Conjugation of 5(6)-Carboxyfluorescein and 5(6)

Home , Fluorescein

Sensors and Actuators B 161 (2012) 93–99

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.elsevier.com/locate/snb

Conjugation of 5(6)-carboxyﬂuorescein and 5(6)-carboxynaphthoﬂuorescein

with bovine serum albumin and their immobilization for optical pH sensing

a b a,c a,c,∗

Sarka Bidmanova , Antonin Hlavacek , Jiri Damborsky , Zbynek Prokop

Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5,

625 00 Brno, Czech Republic

Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

Enantis Ltd., Palackeho trida 1802/129, 612 00 Brno, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: An approach for the immobilization of a pH indicator in optical pH sensors and biosensors was developed.

Received 7 April 2011

Fluorescent dyes 5(6)-carboxyﬂuorescein and 5(6)-carboxynaphthoﬂuorescein were conjugated with

Received in revised form 6 September 2011

bovine serum albumin as a non-enzymatic scaffold protein and the conjugation procedure was optimized.

Accepted 17 September 2011

Fluorescent properties, sensitivity to temperature and photostability of conjugates have been studied and

Available online 6 October 2011

characterized into details. The conjugates were immobilized on glass support by: (i) glutaraldehyde cross-

linking or (ii) entrapment in sol–gel matrix ORMOCER with subsequent glutaraldehyde cross-linking. The

Keywords:

response to pH and leaching potential were evaluated. The sensor layer, based on immobilized conju-

pH indicator

Fluorescence gates of 5(6)-carboxyﬂuorescein and bovine serum albumin, displayed rapid response over the pH range

4.0–9.0, making it compatible with a range of applications such as bioprocessing, clinical diagnostics

Optical sensor

Immobilization or environmental monitoring. Immobilized conjugates of 5(6)-carboxynaphthoﬂuorescein and bovine

Conjugation serum albumin showed an enhanced sensitivity in alkaline pH levels, response for glutaraldehyde cross-

Fluorescein linking ranged from pH 9.1 to 11.0 and from 8.3 to 10.0 for the ORMOCER entrapment, however, control

of the indicator leaching was essential.

1. Introduction possibility of remote sensing and in vivo measurement. pH can be

measured in electrically noisy environments that would interfere

pH is one of the most frequently measured properties of with potentiometric-type electrodes [2–4]. Application of optical

solutions because many biological, chemical and geochemical ﬂuorescence-based pH sensors has been reported in marine

processes are dependent on this physico-chemical property. research [5–7], biomedical diagnostics [8] and monitoring of

Determination of pH is routinely performed using the glass elec- biotechnological processes [9,10]. Moreover, ﬂuorescence-based

trode [1]. Optical pH sensors, particularly ﬂuorescence-based, pH sensors can be used as transducers in enzymatic biosensors

have proven to be an attractive alternative to electrochemical for the determination of glucose [11,12], urea [12–15], creatinine

pH sensing [2]. They are typically constructed by immobilization [12], penicillin [12,16–20], acetylcholine [21], organophosphorous

of a ﬂuorescence pH indicator on the light guide from one or pesticides [22] and halogenated hydrocarbons [23].

more optical ﬁbers, which are used to couple light between the A wide range of indicator dyes are currently available for con-

indicator and the measurement instrumentation [3]. The result- struction of ﬂuorescence-based pH sensors and biosensors. Fluo-

ing sensors offer high sensitivity, feasibility of miniaturization, rescein is the most commonly used dye (Table S1) with a large

variety of technical applications due to high quantum yield and

its large absorption in the visible ﬁeld [24–26]. In most cases,

ﬂuorescein and its derivatives, e.g., 5(6)-carboxyﬂuorescein (CF,

Abbreviations: BSA, bovine serum albumin; CF, 5(6)-carboxyﬂuorescein;

Fig. 1), are sensitive in an acidic or near neutral pH region. Good

CF-BSA, conjugates of 5(6)-carboxyﬂuorescein with bovine serum albumin;

sensitivity in an alkaline pH range is known for another deriva-

CNF, 5(6)-carboxynaphthoﬂuorescein; CNF-BSA, conjugates of 5(6)-

tive 5(6)-carboxynaphthoﬂuorescein (CNF, Fig. 1). Both dyes were

carboxynaphthoﬂuorescein with bovine serum albumin; GA, glutaraldehyde;

MALDI–TOF MS, matrix-assisted laser desorption/ionization–time of ﬂight mass exploited in the construction of ﬁber-optic pH sensors (Table S2)

spectrometry; ORMOCER, ORganically MOdiﬁed CERamics.

and published in the articles [13,23,27,28].

∗

Corresponding author at: Loschmidt Laboratories, Department of Experimental

Selection of an immobilization technique for ﬂuorescent dyes is

Biology and Research Centre for Toxic Compounds in the Environment, Faculty of

a critical step in the development of a pH sensor. The sensors based

Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.

on entrapment often suffer from indicator leaching due to small

Tel.: +420 5 4949 6667; fax: +420 5 4949 6302.

E-mail address: [email protected] (Z. Prokop). size of a ﬂuorophore [29]. Functional groups of the dye are useful

94 S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99

acetone, methanol and toluene of HPLC grade were purchased from

Chromservis (Czech Republic). All other chemicals were purchased

from Sigma–Aldrich (USA). Aqueous solutions were prepared with

MilliQ water obtained using a Water Puriﬁcator Simplicity 185 of

Millipore (USA).

