KR20210105025A - Electrochemical biosensor with microfluidic channel - Google Patents

Abstract

The present invention relates to an electrochemical biosensor coupled with microfluidic channels, and more particularly, by using a surface-modified electrode, sensitivity of a sensor is increased, and with a non-permanent coupling method of a microfluidic channel part and an electrode part, it is possible to detect and then recover a target material that specifically binds to an aptamer or a detection antibody, thereby being used for latter analysis.

Description

Electrochemical biosensor with microfluidic channel

The present invention relates to an electrochemical biosensor coupled with a microfluidic channel.

Exosomes are small endoplasmic reticulum with a size of 30-100 nm secreted from cells and contain various types of proteins and genetic materials derived from cells, from which the state of the tissue can be determined. In particular, secretion is not well under normal conditions, and secretion is increased in disease conditions such as cancer. Exosomes detected in the blood of cancer patients contain about 4500 proteins, Because it contains lipid and genetic information, it is attracting attention as a biomarker for diagnosing these diseases. Recently, the function as a drug delivery system for delivering a drug into cells by inserting a therapeutic protein into the exosome has also been attracting attention, thereby increasing the demand for detection and analysis of the exosome.

For the detection of exosomes, an optical method through fluorescence measurement of a fluorescent material, etc., or a mass-sensitive sensor and micro biochip that analyzes and measures the difference in mass appearing before and after binding to a target material A sensor using the method is used. The optical sensor uses the quenching and FRET effect of two fluorescent dyes having different wavelengths by connecting a fluorescent dye to an aptamer molecule. On a chip), it is far from miniaturization of the sensor.

In addition, in order to integrate the glass chip for detecting the reaction with an electrical signal and the PDMS (Polydimethylsiloxane) chip for injecting the sample, the existing micro biochip is treated with oxygen plasma for a certain time and with a certain power and then bonded through a process. However, the sensor and the channel are permanently bonded through the bonding process by oxygen plasma. This is difficult to harvest after detecting exosomes, so there is a problem in subsequent analysis such as separating exosomes, lysis, separating proteins, genetic materials, etc. and verifying them by PCR.

Republic of Korea Patent Publication No. 2018-0135183 (2018.12.20.)

The present invention is designed to solve the above-mentioned problems, does not require optical equipment, and includes a surface-modified electrode for high-sensitivity detection of a target material and increases the sensitivity of the sensor to detect and quantitatively measure the target material in a wide concentration range An object of the present invention is to provide an electrochemical biosensor.

Another object of the present invention is to provide an electrochemical biosensor that facilitates the downstream analysis of a target material through a non-permanent bonding method between an electrode and a microfluidic channel.

Another object of the present invention is to solve the conventional problem of fluid leakage from excessive pressure by adjusting the strength of the bonding through a non-permanent bonding method between an electrode and a microfluidic channel.

In order to achieve the above object, the present invention provides an electrode unit comprising a working electrode on which an aptamer or a sensing antibody capable of binding to a target material is immobilized and surface-modified with a graphene nanoplatelet complex; microfluidic channel unit; and a junction for coupling the electrode part and the microfluidic channel part, wherein the coupling is a non-permanent coupling method.

In the electrochemical biosensor according to an embodiment of the present invention, the graphene nanoplatelet composite comprises: a graphene nanoplatelet; transition metal chalcogen compounds; and any one or two or more of a thiol-based polymer and an amine-based polymer.

In one embodiment of the present invention, gold nanoparticles may be applied to the top of the working electrode of the electrode unit.

In one embodiment of the present invention, the microfluidic channel unit may include a particle injection unit, a particle moving unit, a reaction unit, and a particle discharge unit.

In one embodiment of the present invention, the reaction unit may be characterized in that a ceiling spaced at a predetermined interval and a side wall connected to the ceiling are formed so that the fluid entering through the particle injection unit can form a vortex.

In one embodiment of the present invention, the non-permanent coupling method of the junction part may be to couple the microfluidic channel part and the electrode part using magnetic force. In addition, the junction part includes an upper cover and a lower cover, and at least one selected from the upper cover and the lower cover further includes a receiving part for accommodating the microfluidic channel part and the electrode part therein, the upper cover and the lower cover Any one or more selected from the upper cover and the lower cover including a magnet may be coupled using a magnetic force.

In one embodiment of the present invention, the target material may be an exosome.

In addition, the present invention provides a kit for detecting exosomes comprising an electrochemical biosensor having the above-described characteristics.

In addition, the present invention provides a liquid sample for analysis; injecting the sample for analysis into a particle injection unit of a microfluidic channel of a sensor for detecting a target material; concentrating the target material of the sample for analysis through the vortex formed in the reaction part of the microfluidic channel; forming a complex by specifically binding the target material with an aptamer or a detection antibody immobilized on a working electrode facing the reaction unit; Including; analyzing the target material in the sample for analysis by measuring the electrochemical signal generated by the complex, wherein the working electrode is a graphene nanoplatelet, and a transition metal chalcogen compound, a thiol-based polymer, and an amine It provides a method for analyzing a target material, characterized in that it contains any one or two or more selected from the group consisting of polymers.

