

6.3.1 Materials

All chemical solutions were made with 18.2 MΩ Milli-Q water (Milli-QUV plus from

Millipore, Billerica, MA). The water thus obtained was then distilled using a quartz

distiller to obtain ultra-pure water. Samples were acidified with trace-select nitric acid

(Sigma) with which pH of the sample was maintained at 1. Lead solutions were prepared

from different dilutions of atomic absorption standard (Sigma). Polypropylene flasks

(Nalge Nunc International, Rochester, NY, USA) were used for storing Pb solutions to

preempt any contamination due to storage in glass containers [16]. Intralipid (Baxter

Healthcare Corp., Deerfield, IL), a 20% lipid fat emulsion was used as the lipid additive for better rotation properties in serum and blood samples [Chapter 2 ,3]. 1 wt% of this in pH 1 nitric acid was used as a lipid additive. Whole blood tested for HIV, HCV, HbsAg and syphilis (Innovative Research Inc., Southfield, MI) was used for blood analysis.

EDTA-Na was used as the anti-coagulant. Acid pre-treated blood was filtered by a 0.45

μm cellulose acetate filter (Corning, NY) and centrifuged (Eppendorf, Germany) to filter out large-sized precipitates. Capillary tubes (A.H. Thomas Co., Philadelphia, PA) having

0.025 mm wall thickness and 0.5 mm i.d. were used to direct the nitrogen jets to rotate

the samples. The air flow rates were fixed at 160 mL/min for all measurements.

6.3.2 Apparatus

100 Electrochemical measurements were made using CH100 electrochemical workstation

(CH Instruments, Austin, TX). Homemade substrates with platinum mini-disc electrodes

were employed. The electrochemical cell was fabricated using Corning glass slide (7.5

cm × 2.5 cm and 0.1 cm thick, from Fisher Scientific, Pittsburgh, PA) as substrate. The

WE was made from 125 μm, 250 μm diameters platinum wire (Alfa Aesar, Ward Hill,

MA). Silicone elastomer (DOW Corning, Midland, MI) was applied to form the

hydrophobic film ring that confines the sample drop into a semi-sphere (fabrication

procedure is described earlier [21]). The electrode center is offset 1.8mm from the center

of the ring. Electrodes were polished on microcloth polishing pad (Buehler, Lake Bluff,

IL) mounted on a Delta 31-120 disk sander (Delta, Jackson, TN). Alumina polishing

paste (Buehler) of 1 and 3 micron sizes were used for polishing.

The Ag|AgCl (3N KCl)reference electrode (BAS, West Lafayette, IN) and gold wire

spiral counter electrode (Alfa Aesar) were placed under the substrate in 0.1 M KNO3, connected to the sample side by a liquid junction filled with 1 wt% agar gel (Sigma).

6.3.3 Procedures

6.3.3.1 Coating electrode with CAP and optimizing thickness for Pb analysis

An acetone solution containing 3 mg/mL of CAP was prepared for coating an Hg pre-

deposited platinum electrode. Hg was pre-deposited ex situ from a 1.5 mM Hg solution in

5% HCl. 3 μL of the CAP solution was placed as if a sample and the solvent was allowed to evaporate. Since this is not done in an atmosphere of acetone vapor, evaporation is

rapid and the resultant CAP membrane thus formed is porous (pin-hole formation).

Electrochemistry on this CAP coated electrode was evaluated by stripping voltammetry

101 of Pb. A stationary sample containing 2.5 ppm Pb acidified to pH 1 by nitric acid was

evaluated on the electrode with and without CAP. Also, the same was evaluated with a

2.5 ppm Pb stationary sample also containing 25% fetal bovine serum. Pre-concentration

times in each case were adjusted such that all produced equivalent stripping currents.

From these results, the CAP membrane thickness was optimized.

6.3.3.2 Pb detection in spiked fetal bovine serum samples and resolving shape change

during rotation of sample

Using optimized CAP membrane parameters, detection capability of the RSS in a 20 μL sample containing 25% serum, lipid additive (for better rotation [Chapter 2, 3]) and Pb in the 75-750 ppb range was investigated. The samples were acidified to pH 1 using nitric acid. Samples were rotated by humidified nitrogen jet with flow rate 160 mL/min. The

same was also repeated for 10 μL samples.

6.3.3.3 Convection properties in hemoglobin samples

Studies in solutions simulating proteins (serum albumin) in serum were conducted to

understand their convective properties [Chapter 2]. However, in blood hemoglobin is the

most significant component [Geigy Pharmaceuticals, 1962], hence it is essential to

understand how its presence alters convective properties of blood vis-à-vis the RSS. A

sample containing 2.5 mM potassium ferrocyanide, 3.25 g/dL hemoglobin (normal blood

hemoglobin is 13 g/dL) was evaluated for in electrochemical response in both stationary

and rotated samples. A control was also run in rotated and stationary sample without hemoglobin or lipid for comparison.

102 6.3.3.4 Pb detection in spiked blood samples

Protocol similar to that done with serum was repeated. In a stationary sample containing

2.5 ppm Pb, 25% whole blood acidified by pH 1 nitric acid, stripping analysis was undertaken. The time taken for the stripping current with and without blood was compared to understand how blood matrix affects Pb detection. Feasibility of Pb detection was undertaken by stripping analysis of 100 ppb total Pb content in a 10% whole blood matrix. Lipid was added in this case to facilitate better rotation of the sample.

The sample was rotated using humidified, anti-parallel nitrogen jets at 160 mL/min.

For trace Pb detection in blood samples, a mixture containing one part (100 μL) whole human blood, one part pH 1 nitric acid and one part containing 100 ppb Pb was filtered using a 0.5 micron filter and a centrifuge. This is equivalent to having 100 ppb of Pb in blood diluted 1:3 with pH 1 nitric acid. The filtrate was then diluted 1:2 by adding lipid

(1 wt% Intralipid) for better rotation of the sample. 10 μL of this sample was rotated by

160 mL/min anti-parallel nitrogen jets.

6.4 Results and Discussion

Feasibility of Pb detection was established in aqueous non-biological samples [Chapter 1].

Trace Pb was detected on an Hg pre-coated Pt electrode. However, analyses in biological matrices require coating the electrode with a spacer membrane to prevent its fouling by proteins, and polymer membrane CAP has been employed for this purpose. Optimization of the CAP based sensing system was conducted in stationary serum and blood samples.

103 Optimization studies also helped in testing the robustness of the CAP coated electrode,

when exposed to serum and blood matrices.

