KR100700713B1 - Miniaturized Electrochemical System Incorporating Novel Polyelectrolyte Reference Electrode and Its Application to Thin Layer Electroanalysis - Google Patents

Abstract

Miniaturized electrochemical systems comprising the novel polyelectrolyte reference electrodes and their application to thin layer electroanalysis are provided. The miniaturized electrochemical system including the polyelectrolyte reference electrode includes A) a thin film deposited working electrode inside an operating chamber, which is a first chamber located in the middle of a traveling path of a microchannel, which is a moving passage of an analytical solution, and B) the micro A counter electrode deposited on the inside of the counter chamber, which is a second chamber positioned in the middle of the channel, and C) a polyelectrolyte salt bridge connected to the working chamber outside the path of the microchannel and through the polyelectrolyte salt bridge. And a polyelectrolyte reference electrode including a reference chamber connected to the working chamber, an internal filling solution filled in the reference chamber, and an Ag / AgCl electrode accommodated in the reference chamber. According to the electrochemical system including the polyelectrolyte reference electrode of the present invention, the polyelectrolyte salt bridge separates the analyte solution from the internal solution of the reference chamber while providing a moving passage of chlorine ions. This eliminated contamination by potential interference anions and significantly suppressed the dependence on chlorine ion concentration as compared to the pseudo reference electrode. The lifetime of the reference electrode was improved, and the electrochemical properties of the small thin-layered cell incorporating the trielectrode were demonstrated by cyclic voltammetry. Various electrochemical techniques such as differential pulse voltammetry could be applied. In addition, the polyelectrolyte reference electrode was similar to the macroscopic reference electrode in terms of operation principle and capacitance. Furthermore, practicality is improved by a simple manufacturing process.

Description

Miniaturized Electrochemical System Incorporating Novel Polyelectrolyte Reference Electrode and Its Application to Thin-Layer Electro-Analysis

1 is a schematic view showing an electrochemical system including a polyelectrolyte reference electrode according to the present invention.

2 is a perspective view showing a preferred embodiment in which the electrochemical system of FIG. 1 is implemented in a microchip in the form of a thin cell.

3 is a graph showing the dependence of chlorine ion concentration between the pseudo reference electrode having a thin Ag / AgCl electrode and the polyelectrolyte reference electrode having a salt bridge. Open circuit potential (OCP) was measured against a commercial Ag / AgCl electrode (3 M KCl).

4 is a graph showing the potential deviation of a polyelectrolyte reference electrode in phosphate buffered saline (PBS). Open circuit potential (OCP) was measured against a commercial Ag / AgCl electrode (3 M KCl). Initial sensitivity is shown in insets.

5 is a graph showing the cyclic voltammogram of two different cells showing different electrochemical properties. Chamber heights h were 10 μm and 35 μm. The diameter of the working chamber and the diameter of the counter chamber were 1 mm. Circulating voltamogram was obtained in a PBS solution containing 10 mM Fe (CN) ₆ ^3- and the potential was scanned at a rate of 25 mV / s. Negative current in the y-axis means reduction of ferricyanide.

FIG. 6 is a graph showing the cyclic voltamogram showing the electrochemical properties of thin cells at various scan rates (h = 10 μM). The data were obtained in a PBS solution containing 10 mM Fe (CN) ₆ ^3- and the potential was scanned from 0 V. Inset shows the relationship between scan rate and peak current.

FIG. 7 is a graph showing the differential pulse voltamogram (DPV) of dopamine in thin cells. Experiments were performed in PBS solution containing 5-50 μM dopamine. Experimental conditions for the differential pulse voltamogram were 0.2 sec pulse period, 0.1 V pulse amplitude, and 1 mV scan rate. Inset is a calibration curve.

The present invention relates to a miniaturized electrochemical system comprising a novel polyelectrolyte reference electrode and its application to thin layer electroanalysis.

The electrochemical techniques triggered by the simplicity of the devices have been widely applied to microchip based analysis systems. Although highly sensitive working electrodes vary widely, the selection options for reference half-cells are very lacking in many applications. In principle, a good reference electrode should not be polarized [1]. In other words, the electrode potential must be governed by the high exchange current across the interface. Ag / AgCl electrodes are one of the practical choices for the reference electrode and occupy a wide market. For electrochemical systems formed on microchips, various attempts have been made to fabricate miniaturized reference electrodes using Ag / AgCl configurations. Current microfabrication techniques employ pseudo-reference electrodes that are in direct contact with the sample solution. However, such an unstable potential of the pseudo reference electrode causes a shift in the working electrode potential and affects the output signal in the ammeter sensor. There is no doubt that a stable reference electrode is more important for electrometer measurements. Therefore, in order to realize better performance, efforts to miniaturize the conventional liquid junction reference electrode are very important.