2.2. Preparation of conjugated pH indicators

Fluorescence pH indicators CF and CNF were covalently coupled

to BSA (Fig. 2) using a procedure described previously [32,33] with

following modiﬁcations. CF (5 mg) was dissolved in 1 ml of 0.1 M

Fig. 1. Chemical structure of 5(6)-carboxyﬂuorescein (CF) and 5(6)-

carboxynaphthoﬂuorescein (CNF). 2-(morpholino)ethanesulfonic acid with 0.5 M sodium chloride (pH

6.0). CNF (2 mg) was dissolved in 0.4 ml of methanol. Cross-linker

for covalent binding to the matrix or other molecules, e.g., proteins, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (25.5 mg for CF

which can be cross-linked to another one. Conjugation of the dye and 8 mg for CNF) and N-hydroxysuccinimide (3.8 mg for CF and

to the proteins, e.g., bovine serum albumin (BSA), offers further 1.2 mg for CNF) were weighed in a glass vial and the dye solu-

beneﬁts compared to direct immobilization of the dye. The advan- tion was slowly added. The reaction components were vortexed

tage comes from the two-step immobilization procedure: (i) and reacted for 1 h (CF) and for 0.25 h (CNF) at room tempe-

covalent binding of the dye to BSA and (ii) formation of the rature. Cross-linker was inactivated by addition of 1.4 ␮l (CF) or

sensor layer. BSA is inexpensive and possesses chemically well- 0.56 ␮l (CNF) of 2-mercaptoethanol. BSA (15 mg) was dissolved in

deﬁned structure. Incorporation of such a component improves 1.5 ml of 0.1 M sodium bicarbonate buffer (pH 8.3). The reactive

reproducibility of the immobilization protocol. Both immobiliza- dye solution (0.9 ml of activated CF and 0.3 ml of activated CNF,

tion steps can be optimized separately without inﬂuencing each unless otherwise noted) was slowly added while the protein solu-

other. Therefore, variations in immobilization of dye conjugates, tion was continuously stirred. The reaction mixture was incubated

e.g., conjugate content or development of the enzyme layer, can for 1 h at room temperature with continuous mixing. The reaction

be performed in a simple way. Although application of conju- was terminated by the addition of 0.15 ml of 1.5 M hydroxylamine

◦

gated pH indicator (CNF) with proteins, BSA and organophosphorus (pH 8.5) to the mixture and incubated for 1 h at 25 C. The mix-

hydrolase, was reported in optical biosensors [30,31], systematic ture was ﬁltered through the Millipore Millex syringe ﬁlter with

investigation of conjugates’ properties has not been described. the pore size of 0.45 ␮m (Millipore, USA). The conjugates were

The objective of this study was to compare and evaluate the separated from non-reacted dye by size-exclusion chromatogra-

differences in physicochemical properties of CF and CNF and their phy using Sephadex G-25 Superﬁne (GE Healthcare, Sweden) as

conjugates with bovine serum albumin (CF-BSA and CNF-BSA). Two the stationary phase and 50 mM phosphate buffer (pH 7.5) as the

methods for immobilization of conjugated ﬂuorescent dyes were eluent. Prepared CF-BSA and CNF-BSA were stored in the solution

tested: (i) direct cross-linking with glutaraldehyde (GA) and (ii) the with addition of sodium azide (ﬁnal concentration of 2 mM) or in

◦

combination of entrapment into ORganically MOdiﬁed CERamics a lyophilized form at 4 C.

(ORMOCER) and cross-linking. These methods are generally appli- CF-BSA and CNF-BSA with different dye-to-protein ratio were

cable for the development of ﬂuorescence-based pH sensors and synthesised using above mentioned procedure. Reactive dye solu-

␮

enzyme-based biosensors. Detailed characterization of immobi- tion (150, 300, 450, 600 and 900 l) was mixed with BSA solution

lized conjugates enables a better understanding of advantages and (15 mg of BSA dissolved in 1.5 ml of 0.1 M sodium bicarbonate

limitations of both immobilization techniques and facilitates their buffer, pH 8.3). Dye-to-protein ratio was determined for CF-BSA

selection for optical sensing. and CNF-BSA eluted in 100 mM glycine buffer (pH 8.3).

Matrix-assisted laser desorption/ionization–time of ﬂight mass

2. Experimental spectrometry (MALDI–TOF MS) measurements were carried out

using an Ultraﬂex III instrument (Bruker Daltonik, Germany). Sam-

␮ ␮

2.1. Materials ples (0.6 l) were pre-mixed with 2.4 l of the matrix solution

composed of 20 mg/ml 2,5-dihydroxybenzoic acid in water, triﬂu-

All chemicals were of an analytical grade and used without oroacetic acid and acetonitrile in a ratio of 79:1:20 (v/v/v). This

␮

further puriﬁcation. Fluorescein, hydroxylamine hydrochloride, mixture (0.6 l) was deposited on a stainless steel MALDI target.