6.4.1 CAP optimization

Stripping analysis in stationary 2.5 ppm Pb was conducted on a bare and CAP coated

electrode; the same was conducted in a 25% fetal bovine serum matrix containing 2.5

ppm Pb. Employing rotated samples is not possible because of the differences in rotation due to the presence of protein molecules at the air-water interface [Chapter 2]. Hence,

comparisons between different samples were made in stationary conditions only. Pb

stripping current was obtained from stationary, non-biological sample with a bare Hg pre-

deposited electrode with 30 s pre-concentration time. It was observed that the CAP

coated electrode needed 200 s and in presence of serum, the CAP coated membrane took

600 s (Figure 6-1A) to obtain the same amount of Pb stripping current.

The CAP solution was then diluted 10 times (0.3 mg/mL in acetone). With a thinner CAP

membrane and for a 60 s pre-concentration time in each case only slight difference in Pb

stripping peaks was observed with and without CAP (Figure 6-1B). However, in a 25%

serum matrix, the Pb stripping peak is reduced for a 60 s pre-concentration time. This

reduction in peak is due to a combined effect of reduction in area due to some adsorption

of protein and due to reduced diffusivity [Chapter 2] in a protein matrix. With the thicker

membrane, we observed that it takes about 3 times longer to get the same amount of Pb

stripping current in a serum matrix as opposed to a sample with no serum. Similarly, the

reduction in peak current in case of sample with serum as opposed to no serum is less by

104 about 3 times. Hence, by two independent measurands viz. current and time, we can infer

that in a 25% serum matrix the area and diffusivity together reduce by a factor of 3. This

also proves that making the CAP membrane 10 times thinner did not affect the ability of

this relatively thinner CAP membrane in affording adequate protection of the electrode.

The thinner, optimized membrane has an obvious advantage of not contributing

significantly to the diffusion layer thickness.

40 40 no CAHP, pre-concentration time = 60 s without CAHP, pre-concentration time = 30s with CAHP, pre-concentration time= 60 s 35 35 with CAHP, pre-concentration time = 200 s with CAHP, 25% serum, pre-concentration time =60s with CAHP & 25% serum, pre-concentration time = 600s 30 30 A B 25 25

20 20

15 15 Differential Current / nA Differential Current / nA 10 10 5 5

0 -200 -300 -400 -500 -600 -700 -800 -200 -300 -400 -500 -600 -700 -800 Potential (vs Ag|AgCl) / mV Potential (vs Ag|AgCl) / mV

Figure 6-1 Anodic stripping voltammetry of 2.5 ppm Pb for optimization of CAP membrane. (A) Comparison of pre-concentration times for bare electrode, CAP coated electrode, and CAP coated electrode containing 25 % serum; pre-concentration times yielding same stripping current. (B) Comparison of stripping currents in the three scenarios as in (A) for same pre-concentration time (60 s); CAP solution employed is 10 times more dilute than in (A). DPV parameters same as before [Chapter 1]. Stripping voltammetry conducted on a 150 μm diameter Pt disc electrode coated with Hg ex situ. Samples were rotated by anti-parallel, humidified nitrogen jets at 160 mL/min.

105 6.4.2 Pb detection in serum samples

Using the optimized CAP membrane, pH 1 acidified samples of 75-750 ppb Pb samples

also containing 25% serum were analyzed. A linear calibration was obtained with a

regression coefficient (r2) of 0.87 (not shown). Although promising, this performance is not as good as that obtained in aqueous samples (r2=0.99) [Chapter 1]. Analysis in

aqueous samples in this concentration range yielded a limit of detection of 14 ppb

[Chapter 1]. One observation made here was that during rotation of the sample, due to its

low surface tension on account of the presence of proteins and lipids at the air-water

interface [Chapter 3], there was distortion of the sample shape during rotation as opposed

to the near-hemispherical shape in case of aqueous samples.

Due to the low surface tension, the surface is unable to withstand the shear caused by the

high nitrogen jet velocities. The sample acquires a shape that can withstand this shear

force and has good mechanical stability. The resultant shape however is not reproducible.

Also, the sample spills beyond the area marked by the hydrophobic ring. We had reported

excellent linearity, repeatability and reproducibility when the coupling between the air-

jets and the sample was kept the same [Chapter 1]. However, due to irreproducibility of

sample shape and subsequently non-identical coupling of the air-jet with the sample, the

results obtained are less ideal in the case of analysis in a 20 μL biological sample.

106

DifferentialCurrent / nA 6

0 -200 -300 -400 -500 -600 -700 -800 Potential (vs Ag/AgCl) / mV

Figure 6-2 Repeatability of detection of 150 ppb Pb in a 10 μL sample containing 25% fetal bovine serum by conducting stripping voltammetry on a 150 μm Pt disc electrode that is pre-coated with Hg. A 900 s pre- concentration time was employed here. DPV parameters are the same as used before [Chapter 1]. Samples were rotated by anti-parallel, humidified nitrogen jets at 101 mL/min.

However, when a 10 μL sample for analysis, using the same substrate as that used earlier for 20 μL samples employed, it was found that the sample was confined in the region enclosed by the hydrophobic ring even during rotation of the sample. Detection of 150 ppb Pb using stripping analyses, we found excellent repeatability with a coefficient of variation of Pb stripping current peaks at 4.7% (Figure 6-2).

107 6.4.3 Convection properties of hemoglobin samples

Hemoglobin is the largest component of whole human blood and as a precursor to trace

Pb analysis using the RSS. Hence, it is important to determine effective rotation of

hemoglobin samples simulating rotation in blood samples. Studying the electrochemistry

of potassium ferrocyanide in rotated hemoglobin samples showed no increase in current

in comparison to the same sample in a stationary condition (Figure 6-3A). However, on

addition of lipid to a rotated sample containing hemoglobin, there was an increase in the

current (Figure 6-3B). This result is analogous to the results obtained with BSA

containing samples. Just as BSA forming a film that causes high surface viscosity,

surface properties of acidified hemoglobin samples have been known to display very high

surface viscosity [Gougerot, 1949]. Surface viscosity implies opposition of the surface to

motion, which in the context of the RSS implies that the surface does not couple the air-

jet to the sample and hence does not rotate it. Electrochemically, the surface viscosity in

the RSS manifests in non-increase of currents even in rotated samples.

The above studies were conducted in acidified samples simulating acid pre-treatment of

blood samples for trace metal analysis; this step helps release complexed metal ions for

analysis [Wang, 1982].

On addition of lipid, increased currents were observed, indicative of effective rotation of

the sample. The lipid required to be added here was 3 times more (3 wt%) than is the case

of the hemoglobin samples than in the presence of only serum proteins. Hence, lipid addition would be necessary and sufficient to produce effective convection in blood matrices using the RSS platform.