Karube and his colleagues used thin film Ag / AgCl to mimic the liquid-to-liquid bonding on glass slides and silicon substrates [2-6]. To increase the lifetime of Ag / AgCl, they only chlorinated the boundaries of the Ag film. The contact between the internal solution and AgCl was limited to the microcavities, and the ends of the junction were sealed using cellulose acetate to prevent leakage of the electrolyte solution. However, to successfully reduce the potential and life dependence on KCl present in the external electrolyte, at least four photolithography processes were required to form five different layers. By elution of AgCl, the electrode life was only about 8 hours. Even when the thick Ag film was bonded, the durability of the electrode potential was hardly improved.

Liquid junctions have also been implemented in microscale systems using Ag gel and electrolytes in the form of acrylic gels. Lowy's team contained Ag wires in an acrylic hydrogel matrix containing various electrolytes [7-9]. The open circuit potential remained stable for more than 20 hours and the average operating time was claimed to be one year. This small reference electrode can be used successfully for small volumes of samples with good stability. However, his application is limited in some fields. Since all active ingredients are contained in the microchip of the eppendorf pipette, the manufacturing process is not compatible with microfabrication techniques. This type of electrode is almost impossible to integrate into a multicomponent microfluidic system, so called lap-on-a-chip.

Recently, schoning et al. Introduced a screen printed Ag / AgCl reference electrode with a thin film containing KCl [10]. The Agar layer was protected by a polyvinylchloride / cellulose nitrate bilayer. The potential was stable for more than 2 months and moderately insensitive to KCl concentration. However, the manipulation of agar can hardly be coupled to microfabrication and batching processes.

It is an object of the present invention to provide an electrochemical system which is excellent in lifespan and stable potential and which can be miniaturized. Specifically, an object of the present invention is to provide a miniaturized electrochemical system including a novel polyelectrolyte reference electrode.

Another object of the present invention is to provide a miniaturized electrochemical system including a novel polyelectrolyte reference electrode which is easy in the manufacturing process and effectively prevents contamination of the internal filling solution of the reference chamber and has an improved lifetime.

According to a preferred embodiment of the present invention, A) a thin film-deposited working electrode inside the working chamber which is the first chamber located in the middle of the traveling path of the microchannel, which is the moving path of the analysis solution, and B) the traveling path of the microchannel. A counter electrode having a thin film deposited inside the counter chamber, which is a second chamber positioned in the middle, and C) a polyelectrolyte salt bridge connected to the working chamber outside the path of the microchannel and connected to the working chamber through the polyelectrolyte salt bridge. An electrochemical system is provided that includes a connected reference chamber, an internal fill solution filled in the reference chamber, and a polyelectrolyte reference electrode comprising an Ag / AgCl electrode received in the reference chamber.

According to another preferred embodiment of the present invention, an electrochemical system is provided wherein said polyelectrolyte salt bridge is formed by poly-diallyldimethylammonium chloride.

According to another preferred embodiment of the invention, a) an inlet for introducing an assay solution, b) a microchannel for providing a passage for the analyte solution introduced from the inlet, c) at least a portion of the assay solution passed through the microchannel At least one outlet, d) a working chamber and a counter chamber located in the middle of the path of the microchannel, e) a working electrode and a counter electrode deposited on the inner surfaces of the working chamber and the counter chamber respectively, and f) of the microchannel. A polyelectrolyte salt bridge connected to the working chamber out of the path, g) a reference chamber containing an internal filling solution connected to the working chamber through the polyelectrolyte salt bridge, and h) an Ag / AgCl electrode housed in the reference chamber. Including, an electrochemical system is provided.

1 is a schematic view showing an electrochemical system including a polyelectrolyte reference electrode according to the present invention, and FIG. 2 is a perspective view showing a microchip in which the electrochemical system is implemented in the form of a thin layer cell.

Hereinafter, the present invention will be described in more detail with reference to FIGS. 1 and 2.