In the target material analysis method according to an embodiment of the present invention, after the analyzing step, the step of recovering the target material; may further include.

In the target material analysis method according to an embodiment of the present invention, the recovered target material is used for any one or more downstream analysis selected from the group consisting of PCR, sequencing, mass spectrometry (MS), gene analysis, and protein analysis. ; may be further included.

In the target substance analysis method according to an embodiment of the present invention, the target substance may be characterized in that it is an exosome. In addition, the concentration of the ^{exosome may be 1×10 2} to 1×10 ⁹ exosome/mL.

The electrochemical biosensor for detecting and analyzing a target material according to the present invention increases the sensitivity of the sensor by using a surface-modified electrode coated with gold nanoparticles, and detects and quantitatively measures the target material in a wide concentration range. .

The present invention provides an electrochemical biosensor capable of high-accuracy quantitative and qualitative detection by concentrating a target material even when a target material is included in a trace amount in a sample by combining a microfluidic channel and an electrode with high sensitivity.

In addition, by non-permanently combining the microfluidic channel and the electrode, it is possible to recover the target material even after detecting the target material, which can be utilized for subsequent analysis of the target material.

In addition, by controlling the strength of the bonding between the microfluidic channel and the electrode according to the non-permanent coupling method, it is possible to provide an effect of preventing leakage of fluid from excessive pressure.

1 is an exploded perspective view of an electrochemical biosensor according to the present invention.
2 shows a state in which the electrode part and the microfluidic channel part of the electrochemical biosensor according to the present invention are combined.
3 shows the disassembled state of the electrode part and the microfluidic channel part of the electrochemical biosensor according to the present invention.
4 is a schematic diagram showing the reaction that occurs in the reaction part of the microfluidic channel part and the working electrode of the electrode part when a liquid sample for analysis is injected according to the present invention.
5 is a schematic diagram showing a process of manufacturing a surface-modified electrode according to an embodiment of the present invention.
6 is a graph confirming the electrochemical signal for each concentration of the exosome through the electrochemical biosensor according to the present invention, showing the result that the current value increases linearly as the concentration of the exosome increases.
7 is a graph showing each electrochemical signal according to the surface treatment in the process of manufacturing the surface-modified electrode according to the present invention, the surface of which is not surface-treated (SPCE; light blue) and graphene nanoplatelets; the treatment electrode (GNP / SPCE; green), a surface treatment with MoS ₂ electrode _{(MoS 2 / SPCE; grey)} , graphene nano plate let-complex (GNP / MoS ₂ / CHT) the electrode surface-treated with (NC / SPCE ; red) and the difference in current and resistance of electrodes (GNS/NC/SPCE; blue) coated with gold nanoparticles after surface treatment with graphene nanoplatelet composites were analyzed by CV (cyclic voltammetry) and EIS (Electrochemical Impedance Spectroscopy) confirmed through Current expresses the degree of transfer of electrons between the electrode and the electrolyte, and resistance is the opposite concept, indicating how much electron transfer is hindered.
8 shows a TEM (transmission electron microscope) / HRTEM (high-resolution transmission electron microscope) image of graphene nanoplatelets, MoS _{2 and graphene nanoplatelet composites.}
9 is a result of surface analysis using an atomic force microscope (AFM) as the surface modification proceeds.
Figure 10 is a calibration curve (Calibration Curve) graph showing the exosome detection ability evaluation results using the electrochemical biosensor (MiNEA) and ELISA according to the present invention.
11 is a graph showing the comparison results of exosome quantification by the system and ELISA method according to the present invention using actual patient blood.

Hereinafter, the electrochemical biosensor according to the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those of ordinary skill in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments is not limited to

Also, unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of effectively describing particular embodiments only and is not intended to limit the invention.

Hereinafter, when it is said that any one component "includes" another component, it is not construed as being limited to only the component, unless otherwise stated, and it is understood that other components may be further included. should be

The present invention relates to an electrode unit comprising a working electrode on which an aptamer or a sensing antibody capable of binding to a target material is immobilized and surface-modified with a graphene nanoplatelet complex; microfluidic channel unit; and a bonding unit for bonding the electrode unit and the microfluidic channel unit, wherein the bonding is a non-permanent bonding method.

In order to measure an electrode potential in a liquid sample containing a target material, basically, the potential difference between two points should be measured using two electrodes. In measuring the potential difference, the electrode to be measured is a working electrode. You can measure the potential difference by connecting another electrode here. For example, the electrode part of the present invention may be composed of a two-electrode system or a three-electrode system by further including any one or more selected from the group consisting of a reference electrode and a counter electrode in addition to the working electrode. have. However, in the present invention, when the electrode for measuring the current and the electrode for measuring the voltage are separated, introducing a three-electrode system consisting of a working electrode, a reference electrode, and a counter electrode is when the resistance of the electrolyte is high or the current is high It is preferable because the error due to resistance can be minimized.