108

2 2 A B 1.5 1.5 Rotated samples

1 1 Current / uA Current / uA 0.5 0.5

0 0 Stationary samples

100 150 200 250 300 350 400 450 500 550 600 -0.5 Potential (Vs Ag|AgCl) / mV 100 200 300 400 500 600 Potential (Vs Ag|AgCl) / mV

Figure 6-3 Convection properties of hemoglobin samples were studied by cyclic voltammetry of 2.5 mM potassium ferrocyanide acidified by pH 1 nitric acid (A) Comparison between stationary ( ___ ) and rotated (-----) 20 μL samples containing 3.25 g/dL of hemoglobin. (B) Comparison between stationary and rotated samples of hemoglobin also containing 3 wt% Intralipid; control (-----) in samples containing neither hemoglobin nor lipid was also done. Voltammetry conducted on a bare 250 μm Pt disc electrode. Cyclic voltammetry conducted between 100 mV (also initial potential) and 600 mV at scan rate of 100 mV/s. Samples were rotated by anti-parallel, humidified nitrogen jets at 160 mL/min.

6.4.4 Pb analyses in whole blood

Similar to the experiments in serum, the efficacy of the CAP membrane that was

optimized for analysis in serum was evaluated by stripping voltammetry of 2.5 ppm Pb in

stationary samples of 25% whole blood (Figure 6-4); a comparison was then made with

sample with no blood. When extrapolated, it was found that it would roughly take time an

order of magnitude more in a blood matrix than the time to obtain equivalent stripping

current without blood. It was learnt that the blood supplied was treated with EDTA as an

109 anti-coagulant. EDTA in addition also forms a complex with Pb ions. This implies an

order of magnitude reduction caused by reduced diffusivity, electrode surface area and

reduced concentration of Pb due to complexation with EDTA. Interference from Cu and

Fe is not seen in our results despite their much higher concentrations in blood, and is

consistent with known results [Maeda et al, 2003].

350 s

5 / nA Current Differential 180 s

Background

0 -200 -300 -400 -500 -600 -700 -800 Potential (vs Ag|AgCl) / mV

Figure 6-4 Detection of 2.5 ppm Pb in a 10 μL stationary sample containing 25% whole human blood by conducting stripping voltammetry on a 150 μm Pt disc electrode that is pre-coated with Hg. 180 s and 350 s pre-concentration times were used. DPV parameters same as used before [Chapter 1]. Background measured in a 25% blood sample with no Pb. All samples were acidified with pH 1 nitric acid.

6.4.4 Trace Pb detection in whole blood

With a 250 μm diameter, Hg pre-deposited Pt electrode 10 μL sample containing a final

Pb concentration of 17 ppb was tested and a Pb stripping peak was observed after a pre-

110 concentration time of 1300 s (Figure 6-5). Since the anti-coagulant used by the blood supplier was EDTA-Na, it may be possible that Pb may have been complexed and the detected amount corresponds to a concentration even lower than 17 ppb. However, for detection of trace Pb that is also clinically significant, these results demonstrate feasibility of the RSS platform and the detection protocols developed.

60 Blood sample with Pb Blood sample with no Pb 50 Background

20 Differential / Current nA

0 -200 -300 -400 -500 -600 -700 -800 Potential (vs Ag|AgCl) / mV

Figure 6-5 Detection of Pb in 10 μL sample containing filtered human blood and lipid addition (1:1) by conducting stripping voltammetry on a 250 μm Pt disc electrode that is pre-coated with Hg and covered with CAP. Pb concentration in the sample tested is 17 ppb; concentration in whole blood before filtration was 100 ppb. Pre-concentration time was 1300 s. DPV parameters same as used before [Chapter 1]. Background measured in a sample with no Pb. All samples were acidified with pH 1 nitric acid.

111 6.5 Conclusions

The analytical capability of the RSS in serum and blood matrices has been demonstrated

by trace Pb detection in microliter sized samples. CAP as a suitable membrane to coat the electrode with to provide protection from protein adsorption was explored, and protocols

to test its suitability were explored. CAP provides with good adhesion to the substrate

while affording adequate protection to the electrode. By allowing for pinholes having

sizes smaller than those of proteins to develop during the coating of the membrane by

rapid evaporation of the solvent, we allowed for good permeability properties. This

enabled application of CAP for the first time as an electrode coating membrane in

addition to previously used membranes such as Nafion.

In order to ensure minimal distortion of the sample during rotation, a 10 μL sample was used for analysis. This was analyzed on the same substrate on which the hydrophobic ring ensures that a 20 μL water sample forms a near hemisphere. The sample surface tension reduces in the presence of biomolecules such as lipids and proteins and this by definition of surface tension makes the surface less taut than would be in case with no lipids, proteins or other surface-active substances. The reduction in sample volume helps providing mechanical stability to the sample and hence ensures that the sample stays confined by the hydrophobic even when it experiences severe shear stresses due to the air-jets. Analyses of Pb in diluted serum matrices demonstrated that repeatable results were possible even in the face of severe distortion of the sample surface caused by the air-jets.

112

Our understanding of how modification of surface properties by lipid addition can be leveraged for better rotation of serum and blood samples. This was demonstrated by the ability of the RSS to detect Pb in microliter samples which would enable for screening of

Pb in children and neonates, who are more severely affected by the toxicity of Pb. We have demonstrated that detection of Pb in blood is possible with acid dilution being the only pre-treatment required. Small size of the RSS platform and its simplicity of operation imply that it can be employed for point-of-care applications. Trace metal analyses in serum samples would also be possible.

We have demonstrated in this work feasibility and a developed a protocol for trace Pb

detection in serum and blood matrices using the RSS. With the help of microfabricated

electrodes it would be possible to achieve improved sensitivity and reproducibility. By

making available glassy carbon, boron doped diamond or pyrolyzed carbon electrodes in

the RSS setup it might be possible to eliminate the need for Hg pre-deposition and associated toxic risks from it. With micro-fabrication techniques, standardization of the electrode membrane coating would be possible and hence, ensure reproducibility of results.

113 Chapter 7

Summary and Future Work

114 7.1 Summary

We have demonstrated the suitability of the Rotating Sample System for diagnostics of microliter sized body fluids. System parameters such as electrode position, air-nozzle

position and air-nozzle size were optimized for optimal system performance. The studies

also yielded information on the secondary flow properties within the sample bulk.

Findings from this study would help validate the hydrodynamic theory being developed

for the Rotating Sample System.

The hydrodynamic studies in biological matrices showed the surface activity of bio-

molecules such as proteins and lipids modulate the hydrodynamics in the RSS. The study

was undertaken with the aim that if the RSS is to be used for the diagnostics of microliter

sized body-fluids, then it is not possible to filter out proteins and lipids and work in a

‘cleaner’ matrix. However, the outcome of this study showed how we can leverage the

interaction between biomolecular components to produce better rotation of the sample.

Another outcome of the research led to demonstration of the use of the RSS as a simple

tool for investigating the air-water interfacial properties such as surface tension and

surface composition. Surfaces of body-fluids need investigation, for example in neonatal

care, the surface tension of amniotic fluid is used to test for lung maturity. The RSS is

hence uniquely placed to be able to investigate both bulk as well as surface properties.