The electrochemical system according to the invention consists of the following components:

A) a thin film-deposited working electrode 101 inside the working chamber 106 which is a first chamber located in the middle of the traveling path of the microchannel 104 which is a moving passage of the analysis solution,

B) a counter electrode 102 thin-film deposited in the counter chamber 107 which is a second chamber located in the middle of the path of the microchannel 104;

C) the polyelectrolyte salt bridge 108 connected to the working chamber 106 outside the path of the microchannel 104 and the reference chamber 109 connected to the working chamber 106 through the polyelectrolyte salt bridge 108. ), An internal filling solution filled in the reference chamber, and an Ag / AgCl electrode 110 accommodated in the reference chamber (109).

The microchip 1 in which the electrochemical system is implemented in the form of a thin layer cell includes: a) an inlet 103 for introducing an assay solution, and b) a microchannel for providing a moving passage of the analyte solution introduced from the inlet 103. Channel 104, c) the outlet 105 of the analyte solution passing through the microchannel 104, d) the working chamber 106 and the counterpart chamber 107 located in the middle of the path of the microchannel 104; and (e) providing a moving passage of thin film deposited working electrodes 101 and counter electrodes 102 and f) chlorine ions on the inner surfaces of the working chamber 106 and the counter chamber 107, respectively; Polyelectrolyte salt bridge 108 connected to the working chamber 106 outside the path of 104, g) a reference chamber containing an internal fill solution connected to the working chamber 106 through the polyelectrolyte salt bridge 108 109, and h) an Ag / AgCl electrode 110 housed in the reference chamber 109. It is done.

1 and 2, each component will be described in more detail as follows. First, the working electrode 101 (working electrode or indicator electrode) and the counter electrode 102 (counter electrode) are formed in a thin film form by vapor deposition. The thin film deposited working electrode 101 and counter electrode 102 contribute to miniaturization of the microfluidic chip according to the present invention.

Inlet 103 is formed to introduce the assay solution. The assay solution introduced through the inlet 103 travels through the microchannel 104 and is discharged through the outlet 105. A working chamber 106 (working chamber or indicator chamber) is formed so as to overlap a predetermined region of the thin film-deposited working electrode 101. At this time, the working chamber 106 is located in the middle of the traveling path of the microchannel 104. In the same manner, the counterpart chamber 107 is formed so as to overlap a predetermined region of the thin film-deposited counter electrode 102. At this time, the counterpart chamber 107 is also located on the traveling path of the microchannel 104.

The working chamber 106 is connected to the polyelectrolyte salt bridge 108. At this time, the polyelectrolyte salt bridge 108 exists outside the path of progress of the microchannel 104. Reference chamber 109 is connected to the working chamber 106 through the polyelectrolyte salt bridge 108. The reference chamber 109 is filled by an internal solution (not shown) and receives the Ag / AgCl reference electrode 110. At this time, the Ag / AgCl electrode 110 is a macroscopic electrode, unlike the working electrode 101 and the counter electrode 102, and is an electrode having a wire or rod shape.

The macroscopic electrode Ag / AgCl electrode 110 contains a sufficient amount of AgCl, which makes it possible to keep the potential stable for a sufficient time. The reservoir to accommodate the internal electrolyte solution has a relatively large area and the outflow of chlorine ions (Cl ⁻ ) is virtually excluded by the polyelectrolyte salt bridge. Therefore, the chlorine ion concentration of the internal electrolyte solution contained in the reference chamber 109 is kept constant for a sufficient time. This improves the lifetime of the electrochemical system and leads to an increase in capacity.

According to the present invention, the reference electrode is implemented by a macro Ag / AgCl electrode 110, a monolithic reference chamber 109 filled with an internal fill solution and a micro polyelectrolyte salt bridge 108. The chemical equilibrium of the Ag / AgCl electrode 110 is stable because its surface area is sufficiently large as compared to the electrochemical cell formed on the microchip. The Ag / AgCl electrode 110 is flexible enough to conveniently connect an external operating electrical circuit to the microchip. On the other hand, the micro polyelectrolyte salt bridge 108 enables electrical connection in the form of a thin cell. As a result, electrical stability and sufficient capacity are provided from the macroscopic electrode system, and miniaturization is possible from the microscopic voltametry system. This combination allows the formation of a more stable, manageable electrochemical system on the microchip, regardless of the shape, location or size of the cell.

