WO2013168835A1 - Electrophoretic chip for electrochemical detection - Google Patents

Abstract

The present invention relates to an electrophoretic chip for electrochemical detection and to a method for monitoring an electrochemical signal using the electrophoretic chip without interferences by an electric field. More particularly, the electrophoretic chip is configured in that a working electrode for detecting the electrochemical characteristics of materials for fluidic analysis is arranged at one point in a fluid channel, an equipotential structure electrically connected to the working electrode is arranged on at least one wall surface of the channel, and a counter electrode is arranged in an equipotential space formed by the equipotential structure and filled with a supporting electrolyte solution, in the outside of the channel arranged adjacent to the fluid channel with the equipotential structure serving as a passage, thus detecting an electrochemical signal generated between the working electrode and the counter electrode. Furthermore, the method for monitoring an electrochemical signal without interferences by an electric field through the working electrode arranged at one point in the fluid channel to which electric field is applied comprises a step of measuring an electrochemical signal between the working electrode and a counter electrode. An equipotential space is formed by an equipotential structure formed on at least one wall surface of the fluid channel and electrically connected to the working electrode. The counter electrode is disposed in the equipotential space separated from the channel by the equipotential structure.

Description

Electrophoretic Chips for Electrochemical Detection

The present invention relates to an electrophoretic chip for electrochemical detection and a method for monitoring an electrochemical signal without interference of an electric field using the same. Specifically, the electrophoretic chip is provided with a working electrode for detecting the electrochemical characteristics of the fluid analyte at one point in the fluid channel, the equipotential structure electrically connected to the working electrode on at least one wall of the channel is provided The electrochemical signal generated between the working electrode and the counter electrode is provided with a counter electrode in the equipotential space formed by the equipotential structure and filled with a supporting electrolyte solution outside the channel adjacent to the fluid channel through the equipotential structure as a passage. It is characterized by detecting. In addition, a method for monitoring an electrochemical signal without interference of the electric field through a working electrode provided at a point in the fluid channel to which the electric field is applied, comprising the steps of measuring an electrochemical signal between the working electrode and the counter electrode, In this case, the equipotential space is formed by an equipotential structure formed on at least one wall surface of the fluid channel and electrically connected to the working electrode, and the counter electrode is disposed in the equipotential space separated from the channel by the equipotential structure. A method is characterized.

Microchip electrophoresis (MCE) has emerged as a method for chemical and biological analysis using miniaturized systems over the last decades. Recently, microfluidic chip-based analytical tools have been applied to integrate a plurality of unit processes such as sample preparation, separation, and detection into a single chip so that non-experts can perform the analysis anytime and anywhere. An essential element for portable systems is the detection method for high performance separation-based systems with low detection limits, fast analysis, high efficiency, low cost, one-time and portability. In practice, miniaturized detectors can provide significant advantages for chip-based analytical tools.

Fourier transformation of infrared light absorption spectra (FT-IR), Raman scattering, nuclear magnetic resonance (NMR), refractive index (RI), thermal lens microscopy (thermal lens microscopy; TLM), microplasma-optical emission spectroscopy (OES), surface plasmon resonance (SPR), electrochemical analysis (EC), chemiluminescence Attempts to integrate detection methods such as CL, mass spectrometry (MS), UV-vis absorption (UV-vis), and laser induced fluorescence (LIF) into electrophoretic devices Coming. In particular, attention is focused on MS, LIF, UV-vis and EC due to compatibility with capillary electrophoresis (CE) without significant loss of quality of detection. Although most of the commercially available CE instruments use UV-vis absorption, the detection method is inherently unsuitable for the analysis of trace species in concentration ranges of nanomolar or sub-nanomol due to poor detection limits. MS detection has been reported to provide high efficiency in combination with MCE, but it requires expensive equipment and has a disadvantage in that it is generally not portable. LIF is a universal detection method in combination with MCE that allows for extremely sensitive detection. However, in order to perform the LIF method, the analyte must be synthesized as a phosphor or a natural fluorescent compound must be selected as the analyte. EC detection consists of a very simple device and a microscale electrode integrated on a microchip, but with excellent sensitivity and selectivity. Therefore, it is widely used as an ideal detection method for microfluidic on-chip separation systems. The biggest problem with this EC detection method is the effect of the high CE voltage applied basically for the CE and the strong electric field the detector receives. This can cause severe noise and make electrochemical detection impossible. In addition, electrical surges can cause serious damage to the EC detector.

Detection methods by current measurement are classified into end-channel type and in-channel type according to the position of the working electrode in the microchannel. End-channel detection is an easy way to measure redox currents from analytes by reducing the effects of CE voltage and current. Woolley et al. Proposed an end-channel detection method on a microchip using a working electrode located at the end of a separation channel gradually widening immediately before the working electrode to minimize interference of a capillary electrophoresis field (CE field). However, measuring the electrochemical current near the outlet of the channel can lead to half-wave potential shift of the redox species, thereby reducing reproducibility due to the sensitivity of the relative position of the working electrode and the channel outlet under electrophoresis. In order to solve this problem, Wang et al. Developed an end-channel detection method called "wall-jet" that maintains a specific distance between the separation channel and the working electrode. However, the structure of the isolation channel connected to the vertically present detection strip limits the miniaturization and diversity of the microchip. Although end-channel detection can provide a way to measure EC currents under electrophoresis, there are still some practical problems, including sample plug dispersions that cause peak broadening and affect separation efficiency. Ertl et al. Improved end-channel detection by using sheath-flow on the microchannels to prevent dispersion of the sample plug. However, the formation of skin flow channels increases the complexity of the channel design. Several theoretical and experimental strategies have been reported for measuring redox currents from analytes in separate channels. Martin et al. Proposed a design with working electrodes arranged at the end of the separation channel but still present. However, the in-channel arrangement requires the exact arrangement of working electrodes within the separation channel to minimize half-wave potential shifts and electrochemical noise. Chen et al. Proposed an in-channel detection method that eliminates potential shifts using a dual-channel configuration, but at this time, the working electrode and the reference electrode are placed on the counter electrode located at the channel outlet to minimize the iR drop. It should be as close as possible. In addition, the dual channel in the detection method requires a complex channel design that inhibits the simplification of the chip pattern and makes it difficult to integrate other elements on the same chip.