The problem of adsorption of proteins onto the electrode surface was addressed and a

suitable membrane for use in the RSS setup was developed. Porous cellulose acetate

115 hydrogen phthalate membrane was found as the most suitable candidate to coat the electrode for electrochemical analyses of body-fluids. A simple electrochemical technique to estimate protein adsorption at low acidic pH and accessible to the RSS setup was developed. Efficient electrochemical desorption of proteins has been reported for the first time in this work. By repeated cyclic voltammetry runs in an acidic medium, we were able to desorb proteins from the electrode surface. This finding presents numerous benefits for biomedical sensing and also in applications where protein adsorption poses a problem (e.g. food and dairy industries).

As an example of the analytical capability of the RSS, and combining results from all of

the above works, trace Pb analyses were conducted in serum and human blood matrices.

Determination of Pb in non-biological samples yielded a limit-of-detection of 260 ppt.

Repeatability and reproducibility of Pb analyses articulate the robustness of this simple

system. Capability of detection of Pb in serum and whole blood samples was

demonstrated.

Analysis of small samples is challenging especially since variations and errors which can usually be ignored in larger systems become significant. Analyzing small samples requires a low tolerance of variation in system parameters, and this can be made available with microfabrication technology. Analyses of biological samples have to deal with only one interface i.e. their contact with the sample. With the RSS, there is another interface to be factored in; this work has helped with a better understanding of it. Proof of concept of

116 use of the RSS platform for diagnostics of microliter samples has been established in this work.

The RSS can be employed as a diagnostic platform for analyses of biological, industrial and environmental samples, but also a simple research tool enabling research into newer areas.

7.2 Future work

7.2.1 Optimizing sample size for analyses of biological samples

It has been observed that biological samples exhibit lower surface tension than water. The original RSS design for a hemispherical drop was conceived for a higher level of surface tension. At high surface tension, the sample is able to withstand the shear caused by high velocity air-jets rotating it. With the lower surface tension in biological matrices, the sample is unable to withstand this shear by the air-jets that lead to deformation of the sample. From results in chapter 1, identical jet-sample coupling is essential for repeatable and reproducible results. The two options that can be explored are either increase the size of the inner diameter of the hydrophobic ring holding the sample for a given sample volume, or simply reduce the volume of the sample employed. Both of these help compensate for the reduction in surface tension and provide for structural stability of the sample to cope with the shear caused by the air-jets. By reducing the sample volume to

10 μL, favorable results were obtained for trace Pb analyses (chapter 6) and surface studies involving surfactants (chapter 3). However, this is not an optimized value and

117 although lower volumes do help cope with the problem of reduced surface tension,

evaporation of the sample becomes an issue to deal with. Hence, future work would

require optimizing the size of the sample or inner diameter of the hydrophobic ring.

7.2.2 Interfacial study using the RSS

This work has opened up a whole area of analysis using the RSS viz. interface study.

Interfacial studies have shown how proteins and lipids compete to occupy the air-liquid interface. From the hemoglobin-lipid and BSA-lipid studies, we have found that they have dissimilar affinities for the interface. Protein-lipid interaction is widely studied since this is the interaction occurring at the surface of a cell; hence the core of biological research. The RSS platform could provide an enabling platform for basic research.

Quantitative information on surface composition could also be deduced. Interfacial applications could also include testing of water quality to check for surfactant or other surface impurities. The small size of the RSS would enable it to be employed as an on- site diagnostic tool.

7.2.3 Desorption for precise protein patterning

Desorption of proteins could be engineered to help pattern proteins. It is possible to

fabricate micro/nano electrode patterns. By selectively desorbing of proteins or by

allowing selective adsorption patterns of proteins and also patterns of cells can be

possible to achieve. State-of-art in protein patterning is imprecise and involves complex

instrumentation. Chemotactic growth of neurons has been shown, but in order to achieve

directional growth it is essential to be able to switch off the chemotactic source. By

118 tethering this source to an electrode and then desorbing it, this can be achieved. This can be adapted to the BioMEMS platform in our laboratory.

7.2.4 Micro-fabricated sensors for trace Pb analysis

We have demonstrated feasibility of trace Pb detection in non-biological matrices, serum

and human blood using home-made sensing substrates. Trace Pb analysis would require

microfabricated sensing substrates. We demonstrated in this work how smaller electrode

area afforded better detection sensitivities. However with analyses in biological samples,

there is loss of electrode area and reduced diffusivity. Hence, in order to compensate for

this, an increase in the electrode surface area without reducing the signal to noise ratio

would be required. This can be realized by employing an array of small electrodes. The

small electrodes ensure that there is no increase in the background signal, but at the same

time having several such small electrodes effectively provide with a larger surface area.

7.2.5 Engineering protein adsorption

Protein adsorption is limited by steric hindrances. From our results in chapter 2 it is clear

that the adsorbed protein layer does not completely block off the electrode and is non-

contiguous. Hence, it should be possible to find a suitable protein that would not

completely block the electrode. However, a monolayer of such a protein would help filter

out other proteins, but at the same time permit passage of the analyte to be analyzed.

Such ‘protein-based’ filter would ease the complications associated with fabrication of polymer coating membranes. There is only monolayer coverage of proteins and would automatically ensure uniformity of thickness. Ensuring uniformity of membrane

119 thickness is currently is the biggest problem facing conventional membranes used to coat electrodes.

7.2.6 Enzyme activity loss due to adsorption

Proteins adsorb onto surfaces and undergo change in conformation. This is true of

enzymes too; however, change in conformation would lead to loss of activity. A unique

feature of the RSS is its high contact surface area to volume ratio. Hence, in the case of

RSS application for enzyme activity measurements it would be important to know the

kinetics of this loss of activity due to change in conformation of the enzyme. It would be

essential to prove that this change in conformation is much slower than the time required

for measuring enzyme activity using the RSS as a micro pH stat.

The above can be demonstrated by allowing the enzyme to adsorb onto the RSS substrate from a solution containing the enzyme only. This solution is then replaced by a blank solution to wash un-adsorbed enzyme molecules. After the ‘wash’ step, the adsorbed enzyme is exposed to a solution containing its substrate. The activity of the enzyme would need to be monitored against time to show how any change in conformation affects the activity.

Also, due to the hemi-spherical nature of the sample, there exists a large surface area of

the sample where the enzyme is exposed to an air-liquid interface. Conformational change can occur even at this interface due to the proclivity of enzymes and proteins to migrate to this interface. Study of the effect of conformational change at the liquid- substrate interface can also be used to provide an estimate of the kinetics of conformation change. This would give us an idea as to how quickly the analysis would have to be

120 completed in without having to factor loss of enzyme activity due to conformation

change at the two interfaces.