According to a preferred embodiment of the present invention, the polyelectrolyte salt bridge 108 is formed by poly-diallyldimethylammonium chloride. Diallyldimethylammonium chloride ("DADMAC") is a water soluble tetravalent ammonium compound, its linear polymer (poly-diallyldimethylammonium chloride, "pDADMAC") is also water soluble, wastewater treatment, paper industry, mining industry. And in biology. In biomedical applications, micro- and nano-particle drug delivery systems for proteins and peptides, and their applications to non-viral vector systems for delivering DNA, RNA to cells or tissues are currently being studied [11]. ]. pDADMAC is a polyelectrolyte hydrogel and strongly absorbs water along the polymer chain by high charge density. Furthermore, the pDADMAC layer is conductive and behaves like a KCl-saturated agar layer commonly used in macroscopic electrochemical systems. pDADMAC fillers can be formed at specific sites on the microchip by ad molecularization techniques [12]. By Kappa-Carrageenan et al., Gamma-radiation promotes the polymerisation of DADMAC and forms an internally permeable polymer network structure [13]. Bebe and colleagues, with the help of a photoinitiator, fabricated a microfluidic system by polymerizing DADMAC monomers with UV light on a mask [14]. Thus, they could selectively form polymer structures only at specific sites of interest. By this polyelectrolyte bridge 108, the influence by chlorine ions is significantly reduced because the Ag / AgCl electrode 110 located in the reference chamber 109 is operated by the polyelectrolyte bridge 108. This is because it is separated from the analysis solution of the electrode 101.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example

1. Material used

Photoinitiator {2-hydroxy-4 '-(2-hydroxyethoxy) -2-methylpropiophenone (DADMAC)} and crosslinking agent (N, N'-methylenebisacrylamide) from Sigma Aldrich Co., Ltd. (USA) Purchased. Ag wire (1.0 mm diameter, 99.99%) and polydimethylsiloxane (PDMS) were purchased from Aldrich Corporation (USA) and Dow Corning (USA), respectively. Negative epoxy-type photoresist (SU8 ^™ ) and developer were purchased from Microchem Inc. (USA) and used to make molds for PDMS channels. To pattern Pt electrodes, sulfuric acid, hydrogen peroxide, acetone, methanol and hexamethyldisilane (HMDS) were analyzed to an analytical grade. Tey. It was purchased from Baker Corporation (USA) and used without further purification. Positive photoresist (AZ5214-E ^™ ) and developer (AZ300MIF ^™ ) were purchased from Clariant Corporation (Switzerland). Photoresist and HDMS were stored at 4 ° C. in the refrigerator.

NaH ₂ PO ₄ (98%), Na ₂ HPO ₄ -12H ₂ O (98%) and KCl (99.5%) were purchased from Junsei Chemical Co., Ltd. (Japan), K ₃ F ₃ (CN) ₆ (99% ) Was purchased from Ajax Chemicals, Inc. (Australia), and dopamine (3-hydroxytyramine hydrochloric acid, 98%) was purchased from Aldrich and used. Deionized water purified using nanopoor diamond was purchased from Barnstead (USA) and used for the preparation and cleaning of electrolyte solution.

2. Fabrication and Structure of Integrated Three-electrode System

2.1 SU-8 Molding Molds for PDMS Channels

Corning 2947 slide glass (75 mm × 25 mm, thickness: 1 mm) was used as the substrate. One glass was washed in a Piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1), then washed with deionized water, acetone (CMOS grade, JT Baker, USA), methanol (CMOS grade, JT Baker, USA) and two sequential washes with deionized water. The washed slide glass was placed on a hot plate at 150 ° C. for 10 minutes to dehydrate, and then cooled to room temperature. Using a spin coater purchased from Wonju Co., Ltd. (Daejeon, Korea), hexamethyldisilazane (HMDS) was spin-coated on the slide glass for 30 seconds at a speed of 4000 rpm as an adhesion promoter, and excess solvent was 150 It was evaporated by baking for 5 minutes on a hotplate at < RTI ID = 0.0 > SU8 was spin coated on the cooled slides at a speed of 1800 rpm for 30 seconds to form a SU8 film with a thickness of 10-11 μm. The SU8-coated slide glass was then baked in two steps on a hotplate: 2 minutes at 65 ° C. and 5 minutes at 90 ° C. After cooling, in a mask aligner purchased from Midas Systems, Inc., after placing the channel-patterned mask, the slide glass was exposed to UV light (365 nm, intensity: 16 mW / cm ² ) for 10 seconds, Baked at 65 ° C. for 1 minute and 90 ° C. for 2 minutes. Thereafter, the cooled slide glass was developed using a SU8 developer. Carefully washed with isopropyl alcohol and deionized water, and the SU8 pattern on the slide glass was cured for 2 hours on a 175 ° C. hotplate to surface adhere.