The decoupling approach is another method to separate the signal by current measurement from the electrophoretic field and to eliminate the diffuse band-wideness observed in the end-channel method. Rossier and colleagues have integrated a decoupler, which consists of an array of microholes located perpendicular to the separation channel. Chen et al. Used a palladium metal electrode as a decoupling device to effectively remove hydrogen bubbles. Although the decoupling method for in-channel detection can separate the EC detector from the separation field and provide another effective way to suppress the band widening that is characteristic of the end-channel method, the decoupling device There is still a band dispersion due to the rapid decrease in electric field strength between the electrode and the working electrode. Another approach using bipolar electrochemistry is in-channel detection without the use of decoupling devices. However, current-based detection method-based bipolar electrochemistry, which modulates the potential of the working electrode by controlling the size of the bipolar electrode, the electrode spacing, or the electric field strength, provides an intrinsic redox potential that depends on the individual redox-active analyte. The variety makes them unsuitable for the detection of redox-activity analytes.

Accordingly, the present inventors have diligently studied a method that can minimize the influence of strong electrophoretic field and enable reproducible electrochemical measurement. As a result, a polymer electrolyte electrically connected to a working electrode in a fluid channel as an equipotential structure on one wall of the fluid channel By introducing a polyelectrolytic gel salt bridge (PSGB), it acts as a pseudo-reference electrode, causing the working electrode and the reference electrode to experience the same potential difference, so that they are in-channel anywhere on the microchannel without being affected by electrophoresis. A novel microchip for capillary electrophoresis that enables detection and a detection method using the same has been identified and the present invention has been completed. The in-channel method is particularly suitable for MCE for the following reasons: (i) In-channel detection can easily be carried out without decouplers. (ii) The production of PGSB by UV exposure is very simple and therefore the PGSB can be placed anywhere in the separation channel. (iii) An electrically isolated detector can eliminate damage to the electronics, thereby minimizing potential fluctuations at the working electrode. (iv) By placing the working electrode in the separation channel, high separation efficiency can be obtained by eliminating the band-wideness observed when using the end-channel detection method. (v) reducing the half-wave potential shift by placing the working electrode and the reference electrode on the equipotential surface and substantially reducing the background noise due to the rapid decrease in resistance of the polymer electrolyte salt bridge region; Thus, a constant potential can be applied to the working electrode even under varying and posibly fluctuating electrophoretic fields.

SUMMARY OF THE INVENTION An object of the present invention is an electrophoretic chip having a fluid channel between both ends and having a fluid channel separating analyte by a difference in moving speed while moving analytes in a sample by the voltage. A working electrode is provided for detecting the electrochemical characteristics of the analyte, and an equipotential structure electrically connected to the working electrode is provided on at least one wall of the channel, and the channel is adjacent to the fluid channel using the equipotential structure as a passage. The present invention provides an electrophoretic chip characterized by measuring an electrochemical signal between a working electrode and a counter electrode by having a counter electrode disposed in an equipotential space formed by the equipotential structure and filled with a supporting electrolyte solution.

Another object of the present invention is a method for monitoring an electrochemical signal without interference of the electric field through a working electrode provided at a point in the fluid channel to which the electric field is applied, the electrochemical signal between the working electrode and the counter electrode And measuring, wherein an equipotential space is formed by an equipotential structure formed on at least one wall of the fluid channel and electrically connected to the working electrode, and the counter electrode is separated from the channel by the equipotential structure. It is to provide a method characterized by being disposed in the equipotential space.

According to the present invention, in the analysis of materials using electrophoresis, an equipotential structure electrically connected to a working electrode for electrochemical detection existing in a separation channel, for example, a polyelectrolyte gel salt bridge, forms an equipotential space and forms the equipotential space. By placing the counter electrode and optionally the reference electrode in the space, the equipotential structure acts as a pseudo reference electrode, allowing electrochemical detection without interference by a strong electric field for electrophoresis.

1 is a diagram illustrating a process of manufacturing PGSB on a glass microchip. UV adhesion is fast and can be applied to glass chips to ensure the stability of the electrodes. PGSB was formed by exposure to UV immediately after adhesion.

2 shows the design of the electrode and the micro channel. (a) shows the photo-mask design for the electrode and micro channel pattern, the scale being as follows: dual-T from injection channel, sample (and sample-waste) reservoir 6 mm to channel, 2 mm from buffer reservoir to dual-T channel; Separation channel length. 16 mm; Effective length, 12 mm; PGSB channel, 350 μm. (b) shows an image of an electrode and a microchip channel. The channel is 80 μm wide and 15 μm deep. The dual-T channel is 80 μm wide, 15 μm deep and contains an injection intersection of 100 μm. PGSB channels are 120 μm wide and 15 μml deep. Au electrodes have a width of 10 or 20 μm. (c) is a microchip photograph for in-channel electrochemical detection.

3 is a schematic representation of a polyelectrolytic gel salt bridge (GSG) -integrated microchip eletrophoresis (MCE).

4 shows the open circuit potential difference as a function of the relative positions of the Au working electrode and the PGSB under various electric fields. The Au working electrode (10 μm wide) was placed at (a) 0 μm, (b) 100 μm, (c) 200 μm, and (d) 400 μm from the PGSB as shown in the inset. Micro channels were filled with 100 mM KNO ₃ solution.

5 shows the potential profile predicted near the PGSB region. Δφ _PGSB PGSB of channels is much smaller than the potential at any place within the microchannel.

FIG. 6 is a cyclic voltammetry from an Au electrode located in front of the PGSB under a capillary electrophoresis field (CE field). The solid line is a graph for 0 V / cm, the dashed line for 200 V / cm and the dashed line for 400 V / cm. Cyclic voltammetry was performed in a 1 mM K ₃ Fe (CN) ₆ solution containing 100 mM KNO ₃ as auxiliary electrolyte. 10 μm wide Au working electrode was used, Ag / AgCl / KCl (3M) was used as reference electrode, Pt line was used as counter electrode, and scanning speed was 100 mV / s.