It is known that there is no aggregation of protein molecules (except insulin). Hence, in

order to preempt enzyme adsorption, the RSS substrate could be pre-treated with a protein solution allowing the protein to adsorb onto it. This protein monolayer would

prevent any adsorption of the enzyme molecule and any subsequent loss of activity due to

conformation change from adsorption. In order to preempt conformational change at the

air-liquid interface due to the migration of enzyme to this interface, we could include

protein and lipid additives that have a greater affinity to the air-liquid interface but do not

cause a change in conformation of the enzyme.

7.2.7 Kinetic measurements using the RSS

The RDE system has been used for measuring electrochemical reaction kinetics. The

basis of such measurements is that if mass-transport can be made fast enough (e.g.

rotation of electrode in RDE), then electrochemical measurements provides information

on the kinetics of electrochemical reactions. We have demonstrated favorable mass-

transport properties in the RSS, comparable to the RDE. Hence, it should be possible to

employ the RSS as a platform for reaction kinetic measurements.

121 Appendix A

Electrode Area and Sample Shape

The RSS hydrodynamic performance was evaluated on the basis of the diffusion layer

thickness (Equation 2, Chapter 1). Calculating the diffusion layer thickness requires

knowledge of the electrode active area, which could be different from the geometric of

the electrode area (given that the electrode is in the form of a circular disc).

Calculating electrode area

Electrode area was measured using two independent electrochemical techniques viz.

Chronoamperometry and Cyclic Voltammetry, and a comparison was made.

In chronoamperometry, the potential of the electrode in a 1mM potassium ferrocyanide

solution (with 0.1 M potassium nitrate as a background electrolyte) is switched from 500

mV (where no electrochemical reactions occur) to 0 mV, where reduction of ferrocyanide

takes place. This is done in an unstirred solution and the current is given by the Cottrell’s

equation [Bard, 2001]

⎛ A ⎞ )( nFDCti 4r (1) = ⎜ 2/1 + e ⎟ ⎝ πDt)( ⎠

2 where, where re is WE disc radius, A (cm ) the active surface, 1mM concentration C

(mol/cm3) of potassium ferricyanide with diffusion coefficient D (D = 7.3×10-6 cm2/s [2]).

Thus, the slope of a linear fit to the current versus inverse square root of time plot also

122 has information regarding active electrode surface area (Figure 1). The

chronoamperometry experiment was also used to calculate the contribution of edge

current to the total current measured. At the electrode edge the current contribution is not entirely planar; since the electrode is small (125 μm dia.), the current intercept of the above fit gives the contribution of the edge current to obtain the corrected value of the planar diffusion layer thickness.

Correcting for edge effects

Edge effects Planar Diffusion

3 sec. Chronoamperometry with no flow -8 x 10 5 500 mV

4.5

4 0 mV 3.5

3 Current (A) 12 sec. 2.5 Current sampling period 2 1.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 t-1/2 (sec-1/2) edge current to be subtracted nFADC δ = ()− ii plateau edge

Figure 1. Chronoamperometry experiment to calculate electrode area and edge current.

The electrode area was also calculated using cyclic voltammetry. The peak current in the

cyclic voltammogram is given by [Bard, 2001]

123 3 1 1 5 2 2 2 i peak ×= 1069.2 CAFDn ν (2)

where n is no of electrons exchanged, A (cm2) is the electrode area, F is the Faraday’s

constant (96500 coulomb/ mol), D (cm2/s) is the diffusion constant, C (mol/ cm3) is the concentration and v is the scan rate (V/s). A plot of the peak current versus the square root of the scan rate provides information on the area of the electrode.

-8 x 10 6

-8 x 10 7 6.5

4 6

5.5

5 2 4.5 Peak Current (A) 4 Slope=f(Area) 3.5

0 2.5 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Scan Rate Scan rate1/2 -2 Current (A)

-4

-6

-8 0.3 0.25 0.2 0.15 0.1 0.05 0 -0.05 -0.1 -0.15 -0.2 Voltage (V) Figure 2. Cyclic Voltammetry to calculate electrode area and edge current. (Inset) plot of peak current versus square root of scan rate.

Areas obtained from each were tallied with the area obtained from the cross-section of

the wire used to fabricate the electrode. The above experiments were done for each

position of the electrode while studying the dependence of the diffusion layer on

electrode position. The results are tabulated in Table A-1 show consistency of the values

obtained from chronoamperometry and cyclic voltammetry when compared to the

124 geometric area, which is obtained from the cross-section of the wire used to fabricate the

electrode.

Table A-1. Comparison of areas obtained using Chronoamperometry, Cyclic Voltammetry with cross-sectional area of the wire used to fabricate the electrode.

Position from center Area Area chronoamperometry Area wire cross section of sample (mm) Cyclic Voltammetry (cm2) (cm2) (cm2) 0.82 1.31 x 10-4 1.67 x 10-4 1.76 x 10-4 1.44 1.71 x 10-4 1.47 x 10-4 1.76 x 10-4 1.53 1.56 x 10-4 1.42 x 10-4 1.76 x 10-4 1.89 1.46 x 10-4 1.51 x 10-4 1.76 x 10-4

Studying sample shape

The shape of the sample is a net result of the surface tension, gravity and its interaction

with the surface. Given the sample volume and contact angle (surface tension) is possible

to predict the shape of the sample drop using Laplace’s capillarity equations [Neumann,

1996] (Figure 3).

4 3.5 3 2.5 2

1.5 (in mm.) 1

0.5

-0.5

-1 -3 -2 -1 0 1 2 3 (in mm.)

Figure 3. Shape of a 20 μL drop calculated using Laplace’s capillarity equation (algorithm courtesy Makoto Yoshida, PhD)

125 However, in case of the rotated sample centrifugal forces come into play. We would need

to approximate the extent of this deformation which would be important to know in the

consideration of the geometry of the drop during formulation of a hydrodynamic theory

for the RSS.

The pressure experienced at the surface due to the centrifugal forces is calculated below:

Centrifugal force = mω2x

x Mass, m = Density x Volume

= ρ x.dθ.dz.dr dr

Pressure = Centrifugal Force Area

x ∫ 22 θω dzdrdr = ρ 0 r xdθdz

x x ∫ ω 22 drr = ρ 0 x

= ρω2 x2 3

where, x is the horizontal cross-sectional radius of the droplet and varies along the axis of

the droplet from ‘0’ to R (contact radius of droplet) and x varies from 0 to r, ρ is the density of the sample, ω is the rotation rate.