To prepare the channel-patterned PDMS layer, 1 part by weight of hardener was added to 10 parts by weight of PDMS. After mixing these, the mixture was degassed in a vacuum chamber for 30 minutes. Then, the PDMS mixture was poured into the SU8 molding die prepared above and cured at 62 ° C. for 2 hours.

2.2 Metal Electrodes and Their Assemblies

Cleaning and HDMS coating on Corning 2947 slide glass (75 mm × 25 mm, thickness: 1 mm) were performed by the method described above. AZ5241-E was then spin coated for 30 seconds at a speed of 2000 rpm and baked for 1 minute on a hotplate at 100 ° C. The photoresist coated slide glass was exposed to UV light (365 nm, intensity: 16 mW / cm ² ) for 5 seconds after placing the electrode patterned mask, and baked at 100 ° C. for 5 minutes. For image reversal, UV radiation was applied to the photoresist on the slide glass for 15 seconds over the entire area. The pattern was developed using AZ300MIF developer and washed with deionized water.

The slide glass was dried by air blowing. Twenty nanometer thick Ti and 100 nanometer thick Pt layers were sequentially deposited using RF magnetron sputters. The Ti / Pt layer was then immersed in acetone and sonicated for 5 minutes using a 3510E-DTH sonicator. The diameters of the working electrode and the counter electrode were 1 mm and 2 mm, respectively. The PDMS layer with Pt-mounted slide glass and microchannel pattern was plasma treated for 30 seconds using an air plasma cleaner / sterilizer (RGD-100, Reflex Analyticals, USA), aligned, and then permanently Combined.

2.3 Polyelectrolyte salt bridge

Diallyldimethylammonium chloride (DADMAC) was selected as the polyelectrolyte salt bridge material. In the presence of 2% by weight photoinitiator and 2% by weight crosslinker, the 65% DADMAC aqueous solution was saturated with KCl. The DADMAC solution was filled in microchannels formed on the PDMS / glass chip. A mask patterned with salt bridges was placed on the chip, and UV light was irradiated for 5 seconds. The exposed DADMAC single molecules polymerized to form poly-DADMAC (hereinafter “pDADMAC”) between the reference chamber and the working chamber. The salt bridge is circular in a small space to prevent the polymer plug from moving during cleaning and sample injection. The unpolymerized DADMAC solution was removed from the channel and the microchannels and chambers were washed several times with deionized water and phosphate buffer.

3. Procedure

Electrochemical measurements were performed in phosphate buffer, adjusted to pH 7.4 by mixing Solution A (0.1M Na ₂ HPO ₄ , 0.15M NaCl) and Solution B (0.1M NaH ₂ PO ₄ , 0.15M NaCl). Variety of chloride ion (Cl ^-) in a concentration to experiments using, and a plurality of producing the solutions A and B in the stated NaCl concentration, the mixing of two solutions, each of the chlorine ion content [Cl ^-] pH is adjusted in The prepared solution.

Impedance spectra of pDADMAC hydrogels were obtained by the Precision Component Analyzer 6440A LCR meter, between two Ag / AgCl wire electrodes. Two Ag / AgCl wire electrodes were immersed in two 3M KCl solutions, respectively, with microchannels (200 μm × 30 μm, 6 mm in length) between them. One pDADMAC plug is present between the two solutions (3M KCl solution), which acts as a salt bridge. As a result, the two Ag / AgCl wire electrodes are electrically connected by the ion current flowing through the pDADMAC plug. Then, the impedance was measured in the frequency region of 20 MHz at 3 MHz.

Electrochemical measurements (cyclic voltametry (CV) and differential pulse voltametry (DPV)) were performed using an electrochemical workstation CHI 660A (CH Instruments, USA). For open-circuit potential (or simply "OCP") experiments, the reference chamber is filled with 1M KCl solution and the potential is measured using a commercial Ag / AgCl electrode (CHI111, CH Instruments, USA). It was.