7 shows the effect of the distance from PGSB to Au working electrodes under various electrophoretic fields. (a) is electrophoresis when the Au working electrode is located at 50 μm distance from PGSB, and (b) is at 150 μm distance. The width of the Au working electrode is 60 μm and a potential of between 0 and +0.5 V for Ag / AgCl is obtained at a scanning rate of 50 mV / s in a 5 mM K ₃ Fe (CN) ₆ solution containing 100 mM KNO ₃ . Cyclic voltammetry was performed by cycling.

8 is an electrophoretic diagram of 200 μΜ K ₃ Fe (CN) ₆ detected at (a) 0 μιη and (b) 50 μιη from PGSB. Performance conditions were as follows: capillary electrophoresis intensity, -150 V / cm; Au working electrode width, 20 μm; Total length, 1.6 cm; Effective length, 1.2 cm; Running buffer, 25 mM sodium borate; Detection potential, +0.15 V vs. Ag / AgCl / KCl (3 M) reference electrode.

FIG. 9 is an electrophoresis diagram of 1.5 μM K ₃ Fe (CN) ₆ detected using an Au electrode located near PGSB. Performance conditions were as follows: capillary electrophoresis intensity, -150 V / cm; Au working electrode width, 20 μm; Total length, 1.6 cm; Effective length, 1.2 cm; Running buffer, 25 mM sodium borate; Detection potential, +0.15 V vs. Ag / AgCl / KCl (3 M) reference electrode.

FIG. 10 is the electrophoresis of catechol (150 μM) and dopamine (100 μM) under high electric fields obtained using PGSB-integrated microchips. The performance conditions were as follows: Au working electrode width, 20 μm; Total length, 5.4 cm; Effective length, 5 cm; Running buffer, 25 mM MES (2- (N-morpholino) ethanesulfonic acid); Detection potential, +0.05 V vs. Ag / AgCl / KCl (3 M) reference electrode.

11 is a view schematically showing an equipotential structure and an equipotential space formed thereby according to the present invention.

In one aspect, the present invention provides an electrophoretic chip having a fluid channel between both ends thereof and having a fluid channel separating analyte by a difference in moving speed while moving analytes in a sample by the voltage. A working electrode is provided at one point to detect the electrochemical characteristics of the analyte, and an equipotential structure electrically connected to the working electrode is provided on at least one wall of the channel, and is adjacent to the fluid channel using the equipotential structure as a passage. It provides an electrophoretic chip characterized by measuring the electrochemical signal between the working electrode and the counter electrode is provided with a counter electrode in the equipotential space formed by the equipotential structure and filled with a supporting electrolyte solution outside the channel.

In another aspect, the present invention is a method for monitoring an electrochemical signal without interference of the electric field through the working electrode provided at a point in the fluid channel to which the electric field is applied, the electrochemical signal between the working electrode and the counter electrode And measuring an equipotential space by an equipotential structure formed on at least one wall surface of the fluid channel and electrically connected to the working electrode, wherein the counter electrode is connected to the channel by the equipotential structure. A method is characterized by being arranged in separate equipotential spaces.

"Electrophoresis" is a method of separating a substance by the difference in the moving speed of the substance to be analyzed under an electric field. The analytes have their own charges and are based on the principle that the electric field will move at different speeds. In a solvent, such as a buffer, the molecules each have a unique charge (for example, DNA is usually negatively charged and proteins are negatively charged, neutral or positively charged depending on pH). They can be separated because they move at different speeds. Electrophoresis in the present invention is preferably capillary electrophoresis. The capillary electrophoresis is a separation method capable of separating neutrals as well as positive and negative ions at the same time by introducing the separation mechanism of electrophoresis and the instrumentation and automation concept of chromatography. When an electric field is applied to the analyte mixture present in the solution, each component moves in a constant direction and speed depending on the charge and mobility. Each component is separated according to mobility, and factors affecting mobility include surface charge characteristics such as particle size, shape, average charge, and pH, concentration, and temperature of an aqueous solution. Capillary electrophoresis uses dozens of μm diameter capillaries and is typically an electroosmotic, an electrophoretic phenomenon rather than a laminar or parabolic flow caused by an external pump. High separation efficiency can be obtained by flow. Another advantage of the electroosmotic flow is that it moves in the same direction regardless of the charge of the analyte. This is because the electroosmotic flow acts significantly greater than the force that the negative ions tend to the anode. Therefore, it moves faster by the combined force of the cation's electrical and electroosmotic flow, the neutral material moves by the electroosmotic flow rather than the electrical movement, and the anion is the anode by the electroosmotic flow which is significantly larger than the electrical movement. Move to the cathode, but at a slower rate than the neutral.

This principle of capillary electrophoresis can be applied to microchips. By preparing an analysis chip having a microfluidic channel having a length of several cm and a width of several tens of μm and applying an electric field to the sock end, a trace amount of sample can be separated and / or analyzed on the same principle as the capillary electrophoresis. However, when the electrophoretic separation using such a microchip, the moving distance that the sample can be separated is short, a strong electric field must be applied to increase the separation efficiency. In addition, a strong electric field may be applied for the injection, transfer, mixing, reaction, separation, detection or post-analysis of the sample. However, these strong electric fields affect the electrochemical detection of analytes.

The “electric field” is expressed as a voltage applied per unit length, and may be applied up to 30 kV in a commercially available capillary electrophoretic system, but in some cases, a high voltage of 60 kV or more may be applied. When a voltage of 30 kV is applied to a 30 cm capillary, an electric field of about 1000 V / cm is applied. However, the voltage applied for the electrophoresis may be selected in consideration of analyte and separation efficiency. For efficient separation and electrochemical detection of analytes, an electric field of 0 V / cm to 1000 V / cm may be preferably applied, and more preferably an electric field of 0 V / cm to 500 V / cm may be applied. However, the present invention is not limited thereto.

