ULTRASENSITIVE HYDRODYNAMIC USING SOUND WAVE DRIVEN MICROSTREAMING E. Kaplan, J. Reboud, A. Glidle and J. M. Cooper* Division of Biomedical Engineering, University of Glasgow, United Kingdom

ABSTRACT In this work we demonstrate the enhancement of the sensitivity of an electrochemical sensor, using surface acoustic wave (SAW) streaming [1][2] to improve mass transfer within a drop of fluid. The system makes use of hydrodynamic streaming within the sample, in a similar fashion as the well-known rotating disc electrode (RDE) [3][4]. In addition to increased sensitivity, as evidenced by a 4-fold increase in the output current of the electrochemical sensor, it also enables the processing of microscale samples on a disposable chip without any moving parts.

KEYWORDS: Surface Acoustic Wave, Biosensor, Electrochemistry, Mass transport

INTRODUCTION Hydrodynamic provides us with a versatile electrochemical technique for the detection of biochemical analytes with high sensitivity [5][6]. In addition, complex samples can be processed, such as opaque blood or turbid samples such as feces that are not easily amenable to other detection techniques. It relies upon the convective enhancement of mass transfer, usually carried out by moving the electrodes or the liquids above them, creating flows that break down diffusion barriers. While in stationary electrochemical detection systems, the detected current (and hence the limit of detection) is constrained by the diffusion of the product of the electrochemical reaction away from the electrodes, to provide new substrate, hydrodynamic systems break down the diffusion layer at the electrode interface, making the reaction more efficient at that location, and hence the system more sensitive. Here we demonstrate that SAW streaming significantly reduces mass transport limitations, leading to a dramatic increase in sensitivity up to 4 fold (±0.413), on a non-contact platform, using a disposable microchip.

THEORY Electrochemical sensors generally involve the interaction of a biological receptor, bound the surface of an electrode [7], with its target bio-molecules and the detection of electrochemical signals generated by the presence of the target, usually through an electrochemical mediator [8]. The product of the electrochemical reaction is generated near the elec- trodes and creates a diffusion layer, which limits the current flow. The application of ultrasonic waves to the sample sig- nificantly increases diffusion limited currents by increasing the mass transport [9]. Sound waves propagating in the liq- uid generate pressure waves that induce fluid streaming inside the sample, in turn disrupting the diffusion layer of product and increasing current flow. The application of an AC electric excitation through an interdigitated transducer patterned on a LiNbO3 piezoelectric material results in elastic deformations that propagate as SAW [10]. Equation (1) below shows that the resonant frequency of a SAW slanted interdigitated transducer (SIDT) device which has non- uniform finger spacing [1], is based on two parameters of the layout; finger spacing (d) and substrate acoustic velocity ( ). The transducer generates a narrow SAW beam propagating away only at a specific location [11], which provides a mixing effect by using fluid streaming [12].

Figure 1: Schematic of the droplet based electrochem- Figure 2: Schematic of the system. While the SAW is be- ical sensor chip. The SAW, propagated from the SIDT ing radiated into the droplet, the potentiostat applies a causes a mixing effect inside the droplet. When the triangle and measures the current flow through SAW only interacts with one side of the drop, it cre- the (WE) while controlling the applied ates a rotational momentum within it that is akin to a potential by a reference electrode (RE). system.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 251 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany EXPERIMENTAL The SAW-enhanced microchip comprises of the sensing electrodes deposited on a 1 mm glass substrate, coupled to an SIDT on a piezoelectric LiNbO3 surface (Figure 1). Both the sensing electrodes and the transducer (8-12 MHz, [1]) were fabricated in the James Watt Nano-Fabrication Center (Glasgow) by metal deposition (10 nm Ti / 100 nm Au) and lift-off in acetone, using S1818 (Shipley, U.K.) photoresist as the mask. The SIDT contained 41 electrodes, with a finger width of 83.25 μm the highest frequency (12 MHz) and 125 μm at the lowest frequency (8 MHz), with an aperture of 1 cm. A 3 µl sample of 20 mM Potassium Ferrocyanide (K4[Fe(CN)6]•3H2O) solution in a 100 mM KCl supporting electrolyte buffer was pinned on the glass surface using a hydrophilic spot (3 mm radius), surrounded by a hydrophobic layer of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (FOTS, Aldrich), using standard photolithography. SAW were generated at 11.7 MHz at different powers (between 50 mW and 4 W), coupled into the glass microchip through a water- based gel interface and radiated in the droplet [13]. In addition to the measurements performed under the forced convection effect, control voltammetry measurements were done without any acoustic streaming. The peak current (Ias), which is indicative of the reaction rate, was measured using CHI760C electrochemical analyzer (potentiostat).