4. Results and review

4.1 Electrical Properties of Polyelectrolyte Salt Bridges

To characterize the polyelectrolyte reference electrode, electrochemical impedance was measured in the frequency region of 20 Hz to 3 MHz. First, the impedance spectrum of the pDADMAC hydrogel itself was recorded. The measurement system consists of a pDADMAC hydrogel channel with a straight 6 mm length, cross-sectional area 6 × 10 ⁻⁹ m ² , and two Ag / AgCl wire electrodes in a 3M KCl solution separated thereby. The test resulted in a balanced impedance of 26 kΩ across the entire frequency range, meaning that the pDADMAC hydrogel is a resistive, frequency independent material. The resistivity of the pDADMAC polymer is determined by Equation 1 below.

ρ (resistance, m) = area × resistance / length

The resistivity was 0.026 mm. The impedance spectrum of the integrated salt bridge based on the pDADMAC hydrogel was then obtained in the microchip system of this study. The impedance was measured between the reference chamber and the working chamber shown in FIGS. 1 and 2. As expected in the resistivity of the pDADMAC hydrogel, the result yielded a balanced impedance of 40 kΩ over the entire frequency range. The impedance spectrum of the pDADMAC hydrogel plug was very similar to that of the small reference electrode based on agar [15].

4.2 Stability of Polyelectrolyte Reference Electrode

In phosphate buffered saline solution, the average potential of the polyelectrolyte reference electrode was 19 mV compared to Ag / AgCl (3 M KCl) and had a confidence region (95%) of 13-26 mV, which was a reproducible result. The measured value of 25 mV due to the difference in the concentration of chlorine ions was in the confidence region.

One of the headaches affecting the performance of the reference electrode is the anion that precipitates when silver ions are present. When the Ag / AgCl electrode is exposed to a solution containing Br ⁻ , I ⁻ , S ^{2 −} , the poor solubility of the corresponding salt causes a significant potential shift in the negative direction (K _sp (AgCl) = 1.8 × 10 ⁻¹⁰ , K _sp (AgBr) = 5.0 × 10 ^-13 , K _sp (AgI) = 8 × 10 ^-17 , K _sp (Ag ₂ S) = 8 × 10 ^-51 ) [2,16].

Table 1 is a table measuring the potential of the polyelectrolyte salt bridge compared to commercial Ag / AgCl (3 M KCl).

Interference 10nM solution NaBr NaI Na ₂ S Average (trusted area) 95% Potential before addition of interfering substance (mV) 21.7 17.4 18.7 19.3 (± 6.6) Potential after addition of interfering substance (mV) 17.1 17.6 18.0 17.6 (± 1.5)

As shown in Table 1, the reactivity of the polyelectrolyte reference electrode did not show a deviation beyond the error region even after exposure to the concentrated anions. Thus, Ag / AgCl wire in the reference electrode is completely present during the negative ion disturbance test. It has been reported that multilayered pDADMAC films are good diffusion barriers to biochemicals such as glycerol, glucose and sucrose. A 40 nm thick pDADMAC film rejected 90% sucrose and reduced the flow of NaCl by 40% [17]. The system has a 500 μm long pDADMAC barrier and the Ag / AgCl wire is located far from the salt bridge. Therefore, precipitation of interfering anions and silver ions is safely prevented.

The potential of the Ag / AgCl electrode is governed by the degree of activation of chlorine ions, and can be expressed by the Nernst equation of Equation 2 below.

^{E = E 0 - 2.303 (RT} / nF) log 10 [Cl -] α

Here, E ⁰ is the electrode standard potential, n is the number of electrons involved in electrochemical reactions, R is molar gas constant, T is absolute temperature, F is Faraday's constant, [Cl ^-] is the concentration of free chloride ion, α is Chloride activation activity constant. 2.303 (RF / nF) is temperature dependent, converted to 59.16 mV at 25 ° C., and α = 1 in dilute solution. Therefore, the equation is represented by the following equation (3).

_{^{E = E 0 - 59.26log 10 [}} Cl -]