According to an embodiment of the present invention, using a working electrode having a width of 10 μm, electrochemical detection was possible even under an electric field of 500 V / cm without interference of the electric field. However, if the width of the working electrode is reduced to 1 μm, the electrochemical detection is possible under an electric field of 8000 V / cm in theory.

Accordingly, in the present invention, an equipotential structure electrically connected to a working electrode for electrochemical detection of analyte located in a separate channel to exclude the influence of a strong electric field for electrophoresis in the electrochemical detection of an electrophoretic analysis using a microchip. It is characterized by allowing to form an equipotential space by introducing a. On the other hand, the equipotential space may be formed by introducing a polymer electrolyte gel salt bridge as an equipotential structure, for example. The introduction of the equipotential structure eliminates the voltage gradient that can occur due to the electric field for electrophoresis between the working electrode and the reference electrode and allows both electrodes to be at the same potential. The equipotential structure may refer to a structure introduced to extend the equipotential surface into a three-dimensional space.

The electrochemical signal may include an electrical signal of an analyte or a chemical signal converted into an electrical signal through an electrode, and may be a current, conductivity or potential difference, but is not limited thereto.

An electrode introduced on the analysis chip for detecting the electrochemical signal may be provided at one wall (in-channel or off-channel detection method) or at an end thereof (end-channel detection method) in the separation channel. The working electrode for performing the in-channel detection method, that is, the intra-channel detection method, may be provided on one wall in the separation channel perpendicularly to the separation channel, that is, parallel to the electrophoretic field, but is not limited thereto. It can be introduced without limitation.

The electrode system for electrochemical detection can be a two-electrode system or a three-electrode system. The two-electrode system includes a working electrode and a counter electrode for the detection of the analyte, while the three-electrode system further includes a reference electrode. In general, three-electrode system is widely used, but is not limited thereto. In the two-electrode system, the counter electrode also serves as a reference electrode. Accordingly, in the present invention, the term "reference electrode" may refer to a reference electrode provided as a reference electrode in the case of a two-electrode system, and a reference electrode provided separately from the counter electrode in the case of a three-electrode system. A counter electrode and optionally a reference electrode may be provided in an equipotential space filled with a supporting electrolyte solution separated from a channel by an equipotential structure such as the polyelectrolyte gel salt bridge. In this case, the equipotential structure serves as a pseudo reference electrode.

In the present invention, an "equipotential surface" means a surface consisting of points having the same potential in an electric field in a dictionary meaning. The separation channel formed on the microchip has a short distance of several cm and a strong electric field (for example, tens to hundreds of V / cm) is applied for separation within the short distance. The physical distance between the reference electrodes causes errors in the detection signal due to the large potential difference on the solution in the channel. Therefore, in an embodiment of the present invention, a polymer electrolyte gel salt bridge is introduced as an equipotential structure on the working electrode and the equipotential surface for electrochemical detection existing in the channel of the electrophoretic microchip, and is separated from the channel by the equipotential structure. An equipotential space was formed in which the space had the same potential as the working electrode.

The polymer electrolyte gel salt, which is the equipotential structure, is preferably an ion conductive polymer, and thus allows free movement of ions between the separation channel and the space in which the reference electrode and the counter electrode exist, while allowing buffer components of other components, such as the separation channel, to separate. The mixing with the supporting electrolyte solution filled in the external channel separated by the polymer electrolyte gel salt bridge may serve to block. That is, the equipotential structure allows free movement of ions, but can prevent leakage of analytical buffer solution to a high concentration of a supporting electrolyte. Since the equipotential structure formed of the ion conductive polymer has a very low resistance, its width is not limited, but is preferably formed as thin as long as it has a function of allowing the movement of ions and blocking the entry and exit of the solution. For example, the resulting polymer electrolyte gel salt bridge may be prepared in a few tens of nm to several hundred μm in width.

Preferably, the equipotential space formed by the equipotential structure is a vertical distance from the outer wall of the separation channel to the reference electrode including the width of the fluid channel A, the passage length toward the equipotential space formed by the equipotential structure, and the separation channel width. When the width of the passageway toward the equipotential space is C, the vertical distance B from the outer wall of the separation channel to the reference electrode may be at least 1.5 times the width A of the fluid channel. The passage may have a length of several μm to several hundred μm, preferably 5 to 50 μm, and more preferably 3 to 10 μm, but is not limited thereto. In this case, the width C of the passage may be several μm to several hundred μm, preferably 1 to 10 μm, whereby the width D of the equipotential space in which the counter electrode and / or reference electrode separated from the channel is present is It may be 1 to 10 times or 1 to 100 times C, but is not limited so long as it does not depart from the description of the microchip (FIG. 11). The width of the equipotential space is also not limited unless it leaves the microchip. The shape of the equipotential space separated from the channel through the passage may be circular or square, but is not limited thereto as long as it can accommodate a counter electrode and / or a reference electrode for detecting an electrochemical signal, and the width of the equipotential space is not limited thereto. The maximum distance between the face on which the passage exists or the face opposite thereto, the width means the maximum distance between the faces perpendicular to the faces, ie parallel to the passage. However, the size of the channel and the structure presented above is only a value created in consideration of the size of the microchip presented in the embodiment can be adjusted according to the size of the microchip itself. In other words, when manufactured in the nanometer or centimeter level analysis chip can be adjusted to a level corresponding thereto.

The polymer electrolyte gel salt bridge is injected with a polymer monomer (monomer) or dimers to depolymers in the channel and the gel is irradiated locally using a photomask on the same position as the working electrode to form an equipotential surface. It can be prepared by curing in the form. In order to induce curing by light irradiation, the polymer monomer or polymer may be reacted by using a material in which a photoinitiator is bound or by additionally adding a separate photoinitiator. Preferably, as the polymer monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) or diallyldimethylammonium chloride (DADMAC) may be used. As photoinitiator, 2-hydroxy-4 '-(2-hydroxyethoxy) -2-methylpropiophenone may be used, but curing When the gel is formed to form a conductive polymer that can allow free access of the electrolyte ions and can be cured by irradiation with light can be used without limitation. In addition, a crosslinking agent may be further added to stabilize the structure of the prepared polymer electrolyte gel salt bridge to form a more rigid structure. As one example of the crosslinking agent, N, N'-methylenebisacrylamide may be used, but any material capable of stabilizing the prepared polymer salt bridge structure may be used without limitation.