2-mercaptoethanol and potassium dihydrogen phosphate were Analyses were acquired in a linear positive ion detection arrange-

purchased from Fluka (Switzerland). Cresyl violet was obtained ment covering the mass range from 2 to 200 kDa.

from AnaSpec (USA), hydrogen peroxide and sulfuric acid from

Penta (Czech Republic). 2-(N-morpholino)ethanesulfonic acid was

2.3. Immobilization of conjugated pH indicators

purchased from Carl Roth (Germany). The solution of ORMOCER

AL657 and photoinitiator Irgacure were from Fraunhofer Institute CF-BSA and CNF-BSA were immobilized on glass slides

for Silicate Research (Germany). Ethanol for the UV spectroscopy, (26 mm × 10 mm × 1 mm, Merci, Czech Republic) as a solid support

Fig. 2. Conjugation of ﬂuorescence pH indicator (F: CF or CNF) with BSA intermediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99 95

Table 1

Experimental conditions for ﬂuorescence measurements with ﬂuorescent dyes and their conjugates.

Fluorescent dye Preparation of sample Spectroﬂuorometer conﬁguration

Solubilization of dye/immobilization Amount of dye (␮l) in ex (nm) em (nm)

15 ml of phosphate buffer

CF Methanol solution (2 mg/ml) 1 491 513

CNF 10 590 662

CF-BSA As puriﬁed on Sephadex G-25 column 50 492 517

CNF-BSA 50 594 668

CF-BSA Immobilized with GA NA 502 523

CNF-BSA NA 621 677

CF-BSA Immobilized with ORMOCER + GA NA 492 525

CNF-BSA NA 615 675

NA – not applicable.

using two methods: (i) cross-linking with GA or (ii) entrapment in maximum emission and excitation wavelength (Table 1). Fluo-

ORMOCER and subsequent cross-linking with GA. rescence quantum yield of free and conjugated pH indicators

was calculated employing the comparative Williams’ method [34].

(i) Cross-linking with GA

Dilute solutions of ﬂuorescent dyes with an absorbance below

Glass slides were treated with 5% (v/v) hydrochloric acid

0.1 at the maximum excitation wavelength were prepared. Cor-

overnight and then with a piranha solution composed of

rected emission spectra were recorded on a spectroﬂuorometer

95.0–97.5% (v/v) sulfuric acid and 30% (v/v) hydrogen perox-

set according to Table 1. Fluorescein in 0.1 M sodium hydroxide

ide in a ratio of 3:1 (v/v) for 30 min. Subsequently, they were

(quantum yield of 0.95) was used as a ﬂuorescence standard for

washed thoroughly with distilled water and dried using paper

CF and CF-BSA, cresyl violet in methanol (quantum yield of 0.53)

cloth. The surface of the glass slides was modiﬁed using a pro-

as a standard for CNF and CNF-BSA [35]. Response to temperature

cedure previously described [33] and based on a 2% (v/v) silane

was measured with dye solutions prepared according to Table 1.

coupling agent 3-glycidoxypropyltrimethoxysilane (GOPTS) in

Both dye solutions and cuvette were equilibrated for 20 min at

◦

95% (v/v) ethanol. Lyophilized BSA (5 mg) and lyophilized CF-

a temperature of 1, 5, 10, 15, 20, 25, 30, 35 and 40 C.

BSA or CNF-BSA (5 mg) were dissolved in 50 ␮l of 50 mM

Two experimental procedures were used to evaluate photo-

␮

phosphate buffer (pH 9.5). This mixture (2 l) was applied on

stability of free and conjugated pH indicators. The short-time

the modiﬁed glass slide with a pipette. The glass slide was kept

photostability was determined by continuous recording of the

◦

in the air for 5 min and then exposed to GA vapours for 30 min

emission spectra for 3 h at 20 C, then the dye solutions were stored

◦ ◦ ◦

at 20 C. The immobilized conjugates were stored overnight in

at 4 C in dark for 24 h. Dye solutions were equilibrated at 20 C for

10 mM phosphate buffer (pH 9.0).

30 min and spectra were acquired for each dye for 3 h, as described

(ii) Entrapment in ORMOCER and subsequent cross-linking with GA

previously. Long-term photostability was evaluated for sterile dye

◦

Glass slides were washed thoroughly with toluene, acetone,

solutions stored at 4 C for 100 days shielded from ambient light.

ethanol and 1 M sodium hydroxide. Then they were washed

Leaching was determined for immobilized conjugates stored in

◦

with distilled water and dried using paper cloth. The ORMOCER

10 ml of 10 mM sterile phosphate buffer (pH 9.0) at 4 C. Leaching

solution was prepared by mixing 1.25 g of ORMOCER AL657 and

of conjugates was measured after 1, 3, 7, 14, 21 and 28 days. The

0.0125 g of photoinitiator Irgacure equilibrated to room tem-

amount of leached conjugates was assessed as the ﬂuorescence of

perature. A well-vortexed mixture (2 ␮l) was applied on the

conjugates in storage buffer after removal of the glass slides.

glass slide using a pipette. The glass slides were placed below

a high-pressure mercury lamp (100 W, Saﬁbra, Czech Republic)

3. Results and discussion

for 60 min for hardening of the immobilized layer. Lyophilized

BSA (5 mg) and lyophilized CF-BSA or CNF-BSA (5 mg) were

3.1. Free and conjugated pH indicators

dissolved in 50 ␮l of 50 mM phosphate buffer (pH 7.5). This

mixture (2 ␮l) was applied on the glass slide with preformed

3.1.1. Fluorescence properties

sol–gel layer. The glass slide was kept in the air for 5 min and

◦

Spectrochemical properties of pH indicators, e.g., spectral ma-

then exposed to GA vapours for 30 min at 20 C. The immo-

xima, pH sensitivity and quantum yield, can be substantially

bilized conjugates were stored overnight in 10 mM phosphate

affected by conjugation. The effects of conjugation on pH-

buffer (pH 9.0).

