Portable Lock-in Amplifier-Based Electrochemical Method to Measure

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Portable Lock-in Amplifier-based Electrochemical Method to Measure an Array of 64 Sensors for Point-of-care Applications Radim Hrdý, Hana Kynclová, Ivana Klepá#ová, Martin Bartosik, and Pavel Neuzil Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00776 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Analytical Chemistry

Portable Lock-in Amplifier-based Electrochemical Method to Measure an Array of 64 Sensors for Point-of-care Applications Radim Hrdý d , Hana Kynclová b , d , Ivana Klepáčová a , d , Martin Bartošík c , and Pavel Neužil a , b * a

Northwestern Polytechnical University, School of Mechanical Engineering, Department of Microsystem Engineering, 127

West Youyi Road, Xi’an, Shaanxi 710072, P. R. China b

Central European Institute of Technology, Brno University of Technology (BUT), Antonínská 1, CZ-616 00 Brno, Czech

Republic c

RECAMO, Masaryk Memorial Cancer Institute, Žlutý kopec 7, CZ-656 53 Brno, Czech Republic

d

Department of Microelectronic, FEEC, Brno University of Technology (BUT), Technická 10, CZ-616 00 Brno, Czech Republic

*

Corresponding

author:

Pavel

Neužil.

Telephone:

+86

150

9133

1869;

E-mail:

[email protected],

[email protected]

ABSTRACT We present a portable lock-in amplifier-based electrochemical sensing system. The basic unit (cluster) consists of four electrochemical cells (EC), each containing one pseudo-reference electrode (PRE) and one working electrode (WE). All four ECs are simultaneously interrogated, each at different frequency, with square wave pulses superposed on a sawtooth signal for cyclic voltammetry (CV). Lock-in amplification provides independent read-out of four signals, with excellent noise suppression. We expanded a single cluster system into an array of 16 clusters by using electronic switches. The chip with an array of ECs was fabricated using planar technology with a gap between a WE and a PRE of ≈2 µm, which results in partial microelectrode-type behavior. The basic electrode characterization was performed with the model case using a 2+

3+

ferricyanide−ferrocyanide redox couple (Fe /Fe ) reaction, performing CV and differential pulse voltammetry (DPV). We then used this system to perform cyclic lock-in voltammetry (CLV) to measure concurrently responses of the four ECs. We repeated this method with all 64 ECs on the chip. The standard deviation of a peak oxidation and reduction current in a single channel consisting of 13 ECs was ≈7.46% and ≈5.6%, respectively. The four-EC configuration in each measured spot allows determination of non-performing ECs and, thus, to eliminate potential false results. This system is built in a portable palmsize format suitable for point-of-care applications. It can perform either individual or multiple measurements of active compounds, such as biomarkers.

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New diagnostic techniques for the detection of biomolecules, including proteins and nucleic acids, are currently of huge interest in biomedical research. Especially appealing are biomolecules serving as cancer biomarkers to indicate infectious diseases including Ebola, avian influenza and severe acute respiratory syndrome (SARS), or food pathogens. For instance, bacterial or viral nucleic acids are mostly identified using polymerase chain reaction (PCR), with subsequent optical detection. A gold standard method used to identify proteins is the enzyme-linked immunoassay (ELISA). It uses two antibody–antigen reactions, with the second antibody being fluorescently labeled. This method has excellent specificity, but is time-consuming and labor-intensive. Electrochemical techniques appear as an interesting alternative as they are fast and relatively 1-3

inexpensive, can achieve high sensitivity, are simple to implement, and can be integrated into complex systems . Voltammetric methods are especially popular, giving an option of simultaneously performing qualitative as well as quantitative analysis of multiple compounds. The typical electrochemical cell (EC) consists of three electrodes: working electrode (WE), reference electrode (RE), and counter (auxiliary) electrode (CE). With a closed-loop feedback system, the electrical current flowing into the RE is practically 4

zero; thus, there is a negligible voltage drop at the interface of the test solution and RE . In the last few decades, and with the development of planar technology for silicon fabrication, a new category of 5

electrochemical devices was introduced that uses optical lithography patterning with subsequent metal etching . This allows shrinking of the electrodes and also introduces a small gap, in the order of micrometers, between electrodes. The EC systems are produced on silicon or glass wafers in large quantities and are used for various applications such as heavy metal 6

7,8

detection and biosensing . Once the fabrication is completed, the wafers are diced into individual chips. There were −

fundamental problems in making a planar RE, such as Ag/AgCl, as it requires a stable concentration of Cl ions in solution 9

surrounding the electrode. It is a rather difficult task to fulfill and, so far, success has been limited . A simple solution to this 10

problem is using a pseudo-reference electrode (PRE) . The PRE is able to fulfill most requirements of a true potential RE despite the unwanted potential drop at its surface and lack of a thermodynamic equilibrium.