In the present invention, the working electrode is an electrode in which a reaction of interest electrochemically occurs, and may be a cathode or an anode according to an oxidation reaction or a reduction reaction. In addition, the reference electrode refers to an electrode that provides a reference potential, and the potential difference may mean appearing between the reference electrode and the working electrode. The counter electrode does not directly participate in the reaction and does not affect the potential value, but refers to an electrode through which current flows.

The electrode can be used without limitation as long as it can be used for a sensor, such as a gold (Au), palladium, silicon (Si), or carbon (C) electrode. As a method of forming the electrode, those that can be used for electrochemical sensors such as sputtering, screen printing, and inkjet printing can be used without limitation.

The working electrode according to the present invention is surface-modified with a graphene nanoplatelet composite, wherein the graphene nanoplatelet composite includes any one of a graphene nanoplatelet, a transition metal chalcogen compound, a thiol-based polymer, and an amine-based polymer Or it may further include two. According to the present invention, when the graphene nanoplatelet further includes any one or two of a transition metal chalcogen compound, a thiol-based polymer, and an amine-based polymer to form a complex, the graphene self-aggregation phenomenon by van der Waals force is prevented. By reducing the degree of adhesion to the electrode, the mechanical properties and electrical properties of the material can be improved.

Transition metal chalcogenides (TMDC) means that two chalcogen elements (X, group 16 of the periodic table) are covalently linked per one transition metal element (M) to have a chemical formula of _{MX 2 .} The present invention forms a composite in which a transition metal chalcogen compound having a bandgap is combined with a graphene nanoplatelet having no bandgap, so that the electrical properties of the electrode can be excellently maintained even when the bandgap is changed through doping or the like.

In the transition metal chalcogen compound (MX ₂ ) according to the present invention, M is any one or more selected from the group consisting of Mo, W, Ti, Tc, Hf, Zr, Re, Pd and Pt, X is S, Se and Te It may be any one or more of them. That is, for example, MoSe ₂ , WS ₂ , WSe ₂ , TiS ₂ , TiSe ₂ , TiTe ₂ , HfS ₂ , HfSe ₂ , HfTe ₂ , ZrS ₂ , ZrSe ₂ , ZrTe ₂ , TcS ₂ , TcSe ₂ , TcTe ₂ , ReS ₂ , ReSe ₂ , ReTe ₂ , PdS ₂ , PdSe ₂ , PtS ₂ , and PtSe ₂ may be at least one selected from the group consisting of. Preferably _{, using MoS 2} is good because of the advantages of high electron transport rate, flexibility, improved mechanical strength, and relatively low toxicity and a large surface area.

In the present invention, the amine-based polymer that may be included in the graphene nanoplatelet composite is specifically, for example, chitosan, chitin, polyaniline, polylysine, polyallylamine, polyethyleneimine, and poly(2-dimethylaminoethyl methacryl). rate) (poly(2-dimethylaminoethyl methacrylate)) may be at least one selected from the group consisting of, but is not particularly limited thereto.

In the present invention, preferably, an amine-based polymer having low solubility in water is good, and specifically, using chitosan has low solubility in water and biocompatible properties, and graphene nanoplatelets and MoS ₂ structures are combined It can act as an adhesive.

In the present invention, the thiol-based polymer that may be included in the graphene nanoplatelet composite may be a polymer including a thiol group in a side branch, and specifically may be a polyamide-based polymer including cysteine, but is not particularly limited thereto. .

When a thiol-based polymer or an amine-based polymer is included in the graphene nanoplatelet composite, it can act as a binder when forming a complex with the graphene nanoplatelet and the transition metal chalcogen compound, and the surface is modified with the graphene nanoplatelet composite. When the gold nanoparticles are applied to the top of the working electrode, the gold nanoparticles can be stably applied to the composite by forming a covalent bond with the gold nanoparticles.

In the graphene nanoplatelet composite of the present invention, the graphene nanoplatelet and the transition metal chalcogen compound may be included in a content ratio of 1:0.1-5 by weight. Preferably it is included in a content ratio of 1: 0.5 to 3, more preferably in a content ratio of 1: 1 to 2.5, since it is good to lower the charge transfer resistance of the sensor surface.

In one embodiment of the present invention, the working electrode surface-modified with the graphene nanoplatelet composite is prepared by preparing an amine-based polymer solution, and mixing graphene and a transition metal chalcogen compound in the solution to prepare a uniform dispersion. Then, the dispersion may be prepared through spin coating or drop coating.

In addition, gold nanoparticles may be applied to the upper end of the working electrode. As the working electrode is coated with gold nanoparticles, an aptamer or a sensing antibody for detecting a target material is more stably fixed, and as the surface is modified with a graphene nanoplatelet complex, the transfer rate of electrons generated as a result of the redox reaction A synergistic effect is exerted in improving the sensitivity of the target material by increasing the

According to the present invention, the application of gold nanoparticles is generally chemical vapor deposition (CVD), atomic layer deposition, sputtering, laser ablation, electrochemical deposition, and electric discharge. It may be performed by any one or more methods selected from the group consisting of arc-discharge, plasma deposition, thermochemical vapor deposition, and electron beam vapor deposition, but is not particularly limited thereto. Preferably, it may be applied through electrochemical welding by performing a constant potential treatment in a sulfuric acid solution and a gold chloride solution for a certain time.