dependent excitation and emission spectra of free and conjugated

2.4. Characterization of conjugated pH indicators indicators CF and CNF were evaluated (Figs. S1 and S2). The spec-

tra exhibited the same shape, in all cases the ﬂuorescence intensity

Fluorescence spectra were measured using spectroﬂuorome- increased with increasing pH. The excitation maximum was almost

ter FluoroMax-4P (HORIBA Jobin Yvon, USA) with a 150-W xenon identical for free and conjugated CF, the emission maximum of

arc lamp as a light source. A square quartz cuvette with a 1-cm conjugated CF was slightly shifted (Table 2). The excitation and

pathlength was used for dye solutions, and a triangular quartz emission maximum of conjugated CNF exhibited a slight shift to

cuvette for immobilized dyes on glass slides. Sample solutions were higher wavelengths in comparison to free CNF (Table 2).

introduced into the cuvette by injection with a syringe. All measure- Effect of dye-to-protein ratio on ﬂuorescence intensity was

◦

ments were carried out in 10 mM phosphate buffer at 20 C (unless investigated and an optimal labelling level was determined for each

otherwise noted) minimally in triplicate. The pH meter ORION Star indicator (Fig. 3). The dye-to-protein ratio signiﬁcantly inﬂuenced

2 (Thermo Scientiﬁc, USA) with pH-electrode Double Pore (Hamil- the ﬂuorescence of conjugates. Moreover, CF-BSA and CNF-BSA

ton, USA) calibrated using three pH reference buffers (Hamilton, revealed a different proﬁle and optima of the dye-to-protein ratio.

USA) was used for pH measurements. The pHs of solutions were Fluorescence of CF-BSA continuously increased with increasing

adjusted by addition of 3 M hydrochloric acid or 3 M sodium dye-to-protein ratio to the highest point at the value 5.4. This ratio

hydroxide. The excitation and emission spectra were recorded at was interpreted as the optimum when CF-BSA exhibited sufﬁcient

96 S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99

Table 2

Fluorescence properties of ﬂuorescent dyes and their conjugates in free and immobilized form in aqueous buffer.

a a

Fluorescent dye Form ex (nm) em (nm) Quantum yield pH sensitivity pKa value

CF Free 491 513 0.93 5.0–7.8 6.36

CF- Free 492 517 0.48 5.0–7.9 6.43

BSA GA 502 523 NA 4.0–6.5; 6.5–9.0 7.69

ORMOCER + GA 492 525 NA 4.0–6.0; 6.0–8.7 7.42

CNF Free 590 662 0.05 6.7–9.1 7.97

CNF- Free 594 668 0.05 6.7–9.5; >9.5 8.09

BSA GA 621 677 NA 9.1–11.0 10.16

ORMOCER + GA 615 675 NA 8.3–10.0 9.30

NA – not applicable.

Spectral maxima were determined in pH 9.0; the only exception was pH 11.0 for immobilized CNF-BSA since at this pH the dye exists in the deprotonated form, yielding

maximal ﬂuorescence signal.

ﬂuorescence for application and the consumption of the material is

still reasonable. Synthesis of CF-BSA with a higher dye-to-protein

ratio produced an excess of unbound indicator. An optimum dye-

to-protein ratio for CNF-BSA (when the conjugate showed the

highest ﬂuorescence) was 1.6. The ﬂuorescence intensity decreased

with a further increase in the amount of indicator molecules.

The decrease in ﬂuorescence of CNF-BSA with increasing dye-to-

protein ratio could be explained by self-quenching, as per previous

observations for ﬂuorescent dyes bound to human serum albumin

[36]. Conjugates with selected optimal dye-to-protein ratio were

used in further experiments.

The ﬂuorescence pH indicators CF and CNF show a signiﬁcant

difference in their ﬂuorescence quantum yield (Table 2). CF exhi-

bited a quantum yield identical to ﬂuorescein (0.93), suggesting

that the extra carboxy group in CF had no discernible effect on

its quantum yield. Conjugation with protein reduced ﬂuorescence

quantum yield to 0.48. In comparison to CF, the quantum yield of

CNF is low (0.05) and conjugation did not affect it. Low ﬂuores-

cence quantum yield of CNF and CNF-BSA can lead to application

problems with insufﬁcient intensity of the produced signal. The

ﬂuorescence quantum yield of CF-BSA is sufﬁciently high for appli-

cation in the biosensors even after the decrease caused by the

conjugation.

The effect of pH on ﬂuorescence response of indicators was

tested with 10 mM phosphate buffer in pH ranging from 2.0 to 11.0

(Fig. 4, Table 2). The pH sensitivity of free dyes and conjugates was

Fig. 4. Fluorescence response of CF and CF-BSA, CNF and CNF-BSA to pH changes.

similar with slight broadening to higher pH, particularly in the case

of CNF-BSA. CF and CF-BSA are sensitive to pH changes in a slightly

acidic and neutral pH region, i.e., pH range from 5.0 to 7.8. The most conjugation procedure did not destroy the optical properties of the

sensitive dynamic ranges of CNF and CNF-BSA occurs in a neutral pH indicator molecules.

and alkaline pH region from 6.7 (Table 2).