126

Hence, accounting for centrifugal forces the Laplace’s capillarity equations [Neumann,

1996] can be written for a rotating drop as follows:

dφ ρω x 32 sin φ ' czb2 ++= − (3) ds 3γ x

dx cos φ= (4) ds

dz sin φ= (5) ds

Graphical explanation of the terms in the above equation is given in figure 4

Figure 4. Definition of the coordinate system for fluid interface. At a point (ri,zi), the turning angle is φ. The arc length, s, is measured along the drop. R1 and R2 are the two principal radii of curvature; R1 turns in the plane of the paper, and R2 rotates in the plane perpendicular to the plane of the paper.

127 Equation (3) is obtained by including the pressure caused due to the centrifugal force to the total pressure experienced at the interface.

With equations 3, 4 and 5 it would have been possible to estimate theoretically the shape

of the sample drop during rotation. However, the curvature b’ is a function of the rotation

and is unknown. This may be possible to evaluate by parameter estimation; but is a non-

trivial task.

Hence, we studied the images of rotated and stationary drops to find the deformation of

the drop during rotation. Subtraction between the images showed the differences between

the stationary and rotated drops. It revealed that during rotation there is lateral widening

of the drop (due to centrifugal forces) and dorsal flattening (effect of constant density

being maintained). This change in shape (Figure 5) is however minimal and hemispherical geometry can be assumed for formulation of the theory

Figure 5. The change in sample drop shape during rotation is summarized by an overlay of the rotated drop (red) on the stationary drop; changes in shape are indicated by arrows

128 Appendix B

Lead Toxicity

Background

Early warnings of poisonous properties of Lead (Pb) extend as far as the second century

B.C. [Waldron, 1973]. The early victims of Pb were mainly Pb workers and wine drinkers. Lead’s sweet flavor made it useful in wine-making. Lead-sweetened wine contained as much as 20 mg of lead per liter and was an important drink of upper class

Romans. It is speculated that the synchronous decrease in fertility and increase in psychosis among the Roman aristocracy implicated Pb poisoning in the fall of Rome

[Gilfillan, 1965]. It has been found that noted composer Ludwig Van Beethoven died of

Pb poisoning [CNN, 2000]. Recent analyses of Beethoven’s hair showed high amounts of

Pb, almost 100 times more than those found in people today. Beethoven’s health problems such as chronic abdominal pain, gout and kidney stones are now being traced to the toxic effect of Pb exposure.

Exposure to environmental Pb is a risk even as of today. Exposure occurs from breathing air, drinking water, eating foods and swallowing or touching dust/dirt that contains Pb.

With the phasing out of Pb in gasoline (early 1970s), Pb in paints, and in soils and dust have become the principal sources of exposure in the United States. Both children and adults are susceptible to the health effects of Pb exposure. However, the damage is more

129 severe in case of children because of the developmental effects of Pb toxicity (even with

low but chronic Pb exposure). This is more serious of a problem in inner-city

neighborhoods due to the presence of Pb based paints.

The incomplete development of the blood-brain barrier in fetuses and neonates increases

the risk of Pb entry into the developing nervous system, which can result in prolonged or

permanent neurobehavioral disorders [American Academy of Pediatrics, 1993]. In addition, children (infants and toddlers) are also likely to come into contact with lead-

contaminated soil/dust on carpets and floors. In addition absorption of Pb is considered

five to ten times greater in infants and young children than in adults [Alexander et al.,

1974, James et al., 1985]. Children are more sensitive to elevated Pb levels because their

brain, nervous systems and organ systems are still developing. Other than developmental

effects unique to children, the health effects experienced from adult exposures are similar

to those experienced by children, although the thresholds are generally higher.

Pb in the body

The absorption and biologic fate of Pb once it enters the human body depend on a variety

of factors. Children and pregnant women for example can absorb up to 70% of ingested

Pb, whereas adults typically ingest 20%. Most of the Pb absorbed in the body is excreted

either by the kidney (urine) or through biliary clearance (feces). Adults retain only about

1% of absorbed Pb, but children tend to retain more. Absorbed Pb that is not excreted is

130 exchanged primarily among three compartments: blood, soft tissue and mineralizing

tissues.

Pb in Bones and Teeth

Most of the Pb retained in the body is deposited in bones. Bones and teeth of adults

contain about 94% of the total Pb burden; this is about 73 % in children [Barry 1975]. Pb

is however not uniformly distributed and tends to accumulate in bone regions undergoing

active calcification at the time of exposure [Auf der Heide et al., 1992].

The biokinetics of Pb, the way it is taken up, distributed and stored throughout the body,

and its dynamic interchange between compartments of the body help explain why past

and current elevated exposures can lead to adverse health effects. An acute, high exposure to Pb can lead to high short-term BLL (BLL - Blood Lead Level) and cause symptoms of Pb poisoning; yet symptoms could appear in the absence of significant current exposure because of past accumulation of Pb.

Pb in Blood

Blood carries only a fraction of the total Pb in the body; it serves as the initial recipient of

absorbed Pb and distributes it throughout the body. The half-life of Pb in blood is

estimated to be about 28 days [Griffin et al., 1975]. About 99% of Pb in blood is

associated with red blood cells with the remaining 1% residing in blood plasma [DeSilva,

1981]. However, Pb in blood (BLL) is an important measure of exposure to Pb.

131 Pb in Soft Tissue

Blood distributes Pb to various organs and tissues. Liver, lungs and kidneys have the

highest soft-tissue Pb concentrations immediately after exposure [Gerhardsson et al.,

1995]. Children retain more Pb in soft tissues than adults; selective accumulation of Pb in

the brain may occur in the hippocampus [Adrinel et al., 2004].

Physiological Effects of Pb

Pb performs no physiological function in the human body and ideally should not be

present in the body at all. Pb toxicity can potentially affect every organ system. On a

molecular level, proposed mechanisms for toxicity include biochemical processes; for e,g. the ability of Pb to mimic actions of Calcium (affecting Ca related or dependent processes) [Adrinel et al., 2004] and also complexation with proteins. The health effects of Pb exposure are discussed below.

Hematologic effects

Pb inhibits the body’s ability to make hemoglobin by interfering with several enzymatic

steps in the heme pathway. Specifically Pb decreases heme biosynthesis by inhibiting δ-

aminolevulinic acid dehydratase (ALA-D) and ferrochelatase activity. Ferrochelatase,

which catalyzes the insertion of iron into protoporphyrin IX is quite sensitive to Pb

[Campagna et al., 1999]. A decrease in the activity of this enzyme results in the increase

of the substrate erythrocyte protoporphyrin (EP) in the blood cells. An increase in blood

and plasma δ-aminolevulinic acid and free EPs are associated with Pb exposure [EPA

132 1986a]. The EPA estimates that with BLLs at about 50 μg/dL (40 μg/dL in the case of children), decrease in hemoglobin manifests. Acute Pb exposure also leads to hemolytic anemia. In chronic exposure, Pb induces anemia by interfering with heme biosynthesis

and reducing blood cell survival. Pb exposure causes disturbances in heme metabolism

(Sakai et al., 2000). The heme synthesis pathway is involved with many other processes in the body including neural, renal, endocrine and hepatic pathways.