In a preferred embodiment of the present invention, the working electrode of the electrode part may be surface-modified with _{graphene nanoplatelets, MoS 2 and chitosan.} At this time, each content may be included in 1: 0.1 to 5: 0.01 to 1 by weight, preferably 1: 0.5 to 3: 0.05 to 0.5. More preferably, 1:1 to 2.5: 0.08 to 0.2 indicates a high reduction current peak, so the sensitivity of the electrode is improved, so it is good to exhibit excellent accuracy in quantitatively analyzing a target material with an electrochemical signal.

In addition, gold nanoparticles may be coated on the upper end of the working electrode. By coating the gold nanoparticles, the detection limit of the surface-modified working electrode is further lowered, so that a highly sensitive electrode sensor can be realized. In this case, it is advantageous to increase the surface coverage of the gold nano solution in the range of 1×10 ^-2 to 5×10 ^{-2 M.} More preferably, 2×10 ⁻² to 4×10 ⁻² M may advantageously act on immobilization of the aptamer or the sensing antibody without inhibiting the interaction with the graphene nanoplatelet complex. In addition, the electrode surface-modified with the graphene nanoplatelet composite exhibits an effect of significantly increasing the surface coverage of gold coating.

The microfluidic channel unit constituting the electrochemical biosensor according to the present invention may include a particle injection unit, a particle moving unit, a reaction unit, and a particle discharge unit. Specifically, the liquid sample for analysis containing the target material is injected through the particle injection unit, enters the reaction unit through the particle moving unit, and after reaction occurs in the working electrode, it is configured to exit through the particle discharge unit.

The reaction unit may be characterized in that a ceiling portion spaced at a predetermined interval and a sidewall connected to the ceiling portion are formed so that the fluid introduced through the particle injection unit can form a vortex. Specifically, the ceiling portion and the sidewall connected to the ceiling portion may form a column as a plurality, and the column may form a comb pattern so that a vortex may be formed while the fluid flows in a changed direction, but a flow path in which a vortex may be formed This is sufficient, and it is not particularly limited thereto.

According to the present invention, a structure having a ceiling portion and a side wall connected to the ceiling portion, in particular, a portion corresponding to a vertex in a comb pattern serves as a deflection point to separate the flow of the opposite fluid, and the flow of the split fluid reacts during movement The number of effective collisions with the aptamer or the sensing antibody immobilized on the working electrode by creating a velocity component in the vertical direction to the fluid in the compartment, generating a local micro vortex, and mixing the target material present in the analyte. to increase Increasing the collision frequency between the target material and the aptamer or the detection antibody by a kind of agitation action may have the effect of improving the detection sensitivity of the sensor as the rate of participation in the reaction among the target material in the fluid increases.

In addition, as in the conventional drop-casting method, the reaction time is shorter than that for the reaction to occur only when the target material floating in the fluid sinks to the surface of the electrode, thereby reducing the time required for measurement and a fouling issue appearing on the sensor surface. ) can be reduced, thereby providing the effect of improving the quality of the sensing signal.

The microfluidic channel unit according to the present invention may be made of any one or more of plastic, silicon, and glass. Specific examples of the plastic may include PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), and polystyrene, etc., but it may be preferably made of PDMS, which has good durability, is light and has good processability, and is easy to mold.

The electrode part and the microfluidic channel part according to the present invention may be coupled by a junction part, and the junction part is a non-permanent coupling method and serves to bond and fix the electrode part and the microfluidic channel part by applying a constant pressure. It may be a fixing method using a clamp or a magnetic force.

More specifically, the junction portion includes an upper cover and a lower cover, and any one or more selected from the upper cover and the lower cover may further include a receiving portion accommodating the microfluidic channel portion and the electrode portion therein. By further including the receiving part, the microfluidic channel part and the electrode part can be fixed in a coupled state, so that the working electrode of the electrode part and the reaction part of the microfluidic channel part do not shift and are coupled to each other so that the aptamer or the sensing antibody is a target It can make it easier to react with a substance.

In addition, any one or more selected from the upper cover and the lower cover may include a magnet so that the upper cover and the lower cover are coupled using a magnetic force. Specifically, for example, the microfluidic channel and the electrode part may be coupled to the upper cover provided with one or more magnets by coupling with the lower cover including the material acting on the magnet. Conversely, the microfluidic channel and the electrode part are combined with the lower cover provided with one or more magnets and the upper cover containing a material that attracts the magnets, or both the upper cover and the lower cover are provided with one or more magnets so that attractive forces act with each other. can be combined. The attractive force due to the magnetic force is sufficient to maintain a constant bonding force to the microfluidic channel and the electrode part, and to prevent the fluid from flowing out of the microfluidic channel.

The magnetic force may be controlled by the number of magnets to prevent fluid leakage due to excessive pressure while sealing the microfluidic channel and the electrode part.