3 is a graph showing the dependence of chlorine ion concentration between the pseudo reference electrode having a thin Ag / AgCl electrode and the polyelectrolyte reference electrode having a salt bridge. As shown in FIG. 3, the potential of the Ag / AgCl pseudo-reference electrode in 0.01-1.0 M NaCl solution varies in the region of 129-27 mV. In the case of the thin film reference electrode, the slope of the electrode potential with respect to log [Cl ⁻ ] was 50 mV. For Ag / AgCl thin films, a 10 μm thick Ag layer was electroplated on the sputtered Pt film and the surface of the AgCl layer was chlorinated for 30 minutes using 1% (w / w) FeCl ₃ aqueous solution. For polyelectrolyte reference electrodes, the effect of [Cl ⁻ ] was significantly reduced because the Ag / AgCl wire electrode located in the reference chamber was separated from the analyte solution of the working electrode. The polyelectrolyte reference system exhibited a limited potential shift of 10 mV over the 1 mM-1 M concentration region. This result was similar to the trend in the liquid junction reference electrode [4, 10]. In order to rule out pH variations, it is to be noted that various concentrations of NaCl solution were obtained by adding a fixed amount of NaCl to 0.1M phosphate buffer. Thus, dilute NaCl solution (<0.1 M) will have similar ionic strength.

4.3 Life of Polyelectrolyte Reference Electrode

The life span of the miniaturized liquid reference electrode is influenced by two main factors. One is the elution of AgCl from the Ag / AgCl electrode and the other is the leakage of Cl ⁻ from the internal electrolyte solution causing a significant change in concentration. Ag / AgCl wires have a sufficient amount of AgCl and can hold a potential for several hours at currents of ˜pA. The reference chamber containing the internal electrolyte solution is relatively very large (˜50 nL) and virtually excludes Cl ⁻ outflow to the external solution by the polyelectrolyte salt bridge described above. Thus, the concentration of chlorine ions in the internal electrolyte solution can be kept constant for an extended period of time.

To test long term stability, 1 M KCl solution was charged to the reference chamber, and the PBS solution was charged to the working chamber and the counter chamber. 4 is a graph showing the potential deviation of the polyelectrolyte reference electrode in phosphate buffered saline (PBS). As shown in FIG. 4, over a period of 30 hours or more, the potential slowly changed between 15 mV and 18 mV. Given that the lifetime of most conventional Ag / AgCl thin film electrodes is several hours in continuous mode, the long-term stability of the polyelectrolyte reference electrode is comparable to that of the macroscopic reference electrode. After 30 hours, the experiment was discontinued because the accuracy of the measurement was not guaranteed for a much longer period of time. To avoid any change in electrolyte concentration during the measurement, the PDMS reservoirs for the working electrode and reference electrode elements were covered with para film. However, sequential evaporation of water was unavoidable because complete sealing was not possible. The electrode potential of the macroscopic reference electrode was changed by the outflow of KCl and the outflow of AgCl from the internal solution.

4.4 Linear Swept Voltametry Applications

Thin layer cells, because of their high accuracy, are the preferred system for bulk electrolysis to quantify the total amount of analyte. This is why thin layer cells have been used extensively in flow injection systems. Furthermore, the thin layer system can provide a lot of information (eg, rate constants) that traditional systems cannot provide. Thin layer cells typically have a solution of 2-100 μm thickness on the electrode surface. Recent microfabrication techniques have the advantage of achieving a high area per unit volume without sophisticated equipment. If the solution thickness is more than a few hundred micrometers, the current-potential curve is asymmetric in an ideal diffusion control system, and the peak current is proportional to the square root of the scan rate. The potential difference between the oxide peak and the reduced peak is about 60 mV for one electron reaction. As the solution thickness is reduced to tens of nanometers or less, the voltammetry curves for oxidation and reduction are axial, and the peak current is linearly proportional to the scan rate [18]. Lateral diffusion causes the curved form of the voltammogram. Therefore, thin film systems on microchips should be designed such that the cell is free of lateral diffusion.

An electrochemical cell with a polyelectrolyte reference electrode was prepared and tested in a PBS solution containing 10 mM Fe (CN) ₆ ^3- . 5 shows a circulating voltammogram having two different heights from the working electrode. The dotted curve showing the circulating voltammogram when the chamber height h was 35 μm was asymmetric and the ΔE of the chamber was 37 mV. On the other hand, a solid curve with a height of 10 μm is a typical result of thin cell, and ΔE was only 4 mV. This result shows that the thickness of the electrolyte layer is critical for the voltametry behavior in the thin layer cell. 6 demonstrates that in a cell with a 10 μm height, the peak current is linearly proportional to the scan rate.

To date, accurate reference electrodes have not been integrated simply because existing miniaturized reference electrodes are still so large and complex that they cannot be fabricated in microfluidic systems. In addition to the new polyelectrolyte salt bridges, thin layer cells were fabricated through a relatively simple process (single-stage photolithography), and a practical design could be applied to multicomponent microfluidic systems.