Measurement of MALDI–TOF mass spectra (data not shown) and

3.1.2. Temperature response and photostability

the evaluation of spectral properties and pH sensitivity conﬁrmed

The spectroscopic properties of ﬂuorophores are temperature-

◦

that CF and CNF were bound successfully to BSA. Moreover, the

sensitive, therefore, the temperature effect ranging from 1 to 40 C

on free indicators and their conjugates was investigated (Fig. 5).

A negligible temperature effect was observed for CF, CNF and

Fig. 3. Fluorescence of CF-BSA and CNF-BSA at the emission maximum depending Fig. 5. Fluorescence response of CF, CF-BSA, CNF and CNF-BSA to temperature

on the dye-to-protein ratio. changes.

S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99 97

3.2. Immobilized conjugated pH indicators

3.2.1. Immobilization

Conjugated pH indicators were immobilized using two

methods: (i) covalent cross-linking with GA and (ii) physical

entrapment in a sol–gel matrix ORMOCER with subsequent

cross-linking with GA.

(i) Molecules of conjugates were cross-linked together via forma-

tion of Schiff bases using GA as the most common cross-linking

agent [37]. When the cross-linked layer was formed on the glass

slides cleared by toluene, acetone, ethanol and sodium hydro-

xide, the layer started to delaminate from the surface upon con-

Fig. 6. Short-term photostability of CF, CF-BSA, CNF and CNF-BSA. Fluorescent dyes

were illuminated for 3 h using 150-W xenon lamp, then stored for 24 h in the dark tact with buffer. The GOPTS-modiﬁed glass slides were used

and subsequently illuminated for 3 h using xenon lamp. to solve this problem. GOPTS provided available epoxy groups

for coupling amine-containing ligands, in this case CF-BSA and

CNF-BSA. The immobilization of conjugates on GOPTS-modiﬁed

surfaces led to a homogeneous layer without any cracks and

CF-BSA. However, CNF-BSA showed a signiﬁcant increase in ﬂuo-

◦ delamination.

rescence up to 190% upon a temperature elevation from 1 to 40 C.

(ii) ORMOCER sol–gel matrix was used for immobilization of con-

For this reason, the proper control of temperature is strongly rec-

jugates due to many advantages, such as superior chemical

ommended when employing CNF-BSA in a sensor.

and mechanical stability, high optical transparency and mate-

The short-term photostability of ﬂuorescence pH indicators

rial versatility thanks to organic components [38]. Fluorescent

towards a 150-W xenon lamp was determined (Fig. 6). Fluores-

dyes can be immobilized in a sol–gel matrix by chemical dop-

cent properties of CF and CF-BSA were similar during illumination.

ing, impregnation or covalent binding. Chemical doping refers

The ﬂuorescence of CF was found unchanged after 3 h of illumi-

to incorporation of the dye during formation of the sol–gel

nation. The ﬂuorescence intensity of CF-BSA remained at 98% of

glass [39]. However, mixing conjugates with an ORMOCER solu-

the initial intensity. The indicator exhibited similar behaviour du-

tion led to loss of ﬂuorescence indicator colour and as a result,

ring the second illumination after a 24 h recovery. CNF underwent

the conjugates did not exhibit any ﬂuorescent properties. The

considerable photobleaching under conditions of continuous illu-

immobilization procedure based on impregnation was tested.

mination. Its ﬂuorescence was reduced to 93% after illumination

The ORMOCER layer was formed ﬁrst and hardened below UV

for 3 h, a further ﬂuorescence decrease to 85% was observed du-

lamp, after which the immobilization mixture containing the

ring the second illumination. Interestingly, ﬂuorescence of CNF-

conjugates was applied on the preformed glass. Conjugates

BSA increased signiﬁcantly up to 152% during the ﬁrst 20 min of

were entrapped in the porous polymeric structure into which

illumination. The ﬂuorescence intensity was even higher (211%)

protons can diffuse. The conjugates were subsequently cross-

after incubation in dark in comparison to the level recorded at

linked using GA to minimize the leaching problem.

the end of the ﬁrst illumination. Further illumination led to slow

decrease of the ﬂuorescence, detecting 202% of the initial intensity

after 3 h. 3.2.2. Fluorescence properties

The indicators and their conjugates were stored for 100 days at Spectral properties and pH sensitivity of immobilized conju-

◦

4 C to determine their long-term photostability (Fig. 7). While the gated pH indicators were studied in detail and compared with

ﬂuorescence of free indicators remained nearly unchanged during properties of conjugates in solution (Table 2). The pH-dependent

the entire tested period, the behaviour of conjugates was different. excitation and emission spectra of immobilized CF-BSA were si-

During storage, ﬂuorescence increased for both CF-BSA and CNF- milar to the respective spectra of CF-BSA in solution (Figs. S1),

BSA. Fluorescence of CF-BSA increased rapidly (to 145%) during the the emission maximum is slightly shifted to higher wavelengths

first 10 days of storage, followed by a gradual fluorescence rise to (Table 2). Significant change of shape of excitation spectra

166% during the following 90 days. A gradual ﬂuorescence increase was observed for CNF-BSA immobilized both by cross-linking

(to 143%) was observed for CNF-BSA during the course of the entire with GA and entrapment in ORMOCER with subsequent cross-

tested period. linking (Figs. S2). The excitation maxima were signiﬁcantly shifted

(Table 2) in comparison to the excitation maximum of 594 nm for

CNF-BSA in solution. The emission spectra of immobilized CNF-

BSA retained its shape, the position of maximum was signiﬁcantly

pH-dependent, the difference between pH 4.0 and 11.0 was about

40 nm. The emission maximum determined at pH 11.0 exhibited

only a slight shift in comparison to that of CNF-BSA in solution.