The upper cover and/or lower cover including one or more magnets according to the present invention may be manufactured by a 3D printing method according to the shape of the microfluidic channel part and the electrode part in order to bond the microfluidic channel part and the electrode part.

The bonding method of the junction using magnetic force according to the present invention is excellent in preventing leakage of fluid by lowering the pressure applied to the microfluidic channel compared to the conventional permanent bonding method using oxygen plasma. In addition, the non-permanent binding method using magnetic force according to the present invention can harvest the target material through an easy separation process of the microfluidic channel part and the electrode part after the detection of the target material, so that the target material is not discarded and can be used for later analysis. It can provide you with available benefits.

In addition, in the case of the conventional permanent bonding method, considering the fluid leakage problem of the microfluidic channel part, there was a limitation that a glass substrate that is easy to bond should be used. and the choice of substrate is free.

The target material of the electrochemical biosensor according to the present invention may include, for example, proteins, carbohydrates, lipids, pharmaceuticals, small molecular substances, organic non-pharmaceuticals, and macromolecular complexes including cells, and the target of the aptamer or sensing antibody is As long as it is a material that can be used without limitation, it may be preferably an exosome.

The present invention may provide a kit for detecting exosomes comprising a biosensor having the above-described characteristics.

In addition, the present invention provides a method of analyzing a target material using the biosensor. Hereinafter, a method for analyzing a target material is according to the biosensor according to the present invention described above, and includes the characteristics of the above-described components, and a redundant description is omitted.

Specifically, providing a liquid sample for analysis; injecting the sample for analysis into a particle injection unit of a microfluidic channel of a sensor for detecting a target material; concentrating the target material of the sample for analysis through the vortex formed in the reaction part of the microfluidic channel; forming a complex by specifically binding the target material with an aptamer or a detection antibody immobilized on a working electrode facing the reaction unit; and analyzing the target material in the sample for analysis by measuring the electrochemical signal generated by the complex, wherein the working electrode is selected from graphene nanoplatelets, transition metal chalcogen compounds, and amine-based polymers It may be characterized in that it further includes any one or two.

The electrochemical signal may be detected using a labeling molecule bound to an aptamer or a detection antibody. Specifically, for example, an exosome is bound to an aptamer, and an aptamer to which a secondary signaling material is bound to the exosome is bound to generate a signal. As an example of the secondary signaling material, there may be silver nanoparticles. Silver nanoparticles are one of the materials capable of transmitting an electric current and are injected as a secondary signal transduction material after the target material binds to it. In addition, it is also possible to quantitatively evaluate a target material through a change in the amount of an electrochemical signal.

In one embodiment of the present invention, after the analyzing step, the step of recovering the target material may be further included. Conventional sensors for detection have had to be limited to qualitatively or quantitatively detecting a target material, but in the present invention, since the electrode part and the microfluidic channel part are joined by a non-permanent coupling method, after the detection of the target substance, the electrode By separating the part and the microfluidic channel part, it is possible to recover the target material.

Therefore, the present invention can provide a target material analysis method further comprising the step of using the recovered target material for a subsequent analysis. The back-end analysis may be amplification, sequencing, etc., and specifically, for example, may be any one or more selected from the group consisting of PCR, sequencing, mass spectrometry (MS), gene expression analysis, and protein analysis.

The target material of the electrochemical biosensor according to the present invention may include, for example, proteins, carbohydrates, lipids, pharmaceuticals, small molecular substances, organic non-pharmaceuticals, and macromolecular complexes including cells, and the target of the aptamer or sensing antibody is As long as it is a material that can be used without limitation, it may be preferably an exosome.

The detectable exosome concentration may be 1Х10 ² to 1Х10 ⁹ exosome/mL. This may mean that detection is possible in a wide concentration range, and is not necessarily limited to these values.

The binding to the target substance is an aptamer or a sensing antibody, preferably an aptamer. Aptamers, also called chemical antibodies, can be easily structurally modified compared to single antibodies, and are easy to synthesize and stable. Also, it can be used to measure small molecules that are difficult to detect with antibodies. The aptamer strongly immobilizes the target material by the G-rich group, which has the advantage of facilitating free binding and separation of the target material by using the property of unwinding the structure by high temperature or high salt concentration.

In one embodiment of the present invention, the aptamer may be an epithelial cell adhesion molecules (EpCAM) aptamer. Preferably, it may be an aptamer having a specific affinity for the exosome.

A predetermined functional group may be attached to the aptamer or the sensing antibody so as to be immobilized on the electrode part, particularly, the gold nanoparticles applied to the working electrode. Specifically, for example, it may be any one or more functional groups selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group, and a thiol group, but is not particularly limited thereto.

The liquid sample for analysis may be any one or two or more selected from the group consisting of blood, urine, intestinal fluid, and saliva.

Hereinafter, detailed contents for carrying out the present invention will be described with reference to the accompanying drawings. The accompanying drawings show the verification results for the electrochemical biosensor and the target material analysis method using the same according to the present invention through the following examples and experimental examples. However, the drawings showing the following examples and experimental examples are only a reference for explaining the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms. And, in the description of the present invention, if it is determined that the subject matter of the present invention may be unnecessarily obscured as it is obvious to those skilled in the art with respect to related known functions, the detailed description thereof will be omitted.