Neurotoxic effects

Beethoven’s neurological illnesses before his death are now being traced to high Pb

levels found in his hair [CNN, 2000]. High Pb levels lead to elevated levels of δ-

aminolevulinic acid (ALA), which leads to several neurotoxic effects (Campagna et al.,

1999). Increased ALA levels cause to disturb the GABAergic system. Being the most

sensitive target of Pb exposure, children are most vulnerable to the neurologic effects of

Pb toxicity because their brains and nervous systems are still developing and the blood-

brain barrier is incomplete. In children acute exposure may produce encephalopathy and its attendant signs (hyperirritability, ataxia, convulsions, stupor, coma or death). BLL of

about 70-80 μg/dL would indicate a serious risk. Children also suffer other neurologic

effects at much lower blood Pb levels such as a decrement in intelligence quotient (IQ).

There is also a probability of hearing impairment in children with increasing BLL and the disruption of balance and impairment of peripheral nerve function; these effects may begin as low as 10 μg/dL. Child neurologic effects may persist in adulthood, although thresholds are much higher (BLL for Pb encephalopathy is approximately 460 μg/dL).

133 Renal effects

Kidneys are the primary exit route for Pb. Acute, high dose of Pb induced impairment of

proximal tubular function manifests in aminoaciduria, glycosuria and pyperphosphaturia.

These effects are reversible, however, continued repetitive exposure can lead to toxic stress on the kidney, which if not relieved can lead to irreversible lead nephropathy. This occurs at BLL of about 60 μg/dL; serum creatinine and creatinine clearance are used as biomarkers of for damage from Pb. Since renal failure can contribute to the severity of hypertension, there exits a correlation with Pb exposure. Pb exposure is also believed to contribute greatly to the onset of ‘saturnine gout’, which may develop as a result of lead- induced hyperuricemia due to decreased renal excretion of uric acid. It is believed that renal disease is more frequent and more severe when associated with saturnine gout than with primary gout [Batuman et al., 1981].

Endocrine effects

Studies have shown that Pb impedes vitamin D conversion into its hormonal form, 1,25-

dihydroxyvitamin D, which is largely responsible for the maintenance of extra cellular

calcium homeostasis. Diminished 1,25-dihydroxyvitamin D in turn may impair cell growth, maturation, and tooth and bone development. This occurs with children having

BLL 60 μg/dL and above. In adults this poses a risk of lower bone densities and

osteoporosis. A weak negative correlation between duration of Pb exposure and thyroxin

and free thyroxin levels has been observed [ATSDR, 1999] suggesting adverse effect on

the thyroid with chronic Pb exposure.

134 Cardiovascular (Hypertension) effects

Low exposures (BLL~30 μg/dL) have shown only a low association of Pb exposure with

hypertension; however, greater exposures increase the risk for hypertensive heart disease

and cerebrovascular disease. Studies have reported an increase in blood pressure with an

increase in Pb exposure [Victery et al., 1988]. It is estimated that on a population mean,

with each doubling of blood Pb can account for a 1 to 2% variance in blood pressure.

Reproductive effects

Studies have shown that increased Pb exposure leads to abnormal sperm counts

[Alexander et al., 1996]; effects are manifest at BLL of 40 μg/dL. Long term exposure has also shown to diminish sperm counts and total sperm motility. With pregnant women, studies have indicated increased abortions, miscarriages and stillbirths [Nordstrom et al.,

1979].

Lead exposure

Most human exposure to Pb occurs through ingestion or inhalation. Pb based paints are a

major source of exposure for children. As Pb paint deteriorates it enters the body through

normal hand-to-mouth activity and inhalation [Sayre et al, 1974]. Pb exposure through

the combustion of gasoline has been reduced; however, industrial discharge does

contribute significantly to Pb exposure through inhalation. Atmospheric pollution contributed to Pb in soil and water. Workers in Pb smelting, refining and manufacturing industries are at highest risk for prolonged high levels of exposure. Pb occurs in water

135 through leaching from Pb-containing pipes that still remain un-replaced. Vegetables may also contain Pb due to uptake from the soil.

Table C-1 Standards and Regulations for Pb (source ATDSR)

1 μg/dL = 10 ppb

More details and references on Pb toxicity is available in ATDSR 2000 report.

136 Appendix C

Micro-pH stat for enzyme activity measurements

Background

A pH-stat is an instrument that is meant to maintain a pre-set pH in a physicochemical, biochemical, or live biological preparation, despite ongoing reactions or metabolic activity that tend to shift the pH away from the pre-set value. This is achieved in conventional pH-stats by continuously monitoring the pH in the specimen while adding a strong acid or base solution at a rate controlled by a feedback system so as to best maintain the pre-set pH at all times.

There is a wide range of experimental problems in the area of biological sciences and biotechnology where pH-stating is a required methodology, to obtain basic understanding of the natural phenomena and processes involved, or to help control man-made processes in an optimal fashion.

Conventionally available pH-stats can handle only relatively large sample volumes, typically in the order of milliliters. This is because they operate on the principle of mechanical (volumetric) delivery of finite volumes of reagents into the sample. The process of pH-stating thus causes continuous sample dilution. This complicates quantitative evaluation of the data. Switching between reagents of different

137 concentrations may be necessary to adapt to processes whose rates vary significantly in time.

Introduction to pH-stating

One experimental methodology that has found important applications in an unusually wide range of biological settings is the pH-stat technique. The first experiment involving a crude pH-stating method was reported in 1923 by Knaffl-Lenz who measured the rate at which esterase splits an ester into acid and alcohol by determining the quantity of base required to keep the pH of the enzyme/substrate preparation constant. His manual technique involved the observation of pH, and recording times and base quantities added, till the pH became relatively constant around the desired value.

To date different instrumental embodiments of the pH-stat principle have been realized, such as the pH-stat workstation made by Radiometer Analytical based on its TitraLab series, the pH-stat titrator of the Titrino series by Metrohm, or the AUT-501 workstation made by Analyticon. These instruments all involve the controlled volumetric addition of an acid or base to the sample whose pH is to be maintained at a preset level. This addition occurs by mechanical delivery of either reagent at a rate that ensures that the pH varies as little as possible around the desired value. Continuous homogenization of the sample is obviously a requirement. There is also a need for a pH sensing scheme, to feed the actual pH back to the control unit that is to determine the optimum delivery rate at any instant based on the actual (and recent) deviations of the observed pH from the set value.