Another simplification comes from combining an RE and CE into one, thus reducing the number of electrodes from three to two

11,12

. A two-electrode system is not as versatile as a three-electrode one, but there is no feedback loop required. The

RE/CE electrode is directly interrogated by a signal generator and the WE is electrically grounded. This RE/CE electrode can be also in the form of a PRE.


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Planar technology-based electrochemical systems give an advantage of forming many ECs adjacent to each other on the same chip for system multiplexing

1,13

. Researchers have demonstrated enormous potential for biochemical applications, 14

mainly due to more reliable statistics, decreased surface variability as well as flexibility and robustness . Nevertheless, the system requires switching individual sensors one by one and, thus, interrogates only a single sensor a time. The number of 15

readouts is limited by electronics and connecting cable complexity . In the early 1990s, a new field of microfluidics was introduced followed by the addition of electrochemical detection systems with microfluidic channels. This combination has advantages, such as even sample distribution, simple operation, and simple instrumentation. Naturally, there were many 8

attempts at using these devices in various combinations . They showed that microfluidic platforms are significantly more 16

economic than parallel single-analyte immunoassays . The lithography technique allows the size to be shrunk so much that the WEs starts to behave differently and the linear diffusion does not play the dominant role anymore leading to microelectrode behavior

17-19

. These microelectrodes can be also fabricated for arrays and systems with multiplexing

capability.

In this work, we introduce an array of 64 ECs integrated with a palm-sized readout system. Each EC consists of two electrodes: one WE and one PRE. Four ECs are clustered into a single unit, with all WEs connected together and PREs separated from each other. We demonstrate that the four-channel lock-in amplification system is capable of simultaneous and independent interrogation of all four ECs in one cluster. The lock-in amplification in electrochemistry is primarily employed in frequency range analysis. However, it has been also used in previous works for the detection of second- and 20

third-harmonic signals in cyclic voltammetry (CV) measurements . Lock-in amplification was employed previously to 2+

3+

demonstrate its capability with reversible Fe /Fe

21

redox solutions . The researchers used conventional three-electrode

configuration, such as gold WE, platinum CE, and Ag/AgCl REF electrodes, without any miniaturization or chip integration. The main advantages of our system are miniaturization, portability, and the option to perform either individual or multiple measurements of active compounds such as biomarkers. We also added four 1 × 16 analog switches to subsequently interrogate 16 clusters. Each cluster has four ECs that are measured simultaneously, but independently of each other. An increased number of ECs improves the robustness of the system as the results from one defective EC can be discovered and suppressed. This electrochemical system configuration is prepared to be combined with a microfluidic chip to detect multiple biomarkers from multiple samples.



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EXPERIMENTAL SECTION Measurement principle 22

We used two-electrode ECs in combination with the pulse technique

23

and signal multiplexing . PREs were powered by

square wave pulses superimposed on a symmetrical sawtooth signal for CV. A simplified schematic is shown in Supplementary S1 (Figure S-1A). The WE was grounded via virtual ground using the first operational amplifier (OA1) in an inverting mode. The OA1 had a feedback resistor with a nominal value of resistance of R0 in the feedback loop, converting the induced WE electrical current IWE into output voltage (VOUT) according to the formula: = − ∙ .