The effect of immobilization on the pH response of indicators

was tested in pH ranging from 2.0 to 11.0 (Fig. 4). The pH sensi-

tivity of immobilized conjugates was different when compared to

conjugates in a solution, especially the pH response of immobilized

CNF-BSA which shifted to very alkaline pH. Immobilized CF-BSA

responds to pH over the range 4.0–9.0, with the most sensitive

dynamic pH range from 6.0 to 9.0. The apparent pKa of immobilized

CF-BSA was one pH unit higher than the pKa value of CF-BSA in solu-

tion (Table 2). The enhanced sensitivity of immobilized CNF-BSA

at higher pH levels was observed. CNF-BSA entrapped in ORMO-

Fig. 7. Long-term photostability of CF, CF-BSA, CNF and CNF-BSA stored in 10 mM

◦

sterile phosphate buffer (pH 9.0) at 4 C. CER exhibited pH sensitivity from 8.3 to 10.0, CNF-BSA cross-linked

98 S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99

immobilization. The immobilized conjugate exhibited substantial

differences in spectral properties and pH sensitivity. The enhanced

sensitivity of immobilized CNF-BSA to a strong alkaline pH can be

particularly useful for pH measurements, however, it will be nec-

essary to eliminate leaching of the dye into the storage solution.

Immobilized conjugates are generally applicable for the deve-

lopment of optical sensors. They can be used for optical ﬁber tip

coating as well as in evanescent wave type sensors. Conjugated pH

indicators can be co-immobilized with enzymes in one layer via BSA

molecules to produce an effective biosensor within a close proxi-

mity of the sensing elements and thus minimal diffusion limita-

tions. Moreover, BSA can facilitate immobilization of enzymes with

a low content of surface residues carrying the amine functional

Fig. 8. Leaching of CF-BSA and CNF-BSA after immobilization by cross-linking with groups.

GA or entrapment in ORMOCER and subsequent cross-linking with GA.

Acknowledgements

with GA from 9.1 to 11.0. The apparent pKa of immobilized CNF-BSA

was signiﬁcantly higher than that of CNF-BSA in solution. Spectral

The authors would like to express their sincere thanks to Dr.

shift and change of pH sensitivity can be most likely attributed to

Ondrej Sedo and Assoc. Prof. Zbynek Zdrahal (Masaryk University,

the effect of immobilization on an indicator microenvironment.

Czech Republic) for MALDI-TOF MS analysis and to Prof. Ken Rear-

don (Colorado State University, USA) for introducing us to the ﬁeld

3.2.3. Leaching

of optical biosensors. The Ministry of Education of Czech Republic

Leaching is an important factor for consideration since the

(MSM0021622412 and CZ.1.05/2.1.00/01.0001) and the Ministry

immobilized conjugates are stored in hydrated form. Leaching of

of Defence of Czech Republic (OVUOFVZ200807) are gratefully

conjugates into the storage solution was monitored for 28 days

acknowledged for the ﬁnancial support of this work.

(Fig. 8). Unexpectedly, the detected amount of leached conju-

gates was dependent on the type of conjugate more than on

the immobilization method. Immobilized CF-BSA was stable with

Appendix A. Supplementary data

slight leaching in comparison to immobilized CNF-BSA. Leaching

of CF-BSA was maximal in the ﬁrst day of storage regardless of

Supplementary data associated with this article can be found, in

the immobilization technique (approximately 10%). The leached

the online version, at doi:10.1016/j.snb.2011.09.046.

conjugate is assumed to result from the fraction of conjugate

which was not covalently cross-linked. The amount of leached

CF-BSA increased only slightly during the following days. The References

effect of leaching can be easily eliminated by reconditioning of the

[1] H. Galster, pH Measurement: Fundamentals, Methods, Applications, Instru-

immobilized layer in buffer solution before using it in sensor mea-

mentation, VCH Publishers, New York, 1991.

surement. Signiﬁcant leaching of CNF-BSA from immobilized layer [2] O.S. Wolfbeis, Fiber Optical Chemical Sensors and Biosensors, 2nd ed., CRC

was observed for conjugates immobilized by cross-linking with Press, Florida, 1991.

[3] N.F. Norman Jr., A. Guiseppi-Elie, pH measurement, in: J.G. Webster (Ed.), The

GA. The amount of leached CNF-BSA was 22% after the ﬁrst day

Measurement, Instrumentation and Sensors Handbook, CRC Press and IEEE

of storage, increasing to 65% after 28 days of storage. The leaching

Press, Florida, 1999.

of CNF-BSA from ORMOCER exhibited a different trend, maximal [4] M. Mehrvar, C. Bis, J.M. Scharer, M. Moo-Young, J.H. Luong, Fiber-optic biosen-

sors – trends and advances, Anal. Sci. 16 (2000) 677–692.

leaching was observed after the ﬁrst day (approximately 50%), and

[5] S. Hulth, R.C. Aller, P. Engström, E. Selander, A pH plate ﬂuorosensor (optode)

the immobilized conjugate was then stable without any further

for early diagenetic studies of marine sediments, Limnol. Oceanogr. 47 (2002)

leaching. 212–220.