In order to bond the polyelectrolyte gel salt to the substrate, 3- (trimethoxysilyl) propylmethacrylate (TMSMA) was added to the channel of the prepared analytical chip for a predetermined time and washed with alcohol. The method of coating can be applied to the inner wall of the fluid channel.

A schematic diagram of an electrophoretic microchip for electrochemical detection prepared by the above method is shown in FIG. 3.

The electrophoretic chip of the present invention is preferably manufactured on a substrate made of glass, quartz or silicon, but is not limited thereto. In addition, poly dimethylsiloxane (PDMS), poly methyl methacrylate (PMMA), polycarbonate (PC), polystyrene, cellulose acetate and polyethylene Polymers such as terephthalate (poly (ethylene terephthalate; PETP) may be used, and may be used without limitation as long as the microfluidic channel may be formed by a known method such as nanolithography. It can optionally be made in the form of a conjugate of two or three different materials.

In the present invention, the working electrode may be a fine band electrode made of a metallic material or a semiconductor material such as gold, platinum, carbon, indium tin oxide (ITO), but is not limited thereto. The fine strip electrode may be patterned on a substrate through a known semiconductor process.

The electrophoretic chip according to an embodiment of the present invention may be prepared by separately preparing and bonding a first substrate having a working electrode and a second substrate having a micro flow path. Preferably, UV epoxy may be used as the material for adhering the substrate.

According to one embodiment of the present invention, as shown in Figure 2, using the film photo-mask in which the electrode and the microfluidic channel pattern is devised as previously reported through the photolithography process, the electrode and the fine on the glass substrate, respectively A first substrate and a second substrate having a fluid channel patterned are manufactured. After that, the electrode is spin-coated with UV epoxy, that is, UV curing resin, on the patterned first substrate and the patterned surface is in contact with each of the first and second substrates so as to place the electrode at a desired position. Align and bond to UV. Additionally, the process may include washing the channel with an organic solvent and circulating the potential using an acid solution to remove contaminants remaining on the channel and the electrode. Preferably, the organic solvent may be acetone, sulfuric acid solution as the acid solution, but is not limited thereto.

Then, in order to prepare the salt bridge, TMSMA is coated on the inner wall of the fluid channel as an adhesive material, and then the fluid channel is filled with a mixture including a photoinitiator or a pure polymer monomer or dimer to a derivatizer and / or a separate photoinitiator. By selective exposure using a photomask, a polymer electrolyte gel salt bridge can be formed and added at a desired position. The mixture may additionally include a crosslinking agent. The excess uncured mixture was removed and the channels and electrodes were washed by electrochemical washing with sulfuric acid solution.

The method of manufacturing a microchip by adhering a substrate using the UV epoxy has an advantage that it can be performed relatively simply. However, in the case of manufacturing a chip using UV epoxy as described above, there is a disadvantage that extra UV epoxy may remain on the electrode and impair the function as a sensor. Therefore, a method of washing with a specific solvent is widely used to remove excess UV epoxy from the electrode, but the use of the solvent may damage the electrode itself. In the present invention, a small amount of acidic solution may be injected into the channel and electrochemically washed by circulating the potential.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1: Compounds and Reagents

A 2.5 M aqueous solution of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) with 1% 2-hydroxy-4 '-(2- 0.5% N, N'-methylenebis as a crosslinking agent for preparing a polymer electrolyte gel with hydroxyethoxy) -2-methylpropiophenone (2-hydroxy-4 '-(2-hydroxyethoxy) -2-methylpropiophenone) Used in combination with acrylamide (N, N'-methylenebisacrylamide). 3- (trimethoxysilyl) propylmethacrylate (TMSMA) anhydrous methanol solution was used as coating material for attaching the polymer electrolyte gel on the channel surface. Potassium ferricyanide stock solutions of various concentrations were prepared with 100 mM potassium nitrate as a supporting electrolyte for potentiometric measurements. 25 mM sodium borate was used as an electrophoretic buffer of potassium ferricyanide. A solution containing neurotransmitters such as dopamine and catechol in 25 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer at pH 6.5 Was prepared. All reagents were purchased from Sigma-Aldrich (USA).

Example 2 Fabrication of Microchips Incorporating Polyelectrolytic Gel Salt Bridge

A method of making a microchip electrophoresis (MCE) device using novel photopolymerization of a polymer electrolyte gel plug by traditional photolithography, UV epoxy adhesion and UV exposure is shown in FIG. 1. Patterning of the channel and gold electrodes was made by the inventors similar to previous studies. Briefly, to form a microchannel pattern, a glass cover slide (Cat. No. 1000412, Paul Marienfeld) was first used with a piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1). GmbH & Co. KG, Germany) (Note: Piranha solutions are strong oxidants which react violently with organic compounds and must be handled with special care). A photoresist (AZ4620, Clariant, Switzerland) was spin coated onto a glass slide at 7000 rpm for 30 seconds. Subsequently, the photolithography process was sequentially performed, including soft baking, UV exposure, development, hard baking, and wet etching.

The design of the microchannels and the electrode images were produced using autocad (FIG. 2 (a)). Microchips were fabricated through a photolithography process as previously reported using film photo-masks created from AutoCAD drawings of channel and electrode patterns (Joo, S .; Kim, KH; Chung, TD, Biosens. Bioelectron., 2010, 25: 1509-1515). 2 (b) and 2 (c) show photographs of the manufactured microchip structure. Microchips consist of glass substrates with patterned channels and Au-deposited glass substrates. The microchip pattern and fabrication results are shown in FIG. 2. Au electrodes can be manufactured and used in various sizes for electrochemical studies under various electrophoretic conditions with the electrode scale shown in FIG. 2.