1 is an exploded perspective view of an electrochemical biosensor 100 including a microfluidic channel part 200 , an electrode part 300 , and a junction part 400 according to the present invention. The junction part 400 includes an upper cover 410 and a lower cover 420 , and the upper cover 410 and the lower cover 420 include one or more magnets 412 and 422 . Although not shown in the drawing, the space in which the magnet is not disposed in the upper cover and the lower cover is a space opposite to the space in which the particle injection part and the particle discharge port of the microfluidic channel part 200 are formed, such as a tube communicating with the outside. This can be installed.

In addition, although not shown, the upper cover 410 and the lower cover 420 are accommodated according to the size so that the combination of the microfluidic channel part 200 and the electrode part 300 is fixed and cannot move by using the 3D printing method. It may have a configuration that includes parts.

2 and 3 are a combined perspective view and an exploded perspective view of the electrochemical biosensor 100 including the microfluidic channel part 200 and the electrode part 300 according to the present invention, respectively. With reference to this, the microfluidic channel part 200 and the electrode part 300 will be described in detail.

An object of the present invention is to improve the performance of the electrochemical biosensor 100 by bonding the microfluidic channel part 200 and the electrode part 300 for controlling the flow of a fluid containing a target material. The microfluidic channel part 200 is provided with a reaction part 210 consisting of a side wall and a ceiling part surrounding the working electrode 320 of the electrochemical biosensor 100 . The ceiling portion of the reaction unit 210 is formed with a plurality of comb-tooth-shaped grooves that cause turbulence in the fluid flowing in the reaction unit 210 . An enlarged form of this is shown in FIG. 1 .

In addition, the microfluidic channel unit 200 is provided with a particle injection unit 230 and a particle discharge unit 240 communicating with the reaction unit 210 , and a fluid including a target material is injected into the particle injection unit 230 for reaction. After causing a mutual reaction with the aptamer or the sensing antibody on the working electrode 320 in the unit 210, it is configured to exit through the particle discharge unit 240 .

6 is a result of analyzing the exosome detection ability using the electrochemical biosensor according to the present invention. As the pulse height increases, the signal peak of the exosome increases, and FIG. 6(b) shows the exosome in proportion to the concentration of the exosome. It can be seen that the signal of ^{SOM increases with high linearity (R 2} =0.981). Through this, it can be confirmed that the detection of exosomes is possible in a wide concentration range ranging from 1×10 ² to 1×10 ^{9 exosome/mL.} In addition, from the results that can detect up to 17 exosomes/mL, it can be confirmed that the electrochemical biosensor according to the present invention lowers the limit of detection (LOD) and exhibits significantly improved sensitivity.

_{8 is a MoS 2} - graphene nanocomposite used for surface modification in the present invention and shows a TEM photograph of the structure. Through TEM analysis, it can be confirmed that the nanocomposite is composed of two or more different sheets, and the form of _{graphene nanoplatelets and MoS 2 which are the constituents of the nanocomposite can be confirmed using a high-resolution electron microscope.}

9 is a sequential confirmation of the surface treatment process in FIG. 5 using an atomic force microscope. Specifically, step (I) is the surface state of the gold nanoparticles/graphene nanoplatelet composite/electrode (GNS/NC/SPEC), and step (II) is the state in which the aptamer is bound in step (I) (Aptamer/GNS/ NC/SPEC), step (III) is a state in which the electrode is passivated by treating 6-mercapto-1-hexanol (MCH) in step (II), (IV) ) indicates a state in which the exosomes are bound after step (III).

9 shows that the degree of surface modification was physically verified by measuring a _{vertical distance (V D} ) and average surface roughness ( R _{A ).} That is, as the surface treatment progresses step by step, the vertical distance increases sequentially, and it can be seen that the surface roughness gradually becomes gentle as the material is accumulated. It can be seen that the sensitivity is improved. This is a result confirming that the surface-modified electrode according to the present invention can show excellent performance in detecting a target material.

11 is an experiment for detecting exosomes from the blood of an actual breast cancer patient using the electrochemical biosensor according to the present invention, and comparing the results of quantitative analysis of exosomes through the intensity of electrical signals with quantitative analysis using ELISA was indicated. As a result of the experiment, it can be confirmed that the amount of exosomes detected in stage 1-2 breast cancer patients increased by about 2 times compared to healthy blood samples, and in patients with stage 3-4 breast cancer, it was confirmed that it increased 1.5 times compared to stage 1-2 patients. . Through this, it can be confirmed that the number of exosomes increases as cancer progresses. In addition, it can be confirmed that the sensitivity and accuracy of the electrochemical biosensor according to the present invention are remarkably improved when looking at the result of detecting exosomes at higher concentrations compared to quantitative analysis through ELISA.