4.5 Differential Pulse Voltametry Applications

Differential pulse voltammetry (DPV) is a useful tool for the microanalysis of organic and inorganic molecules, since charge currents are excluded and Faraday currents dominate sensitivity. Since DPV has been widely used in the environmental, biological research [19-22] and food [23] fields, various miniaturized sensors based on various electrode materials have been tested. In most cases, however, these new sensors were tested in bulk solution with macroscopic reference electrodes. Only a few references have used DPV in microchip based electrochemical cells [24].

5 micrometers of dopamine in PBS buffer was clearly detected by DPV. The calibration curve was linear in the region of 5-50 μM and the detection limit was calculated to be 2 μM based on the standard deviation of three background signals (FIG. 7). Since the resistance of the reference electrode system including the polyelectrolyte salt bridge was flat at 40 kΩ, it is believed that the potential pulse can be stably applied to the working electrode without significant error.

Polyelectrolyte salt bridges have been introduced into microfabricated electrochemical cells as ion current paths and diffusion barriers for salt bridges. Polyelectrolyte salt bridges worked as well as traditional salt bridges and showed little delay in response time. In a three-electrode electrochemical cell fabricated in a microchip, a polyelectrolyte salt bridge, preferably a pDADMAC hydrogel, separated the analyte from the internal solution of the reference chamber while providing a passage for the chloride ion. This eliminated contamination by potential interference anions and significantly suppressed the dependence on chlorine ion concentration as compared to the pseudo reference electrode. The life of the reference electrode was improved, and even after continuous use, the electrode potential was stable even after 30 hours.

The electrochemical characterization of small thin-layer cells incorporating triodes has been demonstrated by cyclic voltammetry. Differential pulse voltammetry (DPV) was applied to the electrochemical cell and several micrometers of dopamine were successfully detected. The polyelectrolyte reference electrode is similar to the macroscopic reference electrode in terms of operation principle and capacitance. Therefore, by employing the small electrochemical cell according to the present invention, most electrochemical techniques can be performed.

In addition to the improved performance of the polyelectrolyte reference electrode, another important advantage is that the polyelectrolyte reference electrode can be manufactured by a simple manufacturing process, that is, two-stage lithography. By virtue of their practical design, microchips comprising polyelectrolyte reference electrodes can be used in combination with fluid components such as micropumps, valves and flow sensors for miniaturization of electrochemical analyzers.

While the invention has been shown and described in connection with specific embodiments, it is conventional in the art that various changes, modifications and variations are possible without departing from the spirit and scope of the invention as indicated by the appended claims. Anyone who knows the knowledge of is easy to know.

- references -

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Claims (3)

A) a thin film-deposited working electrode inside the working chamber, which is a first chamber located in the middle of the traveling path of the microchannel, which is a moving passage of the analysis solution, B) a counter electrode having a thin film deposited inside the counter chamber, which is a second chamber located in the middle of the path of the microchannel, and C) a polyelectrolyte salt bridge connected to the working chamber outside the path of the microchannel, a reference chamber connected to the working chamber through the polyelectrolyte salt bridge, an internal filling solution filled in the reference chamber, and the reference chamber An electrochemical system for electroanalyzing analytical solution, comprising a polyelectrolyte reference electrode comprising an acceptable Ag / AgCl electrode. The electrochemical system of claim 1, wherein the polyelectrolyte salt bridge is formed by poly-diallyldimethylammonium chloride. An electrochemical system according to claim 1 is implemented as a microchip in the form of a thin layer cell, a) inlet for introducing the assay solution, b) a microchannel providing a passage for the assay solution introduced from said inlet, c) at least one outlet of the assay solution through the microchannel, d) an operating chamber and a counter chamber located in the middle of the path of the microchannel; e) a working electrode and a counter electrode deposited on the inner surfaces of the working chamber and the counter chamber, respectively; f) a polyelectrolyte salt bridge connected to the working chamber outside the path of travel of the microchannel, g) a reference chamber containing an internal filling solution, connected to said working chamber via said polyelectrolyte salt bridge, and h) an Ag / AgCl electrode housed in the reference chamber, A polyelectrolyte salt bridge connected to the working chamber outside the path of the microchannel, a reference chamber connected to the working chamber through the polyelectrolyte salt bridge, an internal filling solution filled in the reference chamber, and received in the reference chamber. A microchip in which a polyelectrolyte reference electrode is implemented by an Ag / AgCl electrode.

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