[6] C.R. Schröder, B.M. Weidgans, I. Klimant, pH ﬂuorosensors for use in marine

systems, Analyst 130 (2005) 907–916.

4. Conclusions

[7] Q. Zhu, R.C. Aller, Y. Fan, Two-dimensional pH distributions and dyna-

mics in bioturbated marine sediments, Geochim. Cosmochim. Acta 70 (2006)

4933–4949.

Conjugates of CF and CNF with BSA are excellent candidates for

[8] S.A. Grant, K. Bettencourt, P. Krulevitch, J. Hamilton, R.S. Glass, In vitro and

use as ﬂuorophores in optical pH sensors and biosensors. CF-BSA

in vivo measurements of ﬁber optic and electrochemical sensors to monitor

had similar ﬂuorescence properties to free CF, the ﬂuorescence brain tissue pH, Sens. Actuators B Chem. 72 (2001) 174–179.

[9] V.I. Agayn, D.R. Walt, Fiber-optic sensor for continuous monitoring of fermen-

quantum yield decreased after conjugation being the only dif-

tation pH, Nat. Biotechnol. 11 (1993) 726–729.

ference between the free and conjugated form. On the contrary,

[10] J.A. Ferguson, B.G. Healey, K.S. Bronk, S.M. Barnard, D.R. Walt, Simultaneous

CNF-BSA differs from free CNF substantially in photostability, pH monitoring of pH, CO2 and O2 using an optical imaging ﬁber, Anal. Chim. Acta

340 (1997) 123–131.

and temperature sensitivity.

[11] W. Trettnak, M.J.P. Leiner, O.S. Wolfbeis, Fibre-optic glucose sensor with a pH

Two methods were investigated for immobilization of conju-

optrode as the transducer, Biosensors 4 (1989) 15–26.

gates on an optical transducer which is a crucial factor for obtaining [12] C. Müller, B. Hitzmann, F. Schubert, T. Scheper, Optical chemo- and biosensors

for use in clinical applications, Sens. Actuators B Chem. 40 (1997) 71–77.

a high ﬂuorescence signal, fast response and good stability. Both

[13] T.D. Rhines, M.A. Arnold, Fiber-optic biosensor for urea based on sensing ammo-

the cross-linking of CF-BSA and entrapment of CF-BSA, followed by

nia gas, Anal. Chim. Acta 227 (1989) 387–396.

cross-linking, is useful for biosensing applications due to minimal [14] X. Xie, A.A. Suleiman, G.G. Guilbault, Determination of urea in serum by a ﬁber-

spectral changes, negligible leaching and good mechanical stability. optic ﬂuorescence biosensor, Talanta 38 (1991) 1197–1200.

[15] M. Busch, F. Gutberlet, W. Höbel, J. Polster, H.-L. Schmidt, M. Schwenk, The

The sensitivity in acidic and the near-neutral pH region is com-

application of optodes in FIA-based fermentation process control using the

patible with a range of applications such as bioprocessing, clinical

software package FIACRE, Sens. Actuators B Chem. 11 (1993) 407–412.

measurements or environmental monitoring. CF-BSA cross-linked [16] T.J. Kulp, I. Camins, S.M. Angel, C. Munkholm, D.R. Walt, Polymer immobi-

lized enzyme optrodes for the detection of penicillin, Anal. Chem. 59 (1987)

with GA has already been used for construction of enzy-

2849–2853.

matic pH biosensor for the detection of halogenated compounds

[17] M.R.S. Fuh, L.W. Burgess, G.D. Christian, Single ﬁber-optic ﬂuorescence enzyme-

[23]. The performance of CNF-BSA was affected signiﬁcantly by based sensor, Anal. Chem. 60 (1988) 433–435.

S. Bidmanova et al. / Sensors and Actuators B 161 (2012) 93–99 99

[18] W. Höbel, A. Papperger, J. Polster, Penicillinase optodes: substrate determi- suitable for labeling proteins and amino-modiﬁed DNA, J. Chromatogr. B 793

nations using batch, continuous ﬂow and ﬂow injection analysis operation (2003) 83–92.

conditions, Biosens. Bioelectron. 7 (1992) 549–557. [37] I. Migneault, C. Dartiguenave, M.J. Bertrand, K.C. Waldron, Glutaraldehyde:

[19] X. Xie, A.A. Suleiman, G.G. Guilbault, A ﬂuorescence-based ﬁber optic biosen- behavior in aqueous solution, reaction with proteins, and application to enzyme

sor for the ﬂow-injection analysis of penicillin, Biotechnol. Bioeng. 39 (1992) cross-linking, Biotechnology 37 (2004) 790–802.

1147–1150. [38] H. Podbielska, A. Ulatowska-Jarza, Sol–gel technology for biomedical enginee-

[20] B.G. Healey, D.R. Walt, Improved ﬁber-optic chemical sensor for penicillin, Anal. ring, Bull. Pol. Acad. Sci.: Technol. Sci. 53 (2005) 261–271.