(1) 6

The resistor R0 had the nominal value of ≈3.3 MΩ, resulting in a conversion factor ≈−3.3×10 . The amplitude of VOUT was subsequently amplified by the second operational amplifier (OA2), with gain (G) set to −100 as it was also in an inverting mode, demultiplexed, and filtered by a low-pass filter. The VOUT was given by the formula: = ∙ ∙ ∙ ,

(2) 24

where T is the transfer function coefficient of the lock-in amplifier with an approximate maximum value up to ≈0.72 . The 8

−1

system conversion factor VOUT/IWE = R0·T·G can be as high as ≈2.38×10 V·A . This technique of interrogation by pulses with subsequent demultiplexing and filtering is called lock-in amplification. In comparison with known electrochemical techniques, results here were different, but similar to those produced by the differential pulse voltammetry (DPV) as well as square wave 25

voltammetry (SWV) . In our system, we used four voltage pulse generators, each operated at a different frequency (f), 26

enabling the output composite signal to be demultiplexed into four individual signals each corresponding to a different EC . Only the signal of the interrogating f passed through the system, making it highly immune to external noise. We used a personal computer (PC) application connected to the printed circuit board (PCB) by an RS232 interface to select a single cluster with four ECs from the array.

Testing platform The four-channel lock-in amplifier was equipped with a 1-out-of-16 analog selector (switch) in each signal path, expanding the system to be capable of measuring 64 sensors. We added a sawtooth shape wave generator to an attenuator input, thereby superimposing a pulse signal VP with amplitude of ≈200 mV on the sawtooth wave VST (Figure S-1B and C). The


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platform was controlled by two independent programs from the PC. The first program controlled the lock-in amplifier parameters such as the interrogating f of VP of each channel and corresponding duty cycle. The second program controlled switching of the 1-out-of-16 analog switch. We were able to scan all 64 sensors and conduct cyclic lock-in voltammetry (CLV) at selected blocks of four ECs.

Microelectrodes and their fabrication Lithography-based fabrication allows the relatively simple introduction of small-dimension WEs, thus leading to a microelectrode-type behavior, which is especially interesting for high sensitivity systems using an advanced layout generating tool

28

17,27

. The EC chips were designed

and fabricated using single metal technology. The fabrication process

(Supplementary S2) was carried out on glass wafers with diameters of ≈100 mm. Substrates were covered with an ≈20 nmthin CrNi adhesion layer and an ≈200 nm-thick gold layer deposited by physical vapor deposition (PVD) (Figure S-2A). Wafers were then covered with an i-line (365 nm) positive photoresist (PR) using the spin coating technique, resulting in a photoresist thickness of ≈2 µm. Wafers were pre-baked at ≈110 °C for ≈60 s and then exposed using a 5:1 projection stepper, post-baked again at ≈110°C for ≈60 s and developed for ≈60 s in tetra-methyl ammonium hydroxide (TMAH)-based developer. The WEs had a donut shape, with external and internal radii of ≈100.5 µm and ≈89.5 µm, respectively. The WE was surrounded by a PRE, also in the shape of a donut, giving the entire EC circular symmetry a gap between the WE and PRE of ≈1 µm. The metal sandwich layer was removed by an ion milling technique using a secondary ion mass spectroscopy (SIMS)-controlled process. Photoresist was removed by O2 plasma and wafers were cleaned with acetone and isopropyl alcohol, and then blown dry with general purpose N2 (Figure S-2B). We subsequently deposited a SiO2 layer by plasmaenhanced chemical vapor deposition (PECVD) with a thickness of ≈0.5 µm at temperature of ≈180 °C. Wafers were covered with a self-assembly monolayer (SAM) of bis(trimethylsilyl) amine, also known as hexamethyldisilazane (HMDS), which was used as cross-linker to improve adhesion of the subsequently spin-coated photoresist. After the second PR spin coating, wafers were again exposed using a stepper with alignment precision of ≈40 nm and a SiO2 layer was etched by CHF3-based plasma (Figure S-2C and detail in D). The edges of all electrodes were overlapped by ≈0.5 µm with SiO2, thereby eliminating any contact between the electrolyte and the Au/CrNi and avoiding an electrochemical corrosion of the CrNi layer underneath the Au layer. The effective gap between the WE and PRE was then increased to ≈2 µm. The WE and PRE had total designed 2

2

areas of ≈5969 µm and ≈162879 µm , respectively.



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The entire wafer is shown in Figure 1A. Each chip consisted of a 4 × 4 array of clusters, i.e., 64 electrochemical sensors. One cluster formed by four ECs is shown in Figure 1B, a single EC in Figure 1C, and details of the WE in Figure 1D. Sensors in each array block were placed with a pitch of 1000 µm, which is five times greater than the WE outer diameter, to minimize the 29

shielding effects caused by the overlapping of the diffusion layers of adjacent electrodes . Wafers were then diced into individual chips with the size of ≈18 mm × ≈18 mm using a diamond blade dicing saw.