Example 1. Graphene Nanoplatelet-MoS ₂ -Production of chitosan surface-modified electrodes (NC/SPCE)

A mixture of graphene nanoplatelets (Sigma Aldrich) and MoS ₂ (Graphene-supermarket www.graphene-supermarket.com, USA) was sonicated for 20 minutes with a sonicator, and MoS ₂ : graphene nanoplatelets : Chitosan (Sigma Aldrich) was added so that the ratio of chitosan (CHT) was 2:1:0.1 and sonicated for 20 minutes again to form a graphene nanoplatelet composite (NC). Then, _{the surface of the SPCE electrode was washed with a 0.5 MH 2} SO ₄ solution, and a voltage of 0.1-0.7 V was applied. After drop-casting 20 μl of a graphene nanoplatelet composite (NC) solution on the surface of the SPEC electrode, it was dried at room temperature for 1 hour _{to prepare a graphene nanoplatelet-MoS 2} -chitosan surface-modified electrode.

Example 2. Gold Nanostructure-Graphene Nanoplatelet-MoS ₂ -Production of chitosan surface-modified electrodes (GNS/NC/SPCE)

Electrochemical deposition by treating the electrode surface-modified with the graphene nanoplatelet composite (NC) prepared according to Example 1 under a ^{constant potential of -0.2V in 3×10 -2} M gold chloride (HAuCl _{4 ) solution for 120 seconds} A gold nanostructure-graphene nanoplatelet composite electrode (GNS/NC/SPCE) was prepared by applying gold nanoparticles through the method. At this time, the gold-coated surface coverage was 62.89%.

Example 3. Immobilization of EpCAM Aptamer on Surface of Surface Modified Electrode

The disulfide bond of the thiolated aptamer was reduced by incubating 20.0 μl of 6.25Х10 ⁶ M aptamer with 5.0 μl of 1.5Х10 ^-3 M TCEP (Tris(2-carboxyethyl)phospine) solution in the dark for 1 hour. The electrode surface-modified with the gold nanostructure-graphene nanoplatelet composite prepared according to Example 2 was washed with deionized water and dried using nitrogen gas. Thereafter, 7.0 μl of an aptamer with reduced disulfide bonds was dropped onto the washed electrode and maintained at 4° C. in a humid condition overnight. After immobilizing the aptamer on the electrode, ^{7.0 μl of a 1Х10 -3} M MCH (6-mercapto-1-hexanol) solution was dropped on the electrode and left for 1 hour to immobilize the electrode. Before use, the electrode was washed with PBS and maintained in a humid condition at 4°C.

Example 4. Preparation of electrochemical biosensor coupled with microfluidic channels

The microfluidic channel was prepared using a PDMS material. As a photoresist material, SU-8 (SU-8 3035, MicroChem Corp., Newton, MA, USA) was spin-coated onto a silicon wafer at 500 rpm for 15 sec and 1000 rpm for 40 sec. Thereafter, a soft bake process was performed on hot plates at 65°C and 95°C. The template was subjected to exposure treatment to form a pattern. A main channel and a comb-shaped channel were prepared by using the photoresist.

The magnetic coupling part was designed according to a CAD program (Solidworks, Dassault Systemes, France) and manufactured through a 3D printer (3DISON MULTI, Rokit, Korea). An electrochemical biosensor immobilized with an aptamer according to an embodiment of the present invention was prepared by combining the electrode prepared according to Example 3 and the prepared microfluidic channel using a magnetic coupling part.

Experimental Example 1. Confirmation of the physical properties of the surface-modified electrode

Cyclic voltammetry for bare carbon electrodes (bare SPCE), MoS ₂ /SPCE, graphene nanoplatelets/SPCE, graphene nanoplatelet composites/SPCE and gold nano/graphene nanoplatelet composites/SPCE electrodes The results of (cyclic voltammetry: CV) and electrochemical impedance spectroscopy (EIS) are shown in FIG. 7 .

Figure 7(a) shows the greatest reduction peak separation in the gold nano/graphene nanoplatelet composite/SPCE electrode, and FIG. 7(b) is the charge transfer resistance value in the gold nano/graphene nanoplatelet composite/SPCE electrode. (charge transfer resistance) shows the largest measured result. From the above results, as the raw carbon electrode (bare SPCE), graphene nanoplatelet composite/SPCE and gold nano/graphene nanoplatelet composite/SPCE electrodes go, the reduction peak separation of CV and the charge transfer resistance value of EIS gradually increased. Through the increased results, it can be confirmed that the effective surface area is increased, so that the surface of the sensor is well formed according to the stage in which the electrode is manufactured.

Experimental Example 2. Exosome detection limit evaluation

Exosome detection limit evaluation was performed using the electrochemical biosensor according to the present invention. As a result of measuring the electrochemical signal by varying the exosome concentration, as shown in FIG. 6 , a current peak was confirmed in the range of ^{1×10 2} exosomes/ml to 1×10 ^{9 exosomes/ml.}

Experimental Example 3. Clinical evaluation of exosome detection ability

Clinical evaluation of exosome detection ability was conducted using 10 breast cancer patients as an experimental group and 3 healthy normal subjects as a control group. Sample recruitment was done according to the approved protocol of the Yonsei University Severance Hospital IRB. For breast cancer patients, plasma samples were collected from the experimental group of 3 patients in stage 1, 2 patients in stage 2, 2 patients in stage 3 and 3 patients in stage 4, and the control group of 3 normal subjects according to the stage of cancer progression.