Bottom glass slides were also made by applying a similar photolithography protocol. The glass slides were washed with piranha solution and coated with hexamethyldisilazane (HMDS, Clariant, Switzerland), followed by spin coating at 4000 rpm for 30 seconds with AZ5214 (Clariant, Switzerland). Prebaking at 100 ° C. for 1 minute, first exposure to 17 mJ / cm ² for 5 seconds, reversal baking at 100 ° C. for 5 minutes, and then exposure to 17 mJ / cm ² for 5 seconds to AZ300MIF (Clariant, Switzerland). A metal film was sputtered onto the patterned glass by using a DC / RF electron sputter (Atek, Korea). A titanium (Ti) adhesive layer was sputtered to a thickness of about 350 mm 3 and a gold (Au) thin film of 5000 mm thick and 10 to 20 μm wide was deposited thereon at 5 mm / s. Gold-patterned glass was soaked in acetone (JT Baker, USA) to remove residues of the patterned Au / Ti layer. UV exposure was then used for the adhesion process and PGSB integration. After washing with air of piranha solution and air blowing, the gold electrode pattern was spin-coated with UV curing resin (LOT No. A10K01, ThreeBond Co., Ltd., Japan) at 1500 rpm for 30 seconds. The substrate was exposed to ultraviolet light of 365 nm wavelength at 17 mJ / cm ² for 12 seconds. The channel was then washed for 30 seconds with acetone. The bottom glass slide was electrochemically washed with Ag / AgCl and platinum wire as 0.5 and 0.5 M sulfuric acid solutions, respectively, at a circulation potential between +1.0 and +0.2 V. The pattern design of the microchip and the optical image of the manufactured chip are shown in FIG. 2.

The final chip was made by adhering the cover and the bottom glass slide. After washing with sulfuric acid, the surface area of the gold electrode exposed to the microchannel was confirmed experimentally whether the gold thin film acted as an electrode and is stable. Gold surface area was measured by cyclic voltammetry in a 5 mM K ₃ Fe (CN) ₆ solution, which is consistent with the area measured using a video microscope system (ICS-305B, Sometech, Korea). PGSB in the channel was made using UV exposure. Briefly, the microfluidic chip internal channels were filled with 0.1 M TMSMA solution and left at room temperature for 20 minutes. Thereafter, the solution was removed in vacuo, washed with anhydrous methanol, filled with 2.5 M AMPSA monomer solution, and cured by exposing the microfluidic chip to ultraviolet light at 17 mJ / cm ² for 35 seconds through a photomask. Finally, the gold surface was electrochemically cleaned by cycling the potential between +1.0 and +0.2 V with respect to the Ag / AgCl electrode in 0.5 M sulfuric acid solution until a reproducible cyclic voltammetry was obtained. In order to confirm the effect of washing the gold electrode surface with sulfuric acid, electrochemical behavior was observed using a TMSMA-modified gold substrate as a control. The cyclic voltammetry was confirmed to indicate that the gold surface was cleaned by sulfuric acid washing.

Example 3: Instrument

Electrochemical experiments of needle-shaped gold electrodes were performed using a conventional potnetiostat (Model CHI660A, CH Instruments Inc.) using Ag / AgCl and platinum wires as reference and counter electrodes, respectively. Electrophoresis was performed using a DBHV-100 high voltage supplier (Digital Bio Technology, Korea) operated by a six-channel voltage regulation program written in LabVIEW software version 8.2 (National Instruments, Austin, TX). The constant potential is isolated from the external electrical outlet by a custom-made DC power supply that switches from DC 9V batteries to DC +5, +15 and -15V to match the output voltage of the internal power module of the constant potential. It became. Detection by current measurement was performed in a Faraday cage equipped with a picoamp booster (Model CHI200, CH Instruments Inc.).

Example 4: Electrophoresis

In order to optimize the surface conditions of the microchannels, the glass chip channels were treated by the following procedure. First, the channels were washed with deionized water, 0.1 M HCl, 0.1 M NaOH and 25 mM Na ₂ B ₄ O ₇ (or 25 mM MES) for 10 minutes each. The effect of the position of the gold working electrode relative to PGSB was evaluated by electrophoretic separation of ferricyanide species. A 200 μM potassium ferricyanide solution was placed in the sample reservoir and loaded in pinched injection mode by applying +150 V for 20 seconds to the sample waste reservoir; The buffer and waste reservoirs are both floating while the sample reservoir is grounded. For sample injection and separation, voltages of +250 and +100 V were applied to the buffer-waste reservoir and sample reservoir / sample-waste reservoir, respectively, and the buffer reservoir was grounded. As shown in FIG. 3, a detection potential of +0.15 V was applied to the gold working electrode for the Ag / AgCl / KCl (3M) reference electrode located in the chamber beyond the PGSB.

In order to confirm successful electrophoresis and amperometric detection under high electric fields, two biological compounds, 100 μM dopamine and 150 μM catechol, were mixed in 25 mM MES buffer. Similar to the method used to load and separate ferricyanide, the sample plug was loaded by applying +150 V for 20 seconds between the sample and the sample waste reservoir using pinched injection. Separation was performed under various electric fields (50-500 V / cm).

<Experiment Result>

Open circuit potential under external electric field

The open circuit potential (OCP) between the gold working electrode and the PGSB was measured under various electric fields. 4 shows OCP data obtained with a space of −0, 100, 200 and 400 μm between the gold working electrode and the PGSB. The electric field gradient (Δ V _s ) applied along the microchannels for electrophoretic separation was varied from 30 to 400 V / cm. OCP data were clearly dependent on Δ V _s as well as the distance between the gold working electrode and PGSB. OCP increased with increasing distance from Δ V _s . The results indicate that there is an important problem to be solved in order to perform electrochemical detection using an electrode located in the middle of a microchannel subjected to a high electric field for electrophoresis.