Chem. 67 (1995) 4471–4476. [39] O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov, J. Gun,

[21] R.-A. Doong, H.C. Tsai, Immobilization and characterization of sol–gel- Organically modiﬁed sol–gel sensors, Anal. Chem. 67 (1995) 22A– 30A.

encapsulated acetylcholinesterase ﬁber-optic biosensor, Anal. Chim. Acta 434

(2001) 239–246.

Biographies

[22] K.R. Rogers, J.C. Cao, J.J. Valdes, A.T. Eldefrawi, M.E. Eldefrawi, Acetyl-

cholinesterase ﬁber-optic biosensor for detection of anticholinesterases,

Fundam. Appl. Toxicol. 16 (1991) 810–820.

Sarka Bidmanova is a Ph.D. student of Environmental Chemistry at Masaryk Uni-

[23] S. Bidmanova, R. Chaloupkova, J. Damborsky, Z. Prokop, Development of enzy-

versity in the Czech Republic. She received her M.Sc. degree in Microbiology in 2007

matic ﬁber-optic biosensor for detection of halogenated hydrocarbons, Anal.

from Masaryk University and is currently working on the development, optimiza-

Bioanal. Chem. 398 (2010) 1891–1898.

tion and characterization of optical enzyme-based biosensors for military-defence

[24] M.J.P. Leiner, P. Hartmann, Theory and practice in optical pH sensing, Sens.

and environmental applications. Her research interests also include immobilization

Actuators B Chem. 11 (1993) 281–289.

and characterization of enzymes and ﬂuorescence pH indicators.

[25] C. McDonagh, C.S. Burke, B.D. MacCraith, Optical chemical sensors, Chem. Rev.

108 (2008) 400–422.

Antonin Hlavacek is a Ph.D. student of Biochemistry at Masaryk University in

[26] R.W. Sabnis, Handbook of Acid–Base Indicators, 1st ed., CRC Press, Florida, 2008.

the Czech Republic. He obtained his M.Sc. degree in Biochemistry in 2007 from

[27] A. Song, S. Parus, R. Kopelman, High-performance ﬁber-optic pH microsensors

Masaryk University. His research is mainly focused on the immobilization of

for practical physiological measurements using a dual-emission sensitive dye,

biomolecules and the synthesis of quantum dot bioconjugates for bioanalytical

Anal. Chem. 69 (1997) 863–867.

applications.

[28] O.S. Wolfbeis, N.V. Rodriguez, T. Werner, Led-compatible ﬂuorosensor for mea-

surement of near-neutral pH values, Microchim. Acta 108 (1992) 133–141.

Jiri Damborsky is the Josef Loschmidt Chair Professor at the Masaryk University

[29] J. Lin, Recent development and applications of optical and ﬁber-optic pH sen-

in the Czech Republic. He obtained M.Sc. degree in Microbiology in 1993 and Ph.D.

sors, Trends Anal. Chem. 19 (2000) 541–552.

degree in Microbiology in 1997 from Masaryk University. His research group deve-

[30] L. Viveros, S. Paliwal, D. McCrae, J. Wild, A. Simonian, A ﬂuorescence-based

lops new concepts and software tools, e.g., CAVER and HOTSPOT WIZARD, for pro-

biosensor for the detection of organophosphate pesticides and chemical war-

tein engineering, and uses them for the rational design of enzymes for biocatalysis,

fare agents, Sens. Actuators B Chem. 115 (2006) 150–157.

biodegration and biosensing. He has published more than hundred original articles

[31] M. Ramanathan, A.L. Simonian, Array biosensor based on enzyme kinetics mo-

in the ﬁeld of protein engineering and enzymology. He is a co-founder of the national

nitoring by ﬂuorescence spectroscopy: application for neurotoxins detection,

Centre for Biocatalysis and Biotransformation and biotechnology spin-off company

Biosens. Bioelectron. 22 (2007) 3001–3007.

‘Enantis Ltd’.

[32] D. Sehgal, I.K. Vijay, A method for the high efﬁciency of water-soluble

carbodiimide-mediated amidation, Anal. Biochem. 218 (1994) 87–91. Zbynek Prokop is a team leader in the Loschmidt Laboratories at the Masaryk Uni-

[33] G.T. Hermanson, Bioconjugate reagents, in: G.T. Hermanson (Ed.), Bioconjugate versity in the Czech Republic. He received M.Sc. degree in Environmental Science

Techniques, Academic Press, Maryland Heights, 2008, pp. 215–739. and Ecotoxicology in 1998 and Ph.D. degree in Environmental Chemistry in 2001

[34] A.T.R. Williams, S.A. Winﬁeld, J.N. Miller, Relative ﬂuorescence quantum yields from Masaryk University. Zbynek Prokop extended his expertise in biochemistry

using a computer-controlled luminescence spectrometer, Analyst 108 (1983) and biophysical chemistry during his research stays at the University of Ghent in

1067–1071. Belgium, at the University of Groningen in the Netherlands, at the European Molecu-

[35] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Aca- lar Biology Laboratory and at the University of Hannover in Germany. Zbynek Prokop

demic/Plenum Press, New York, 1999. is the co-author of twenty nine publications and two international patents in the

[36] B. Wetzl, M. Gruber, B. Oswald, A. Dürkop, B. Weidgans, M. Probst, O.S. Wolfbeis, ﬁeld of biocatalysis and protein engineering. He is a co-founder of the biotechnology

Set of ﬂuorochromophores in the wavelength range from 450 to 700 nm and spin-off company ‘Enantis Ltd’.