RESULTS AND DISCUSSION Electrochemical response to model systems First, we tested the fundamental behavior of the EC system using a commercial potentiostat and performing CV and DPV with a mixture of K3[Fe(CN6)] and K4[Fe(CN6)] solutions with concentrations of ≈2.5 mM, ≈5 mM, and ≈10 mM for both compounds, in a buffer solution with concentration of ≈0.1 M KCl. The EC chip was electrically connected using a probe station equipped with micromanipulators and placed in a Faraday cage. The testing solution was dispensed manually using volume of 6 µL for each EC for each cluster. The CV was performed with a scan rate (v) in a range with set values from 5 −1

−1

−1

mV·s to 25 V·s and connecting one EC at a time (Supplementary S3 Figure S-3A and B). At low v (up to ≈100 mV·s ), the CV curves had a sigmoid shape typical for microelectrodes. An amplitude of electrical current, typical for microelectrodes at high −1

v, was detectable with v set above 200 mV·s .

DPV was performed with the following set parameters: step potential of 0.5 mV, modulation amplitude of 100 mV, −1

modulation time (pulse duration) of 25 ms, interval time of 75 ms, and v of 6.667 mV·s . The DPV results in potential range from ≈−0.8 V to ≈0.8 V demonstrated an excellent response of the EC (Supplementary S4 Figure S-4A). The amplitude of IWE −1

was linearly proportional to the solution concentration with a value of (1.212 ± 0.016) µA·mM (mean ± standard deviation) (Figure S4B). The potential peak had amplitude of ≈−40 mV.

Once the preliminary study was concluded, chips were mounted on PCB and wire-bonded to the PCB using aluminum wire with a diameter of ≈25 µm, and tested with a simple IWE → VOUT converter at the PCB (Figure 2A). These wires were covered with thixotropic epoxy for mechanical and electrical protection (Figure 2B). We first performed the CV measurement in ≈2.5 2+

3+

−1

−1

mM Fe /Fe solution with a volume of ≈6 µL with scan rate in the range from ≈5 mV·s to ≈1000 mV·s (Figure 2C). We


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extracted peak amplitudes as functions of square root of the √ (Figure 2D) to determine the electrochemically active area A 30

of the WE using the Randles–Sevcik equation :

= 0.4463 ∙ ∙ ∙ ∙

∙∙∙ ∙!

", slope # =

$%&' $√

= 0.4463 ∙ ∙ ∙ ∙

∙∙ ∙!

" and =

(

)*+* ∙,- √∙.

,

(3)

where n is the number of electrons transferred in the redox reaction, F is the Faraday constant, D is the diffusion coefficient, C is the concentration (analyte activity), R is the universal gas constant, T is the thermodynamic temperature, and the slope s is the first derivation of IWE with respect to the square root of the √. The amplitude of s was found to be nearly constant in the entire tested range, with a value of (2.079 ± 0.011) µA·V

−0.5 0.5

·s

(mean ± standard deviation). Here, we found that a single

2

cluster had a WE with total effective area of ≈144 000 µm . That is ≈6.03 times larger than its total geometrical area of 2

≈23876 µm . Together with surface roughness, the increased effective area with respect to the geometrical one is probably caused by its microelectrode-type behavior. In addition, the CV curve shape seems to be a combination of a conventional 17

electrode with a microelectrode as they exhibited conventional behavior when superimposed on a sigmoidal shape. The potential peaks shift ∆V (Figure 2C) is caused by current density at the electrode J with A and resistance of the solution RS and its amplitude is J·A·R. It is a typical phenomenon for two-electrode system configurations and can be corrected by subsequent signal processing

31,32

.

The electrical current recorded with an ultra-microelectrode array (UMEA) is given by the sum of steady-state currents of individual microelectrodes and can be calculated using the following equation / = 0 ∙ ∙ ∙ 1 ∙ ∙

23 (56$)

19,33,34

,

:

(4)

89:*($65)/($

Portable Lock-in Amplifier-Based Electrochemical Method to Measure

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