138

Obviously, for pH-stating to make sense, some type of a reaction has to go on in the sample whose pH otherwise would not tend to vary. The rate of this reaction is equivalent to the acid or base delivery rate that fully compensates for the effects of the reaction on sample pH. One aim of pH-stating is precisely the determination of such reaction rates, and their variations over time. An example is the monitoring of the activity of an enzyme whose operation would shift the pH up or down if no pH-stating took place [Taylor et al.,

1985]. Another possible aim is to maintain some type of optimal pH for the given reaction. This is often needed when enzymatic reactions are studied, so for such systems a pH-stat simultaneously achieves two separate aims. There are experimental settings, however, where only one, or the other, effect of pH-stating is needed and used. In some bioreactors, or in the culture of certain microorganisms, the only aim is to maintain the right pH so as to achieve the highest efficiency for the reaction or culture [Kobayashi et al., 2000], and precise monitoring of an overall reaction rate is not needed. An example for the other aim is when pH-dependent operation of an enzyme system is to be assessed, where pH-values other than optimal are maintained so that the reaction rate at those pH levels can be measured [Yang et al., 1998].

In biology, pH-stating can be used to monitor any biochemical reaction that is accompanied by the production or consumption of protons (more precisely, hydronium ions) or hydroxyl ions. However, a pH-stat can be useful also when carbon dioxide, or bicarbonate ions are affected [Ficara et al., 2003] since they participate in equilibria that determine the pH of the sample.

139

Thus, the range of biological problems where a pH-stat is needed, or would be useful, encompasses the study of enzyme reactions such as the assessment of enzyme activities of lipases, esterases, or proteases. Another major area of application is the analysis of neutralization capacities or buffer capacities of substances often encountered in pharmaceutical, environmental, and agricultural sciences [Giger-Reverdin et al., 2002].

Further examples are: degree of hydrolysis (closely related to nutrient value) of different animal foods including those of marine aquatic animals [Ezquerra et al., 1997]; assessing the efficacy of antacids [Radiometer application notes]; or determining pH stability, total acidity, and/or alkalinity of natural surface waters [Radiometer application notes].

Theoretical considerations

An alternative to adding base convectively to samples that tend to spontaneously acidify can be electrolytic generation of hydroxyl ions from water, provided that the accompanying production of hydronium ions is separated from the sample by a salt bridge. This approach has been used to maintain pH at an enzyme based biosensor, and in some biological studies of H+ fluxes in various tissues as well as in a few research publications several decades ago involving enzyme samples in the order of 100 mL

[Adams et al., 1976]. Its general application to pH-stating for analysis of small samples of any origin has so far evaded interests. However, it has been envisioned that the technique can be developed for general analytical applications using the platforms already available in his lab for microliter sample analyses.

140

Thus, an alternative to mechanically adding acid or base to stat the pH is by altering the pH of the test solution based on the following electrochemical half cell reactions:

+ - at the anode: 2H2O → 4H + O2 + 4e

- - at the cathode: 4H2O + 4e → 4OH + 2H2

Figure 1. pH-stat realization for the Rotating Sample System, RSS (schematic)

Therefore, provided that the cathode and anode are separated by a salt-bridge, pH in the test solution can be increased or decreased by injecting current in the proper direction

(Figure 1). We note that reactions other than those shown above may contribute to

141 current and thus, interfere with the scheme. We will discuss these potential problems

later; at this time we focus on using the cathodic reaction for injecting hydroxyl ions

while at the other electrode, that is “beyond” a liquid junction, some type of a residual

current may flow which has no effect on sample pH.

We have visualized another experiment using the RSS to increase, and then decrease back, the pH of a 20 μL droplet by injecting first a cathodic, and then an anodic current

(100 μA) via a Pt microelectrode (100 μm diameter) embedded in the substrate holding the droplet. The color of the indicator dye (Bromthymol blue) in a pH 4.5, 0.1 M

Potassium Nitrate solution indeed changes from orange (acidic, pH=4.5) to dark blue

(basic, pH=8), and then back to orange (Figure 4) in the course of the experiment. The droplet was rotated deliberately at a very low rate to make the path of the generated base streak visible.

Figure 2. Rotating Sample System (RSS) with current injection in a 20 μL sample

These experiments prove the feasibility of the main concepts of this proposal and their

applicability for microliter size specimens. How close the results obtained with Faraday’s

142 law from current can be to the true base delivery will be tested in this project. In case of little non-Faradaic current, a calibration-free system may become a reality.

Sensor design for pH stating pH sensing is done potentiometrically, i.e. change in H+ concentration causes a change in potential at the pH sensitive electrode, which is measured against a constant potential electrode (the two constitute a pH sensing system). The pH stat proposed for microliter samples employs coulometric addition of acid/base for enzyme activity measurements.

Due to finite resistance of the sample, the injection of current causes a voltage drop in the sample known as the ‘iR’ drop. This voltage drop results in an artificial change in the pH meter reading resulting in an error in pH measurement. A change in sensor design helped mitigate this problem.

The original RSS setup (Figure 1-1, Chapter 1) had a single gel junction that connects the compartment housing the working electrode and the compartment housing the counter and reference electrodes. The micro pH electrode requires a reference electrode of its own, which was originally placed in the same compartment as the counter electrode.

Current flow between the working and the counter electrodes causes an ‘iR’ drop in the junction; this causes a fictitious change in the pH reading not indicative of a corresponding change in sample pH. To overcome this ‘iR’ drop problem, we proposed to use a system with two junctions, each connecting the sample with two separate compartments housing the reference and the working electrode. We thus separated the current path from the reference path (i.e. reference electrode for the pH electrode). When

143 a current is injected into the sample the above ‘iR’ drop effect in the junction is invisible to the reference electrode since the current going through the junction connecting the sample to the compartment housing the reference electrode is zero. The reference electrode is still exposed to ‘iR’ drop in the sample. However, on account of the large conductivity in the sample, this drop is negligible. Any change in observed pH is hence direct result of pH change due coulometric acid/base injection.

pH electrode

Gel junction

Reference electrode Counter electrode compartment compartment

Figure 3. 2-junction sensor design to mitigate ‘iR’ drop problem

144 Preliminary results employing the 2-juntion sensor (courtesy Hung-Yi Hsu, Tzu-

Hsiang Kao)

A LabVIEW based PID controller was designed to achieve pH stating. The controller accepts a pre-set value from the operator and maintains the pre-set value by controlling the voltage outputted to the voltage controlled variable current source. It receives feedback input from the pH meter via the serial port of the computer.

Figure 4. Figure showing performance of PID controller in re-establishing pre-set pH value of 3.5 when a 20 microliter KNO3 sample was spiked with 0.1 μL 5% KOH

145 The above figure shows how the controller helps correct the change in pH caused by addition of 0.1 μL of 5% NaOH.

Hence, it has been shown that pH stating in microliter samples by coulometric injection of acid/base is feasible.

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