Figure 3: Flow profile at a height of 300 µm in a 3μl Figure 4: Velocity at different heights in the droplet; droplet by Micro-PIV. The velocity reached 3.5 from 66 µm to 766 µm. SAWs were propagated at mm/s at an acoustic excitation power of 0.16 W. 0.16 W and 9.2 MHz. Measurements are averages of three independent experiments, while error bars are the standard deviation.

The fluid speed was measured for different heights inside a 3 µl droplet containing fluorescent particles (Fluoro-Max Dyed Red Aqueous Fluorescent particles, R0300 – Thermo Scientific) at a concentration of 4.106 particles/ml in water (SAW generated at 0.16 W and 9.2 MHz), using a TSI High-Speed High-Resolution Particle Image Velocimetry (Micro- PIV) system [12]. This consists in a Nikon eclipse TE2000 , a high frame rate camera (3000 frames per sec- ond at 1024 by 1024 pixels resolution- Photron APX RS), a high repetition rate laser source (10 mJ at 1 kHz) for illumi- nation of the red dyed fluorescent particles and a laser pulse synchroniser.

RESULTS AND DISCUSSION Analysis of acoustic mixing (0.16 W) was measured at different heights of the 3 μl droplet (the surface tension on the surface provides a shape is close to a half sphere). A representative flow profile within the drop (height of 220 μm) is shown in Figure 3, where the highest velocity was 3.5 mm/s (± 0.2 mm/s). While the velocity at a height of 66 μm (the closest measurement to the electrode surface) was very similar at 2.5 mm/s (± 0.32 mm/s), the peak velocity was 6.1 mm/s (± 0.64 mm/s) at 346 μm height (Figure 4). It should be noted that the PIV system does not account for vertical displacement and only provides information on the flow in a horizontal plane. We then quantified the effect of SAW streaming on the electrochemical efficiency of the system. 3 μl drops of sam- ple were processed on microelectrodes fabricated on a disposable glass substrate, which is coupled on to the surface of SAW platform [13]. Figure 5 shows that the peak current increased from 12.1 μA when SAW was not applied to 33.2 μA with SAW (11.73 MHz, 1.3 W) streaming. Figure 6 shows that increasing the SAW power (from 0 to 4 W) leads to an increase in peak current from 11 µA (±1.1µA) to 45 µA (±4.5µA).

CONCLUSION This paper shows that surface acoustic wave based hydrodynamic voltammetry increases the sensing signal outputs by a factor 4. The system has the potential to dramatically enhance sensitivity without any moving parts, on a disposable microchip, as suitable for point-of-care diagnostics. In addition, SAWs have been shown to reduce non specific binding

252 [6] [14] [15] by removing the weakly bound molecules from the sensing surface. In the future, this promising approach will be validated on a model immunoassay platform to detect cancer biomarkers for early diagnostics.

Figure 5: (Scan rate: 1V/s) of 20 Figure 6: Voltammetry (Scan rate: 1V/s) results mM Potassium Ferrocyanide (K4[Fe(CN)6]•3H2O) in without SAW and with SAW (11.73 MHz) using in- 100 mM KCl with SAW (11.73 MHz, 1.3 W) streaming put powers with increasing values from 0 W to 4 W. (Red) and no SAW (Blue). The hydrodynamic effect Each value is the average of 3 experiments and er- improved the peak current by a factor 4. ror bars are the standard deviation.

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CONTACT *J. M. Cooper, Tel: +44 141 330 4931; [email protected]

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