Two main problems are as follows: Bipolar difference in electrochemical potential on the electrode and detection by incorrect current measurement. The first issue is closely related to the bipolar effect observed when the electrodes are exposed to steep electric field gradients. This bipolar electrode behavior can distort the electrochemical signal from the electrode and harm the working electrode surface. The steep gradient of the external electric field in the solution phase (solution phase) can lead to a significant difference in the electrochemical potential between the electrode terminal (Δ V _edge). However, Fermi level through the electrode will not be greatly affected by the field gradient. This heterogeneous electrochemical potential on the electrode surface appears as a function of the width and position of the electrode on the microfluidic chip. The greater Δ V value for the _edge wider than the electrode located away from PGSB within a narrow microchannel were observed. The maximum voltage that can be applied to the microchannels for electrophoretic separation is limited to conditions where no bipolar electrochemical reaction occurs due to an external electric field gradient. In order to satisfy the above condition, Δ V _edge value should be lower than the electrochemical potential window for a given solution and the electrode material (electrochemical potential window).

The potential window is determined through a cyclic voltammetry obtained in the absence of an electric field gradient in solution. For example, an electric field with an intensity of 400 V / cm results in a maximum difference of 0.4 V and 0.8 V, respectively, between the 10 μm and 20 μm wide gold electrode ends. Since the potential window of the gold electrode in the 0.1 M KNO ₃ and MES buffer used in the present invention is wider than 1 V, the electrochemical reaction on the gold electrode due to the external electric field required for electrophoretic separation is negligible. Furthermore, as shown in FIG. 5, the electrical potential gradient is expected to be minimal in front of the PGSB connected to the reference electrode system having a very low resistance. Sikindamyeon electrode is positioned just before the PGSB, reduction in electric potential gradient in the vicinity of PGSB will result in minimal effect polarity that is, inhibition of Δ V _edge. This was confirmed by a significant decrease in OCP fluctuations as shown in FIG. 4 (a).

Another problem is the accuracy of the electrochemical potential applied to the gold electrode for detection by amperometric measurement. This is also a function of the potential drop between the electrode and the _edge Δ V PGSB. Higher resistance between the working and reference electrodes under a steeper external electric field results in a greater potential drop along the solution in the microchannel. Therefore, it is necessary to adjust the position of the working electrode relative to the PGSB for the detection by successful current measurement during the electrophoresis on the microchip. In practice, in the fine channel potential movement between the working and the reference electrode caused by the Δ V _s can be corrected by using a voltage stored hydrodynamic also (hydrodynamic voltammogram). However, the location in the microchannels, which is remarkably desired from the PGSB, is still problematic. First, the electrochemical potential difference Δ V _s due to the electrophoretic electric field is much larger than the potential difference applied between the work-reference electrodes for current detection. Thus, the work is also required for the current detecting small variations in Δ V _s - may result in serious movement in electrochemical potential between a reference electrode. Furthermore, the system comprising: the bipolar effect that is influenced by the Δ V _edge. Due to the significantly reduced electrical potential gradient near the PGSB, the working electrode present to be in electrical connection with the PGSB is expected to be on an almost equipotential surface with the PGSB. Therefore, the solution potential in the working electrode located near the PGSB does not differ significantly from that of the reference electrode (FIG. 5). This is supported by the fact that, even below the 5 mV under high external electric fields (V Δ _s) of the potential variation in the _{OCP (Δ V F) 400 V} / cm ( Table 1). The results are consistent with Klett's report on OCP data obtained on equipotential surfaces. The results indicate that the working electrode located immediately before the PGSB has the same potential as the reference electrode when no electrical potential bias for detection by current measurement is applied. The potential variation was minimized and the noise level was effectively suppressed. Thus, if the electrode is positioned so as electrically connected with PGSB, it is not necessary to correct the detected voltage to compensate for the effects of Δ V _s. As a result, by placing the working electrode close to the PGSB, the electrochemical potential can be accurately applied in the middle of the electrophoretic channel, and continuous current measurement can be performed during the electrophoresis.

Table 1

Cyclic voltammetry under various electric fields

The electrical potential gradient in the middle of the microchannel is proportional to the resistance of the region of interest on the microchip channel. The local resistance in the PGSB region is significantly lower than in other parts of the microchannel because PGSB allows free movement of ions and is an electrical conductor connected to the reference electrode. The potential drop near the PGSB is expected to deform out of the sharp potential drop in other microchannel regions. This was confirmed by the OCP data which is almost free from the influence of the external potential gradient obtained from the electrode placed before the PGSB shown in FIG. 4. The noise generated while measuring the potential difference increased in proportion to the external electric field (Δ V _s ). Nevertheless, the noise level was less than 5 pA in the Faraday cage. The cyclic voltammetry shown in FIG. 6 shows that the electrochemical redox behavior of ferricyanide ions in the electrode remains unchanged except for a few tens of mV potential shift and a slight current decrease under an electric field of 400 V / cm. This potential shift and noise channel during the electrophoretic separation on a micro chip which is caused from the Δ V _s given range - supports the sujunim that can be allowed for within the electrochemical detection.

As the distance between the working electrode and the PGSB increases, EC measurements can intensify the noise and inaccurate electrochemical potential of the electrode, ultimately damaging the working electrode (FIG. 7). Therefore, arranging the working electrode near the PGSB allows the microfluidic chips of various designs to be devised by overcoming the restriction on the detector position in performing the electrophoresis by the current measurement.

Separation on PGSB-Integrated Microchips

Electrophoresis was performed on PGSB-integrated microchips to confirm separation efficiency and detection performance through current measurement reactions. 8 shows electrophoresis from gold microband electrodes located at (a) 0 and (b) 50 μm from PGSB. The electrophoresis was obtained from the separation of 200 μM potassium ferricyanide solution on PGSB-integrated microchips at 150 V / cm. The theoretical number, that is, the separation efficiency is 10700 / m (a) and 11500 / m (b), and the peak currents are 17 nA (a) and 8.0 nA (b). Another role of the polymer electrolyte gel is to prevent the buffer in the separation channel from leaking into the reservoir where the reference electrode is present.