After plasma samples were collected from the experimental group and the control group, they were left at room temperature for 15 to 30 minutes to form a thrombus. Then, the thrombus was removed by centrifugation at 1000-2000 rpm for 10 minutes, the supernatant was transferred to a clean tube, and the exosomes were separated using an exosome separation agent (Total Exosome Isolation Reagent (from plasma), Thermo Fisher Scientific, USA). did. The obtained exosomes were compared and evaluated for their ability to detect exosomes through an electrochemical biosensor and an exosome ELISA kit (Wako Pure Chemical Industries, Ltd., Japan) according to the present invention.

In both methods, the detection amount of exosomes increased as cancer progressed, but the detection amount of exosomes was significantly higher in the case of the electrochemical biosensor according to the present invention compared to the ELISA method. This is believed to be because the electrode through the surface modification of the present invention exhibited high detection sensitivity, and accurate quantification of exosomes through microfluidic channels was possible. In addition, it was confirmed that, according to the present invention, the detection time can be significantly shortened according to the present invention, as it takes 5 hours in the case of the ELISA method, compared to less than 2 hours in the case of the present invention.

In addition, while the amount of sample required for exosome detection was also 100 μl by ELISA, it was possible to detect even 10 μl or less according to the present invention, so that detection accuracy can be increased even with a small sample amount. Confirmed. These results can be confirmed in FIGS. 10 and 11 .

In the above, an embodiment of the present invention has been described, but those of ordinary skill in the art can add, change, or delete components without departing from the scope of the technical idea of the present invention described in the claims. Accordingly, the present invention can be variously modified.

100: electrochemical biosensor 200: microfluidic channel part
210: reaction unit 220: comb pattern flow path
230: particle injection unit 240: particle discharge unit
250: particle moving unit 300: electrode unit
310: substrate 320: working electrode
330: counter electrode 340: reference electrode
400: junction 410: top cover
412: upper cover magnet 420: lower cover
422: lower cover magnet

Claims (14)

an electrode unit including a working electrode on which an aptamer or a sensing antibody capable of binding to a target material is immobilized and surface-modified with a graphene nanoplatelet complex;
microfluidic channel unit; and
Including; a junction for coupling the electrode part and the microfluidic channel part;
The binding is characterized in that the non-permanent binding method, electrochemical biosensor. The method of claim 1,
The graphene nanoplatelet composite is an electrochemical biosensor comprising any one or two or more of a graphene nanoplatelet, and a transition metal chalcogen compound, a thiol-based polymer, and an amine-based polymer. The method of claim 1,
The electrochemical biosensor, wherein gold nanoparticles are applied to the top of the working electrode of the electrode part. The method of claim 1,
The microfluidic channel part electrochemical biosensor including a particle injection part, a particle moving part, a reaction part, and a particle discharge part. 5. The method of claim 4,
The reaction part electrochemical biosensor, characterized in that a ceiling spaced at a predetermined interval and a side wall connected to the ceiling are formed so that the fluid introduced through the particle injection part can form a vortex. The method of claim 1,
The non-permanent coupling method of the junction is an electrochemical biosensor that combines the microfluidic channel part and the electrode part using magnetic force. 7. The method of claim 6,
The junction part includes an upper cover and a lower cover, and at least one selected from the upper cover and the lower cover further includes a receiving part for accommodating the microfluidic channel part and the electrode part therein, and is selected from the upper cover and the lower cover. Any one or more of which is an electrochemical biosensor that includes a magnet and is coupled to the upper cover and the lower cover using magnetic force. The method of claim 1,
The target material is an exosome, an electrochemical biosensor. A kit for detecting exosomes comprising the biosensor according to any one of claims 1 to 8. providing a liquid sample for analysis;
injecting the sample for analysis into a particle injection unit of a microfluidic channel of a sensor for detecting a target material;
concentrating the target material of the sample for analysis through the vortex formed in the reaction part of the microfluidic channel;
forming a complex by specifically binding the target material with an aptamer or a detection antibody immobilized on a working electrode facing the reaction unit;
and analyzing the target material in the sample for analysis by measuring the electrochemical signal generated by the complex;
The working electrode is a graphene nanoplatelet, and a transition metal chalcogen compound, a thiol-based polymer, and an amine-based polymer, characterized in that it further comprises any one or two selected from the target material analysis method. 11. The method of claim 10,
After the analyzing step, recovering the target material; Target material analysis method further comprising. 12. The method of claim 11,
Using the recovered target material for any one or more downstream analysis selected from the group consisting of PCR, sequencing, mass spectrometry (MS), gene analysis, and protein analysis; Target material analysis method further comprising. 11. The method of claim 10,
The target substance is a target substance analysis method, characterized in that the exosome. 14. The method of claim 13,
The concentration of the exosome is 1×10 ² to 1×10 ⁹ exosome/mL of a target substance analysis method.

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