Although polymer electrolyte gels are clearly different materials from glass, the difference between the two areas in terms of separation efficiency is inconspicuous. For example, the theoretical number measured using an electrode located in front of the PGSB is similar to that from other electrodes located 50 μm from the PGSB. On the other hand, the peak current for the electrode located in front of the PGSB is twice higher than that of the electrode located 50 μm from the PGSB. 8 shows that the peak shape of the electrophoresis from the electrode located in the PGSB is very similar to that from the electrode located in the middle of the glass microchannel. The detection limit (S / N = 2) was determined to be 1.5 μM for ferricyanide under −150 V / cm (FIG. 9).

The functionality of the microchip system equipped with PGSB for the separation of neurotransmitters by electrophoresis proposed in the present invention is shown in FIG. It was shown that the electrophoresis of the neurotransmitter mixture consisting of dopamine (100 μM) and catechol (150 μM) can be separated under an electric field in the range of 50 to 500 V / cm. The results shown in FIG. 10 show that the electric field strength is a major parameter in determining separation efficiency. The highest separation efficiency was observed under an electric field of 200 V / cmc (catechol, 10500 / m and dopamine, 8500 / m). As the electric field increased from 50 V / cm to 500 V / cm, the migration time for all compounds decreased. Separation was performed 7 times to confirm the reproducibility of electrophoresis on the microchip integrated with PGSB. Average travel times for dopamine and catechol were 32 ± 0.8 seconds and 36 ± 1.2 seconds, respectively, and peak currents of 750 ± 40 pA and 920 ± 35 pA were observed. The electrophoresis results confirmed the reproducible separation under high electric fields using a PGSB-based detection system.

Claims (21)

1. A voltage is applied between both ends, and the analyte moves in the sample by the voltage, thereby separating the analyte by the difference in moving speed. In the electrophoretic chip having a fluid channel,

   A working electrode for detecting the electrochemical characteristics of the analyte is provided at a point in the fluid channel, and an equipotential structure electrically connected to the working electrode is provided on at least one wall of the channel, and the fluid is formed using the equipotential structure as a passage. An electrophoretic chip characterized by measuring an electrochemical signal between a working electrode and a counter electrode by having a counter electrode disposed in an equipotential space formed by the equipotential structure and filled with a supporting electrolyte solution outside the channel adjacent to the channel.
2. The method of claim 1,

   The electrochemical signal is an electrophoretic chip of the current, conductivity or potential difference.
3. The method of claim 1,

   The equipotential structure is electrophoretic chip, characterized in that to allow the movement of ions.
4. The method of claim 1,

   The equipotential structure is an electrophoretic chip that is a polymer electrolyte gel salt bridge.
5. The method of claim 4, wherein

   The polymer electrolyte gel salt bridge is an electrophoretic chip that is an ion conductive polymer.
6. The method of claim 4, wherein

   The polymer electrolyte gel salt bridge is an electrophoretic chip that is prepared in the form of a gel formed by curing the polymer monomer (monomer) or dimers to depolymers by irradiation with light.
7. The method of claim 6,

   The polymer monomer is a 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) or diallyldimethylammonium chloride (DADMAC) as a unit. Electrophoretic chip.
8. The method of claim 7, wherein

   The polymer monomer is an electrophoretic chip that is a photoinitiator is combined or additionally comprises a separate photoinitiator.
9. The method of claim 8,

   The photoinitiator is a 2-hydroxy-4 '-(2-hydroxyethoxy) -2-methylpropiophenone (2-hydroxy-4'-(2-hydroxyethoxy) -2-methylpropiophenone) electrophoretic chip .
10. The method of claim 6,

    Electrophoretic chip that further comprises a crosslinking agent.
11. The method of claim 10,

    The crosslinking agent is N, N'-methylenebisacrylamide (N, N'-methylenebisacrylamide) electrophoretic chip.
12. The method of claim 1,

    The electrophoretic chip may be made of glass, quartz, silicon, poly (dimethylsiloxane) (PDMS), poly (methyl methacrylate) (PMMA), polycarbonate (PC), polystyrene (polystyrene). An electrophoretic chip selected from the group consisting of polystyrene), cellulose acetate and ethylene terephthalate (polyP).
13. The method of claim 1,

    The working electrode is an electrophoretic chip is an electrode selected from the group consisting of gold, platinum, carbon, indium tin oxide (ITO) and semiconducting material.
14. The method of claim 1,

    The electrophoretic chip is characterized in that the electrophoretic chip further comprises a reference electrode.
15. The method of claim 1,

    When the distance between the inner channel walls of the fluid channel is A, the distance from the inner wall of the fluid channel facing the equipotential structure to the counter electrode is B,

    B is an electrophoretic chip characterized by more than 1.5 times A.
16. The method of claim 1,

    When the length of the equipotential structure located on one wall of the fluid channel is C,

    The equipotential structure has a length (C) of 1 μm to 10 μm, whereby the width (D) parallel to C of the equipotential space separated from the channel is 1 to 10 times C.
17. The method of claim 1,

    The electrophoretic chip is an electrophoretic chip manufactured by bonding a first substrate on which a working electrode is formed and a second substrate on which a micro flow path is formed.
18. The method according to any one of claims 1 to 17,

    The electrophoretic chip is an electrophoretic chip, characterized in that the electrophoretic microchip.
19. A method of monitoring an electrochemical signal without interference of the electric field through a working electrode provided at a point in the fluid channel to which the electric field is applied,

    Measuring an electrochemical signal between the working electrode and the counter electrode;

    In this case, an equipotential space is formed by an equipotential structure formed on at least one wall surface of the fluid channel and electrically connected to the working electrode.

    And the counter electrode is disposed in an equipotential space separated from a channel by the equipotential structure.
20. The method of claim 19,

    The electric field is applied externally for any one process selected from the group consisting of injection, transfer, mixing, reaction, detection and post analysis of a sample.
21. The method of claim 19,

    The electric field is 0 V / cm or more and 10000 V / cm or less.

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