KR20100035275A - Disposable surface plasmon resonance chip having multi-channel - Google Patents

Abstract

PURPOSE: A disposable surface plasmon resonance chip having multi-channel is provided to measure surface plasmon resonance using a transparent polymer material capable of mass production and to inject and discharge the other kind of a bio sample. CONSTITUTION: A disposable surface plasmon resonance chip(100) comprises a lower substrate(30), an upper substrate(10), and a metallic thin film layer(20) interposed between the upper plate and the lower plate. The upper plate includes a plurality of inlets(11) in which a bio sample and a buffer solution are inserted, a recover unit(14), and a plurality of independent channels(12,13).

Description

Disposable Surface Plasmon Resonance Chip having multi-channel}

The present invention relates to a microfluidic delivery chip used in a device for detecting and analyzing protein samples, and more particularly, to compare and analyze a plurality of samples using surface plasmon resonance (SPR). It relates to a microfluidic sensor chip.

The biosensor chip is a tool for confirming the existence of various biological samples such as DNA, RNA, protein, and cells, or performing qualitative or quantitative analysis thereof. In particular, the classical method of performing analysis on biological samples is a blot hybridigation method.

This is a method of attaching DNA, RNA or protein to a membrane filter and forming hybrid molecules using specific intermolecular binding on the filter. In the blot hybridization method, Southern blotting for transferring DNA to a membrane filter and fixing it, Northern blotting for fixing RNA, and Western for fixing protein according to the type of biological sample probe to be immobilized. Western blotting. All of these methods basically use a binding reaction between a ligand and a receptor that selectively binds to the ligand.

However, the above-described methods have to be performed for each sample, and despite the very repeated experiments, it is difficult to perform the experiments under the same conditions, and requires a lot of manpower, time and huge resources.

As an indirect method for analyzing ligand and ligand binding of the biosensor chip described above, a fluorescence assay using a secondary antibody reaction requires the development of a secondary antibody that binds to a specific protein (primary antibody). Proteins containing fluorescent materials that can be bound must be developed. This can only be met if a great deal of labor, time and capital is required. Therefore, much research has been made on analytical methods that do not use fluorescent materials.

Surface Plasmon Resonance (SPR) refers to a phenomenon of optically inducing vibration of electrons occurring on a thin metal thin film. The reason why the surface plasmon resonance is used as a bioreaction detector is that the surface plasmon resonance has a very sensitive characteristic (especially a change in resonance angle or wavelength) occurring on the surface of the metal thin film (or 300 nm or less from the surface). Because. Accordingly, the interaction of various types of biomolecules can be analyzed at the nano level by constructing various sensing interfaces based on chemical surface modification of metal thin films. In addition, surface plasmon resonance does not cause any damage or modification of the sample when measured compared to other methods described above, and does not require the development of a secondary antibody, which is a marker like fluorescence analysis, and can measure biochemical reactions with high sensitivity. Has an excellent advantage.

The apparatus for detecting the bioreaction of proteins using the surface plasmon resonance described above has been commercialized, and the most developed form is equipped with a micro-fluidic system, and according to the initial amount of the biological sample and the reaction experiment. The loss can be minimized.

However, in the case of the conventional surface plasmon resonance sensor chip, the manufacturing cost is high by using a glass substrate, and there is a problem in noise and reproducibility due to reuse through cleaning. In addition, the conventional microfluidic chip has a problem of commercialization due to the need for additional devices for controlling valves and fluids, and to increase in volume, equipment, and sensor chips.

The first object of the present invention for solving the above problems is to measure the surface plasmon resonance phenomenon using a transparent polymer material which is inexpensive and mass-produced, and at the same time a multi-channel capable of injecting or discharging different kinds of biological samples To provide an SPR chip.

More specifically, an object of the present invention is to provide a disposable multi-channel SPR chip having excellent optical properties and easy to mass-produce through a simple manufacturing process using a transparent polymer material having excellent chemical resistance to various chemicals.

In addition, a second object of the present invention is to provide a method for manufacturing a disposable multi-channel SPR chip according to the first object.

In order to achieve the first object of the present invention, in the SPR chip consisting of a lower substrate, an upper substrate and a metal thin film layer interposed at a predetermined position between the lower substrate and the upper substrate, the upper substrate is a biological sample and a buffer solution. A plurality of injection holes which are injected and penetrate the upper substrate; A recovery port for recovering the biological sample and the buffer solution; And a plurality of independent flow paths that open to the lower side of the upper substrate to contact the lower substrate and the metal thin film layer and connect the injection hole and the recovery port via the metal thin film layer. It provides a multichannel SPR chip made of this transparent organic polymer material.

In addition, the present invention provides a method for manufacturing a disposable multi-channel SPR chip comprising the following manufacturing step to achieve the second object.

a) forming a metal thin film layer on a predetermined position of a lower substrate made of a transparent organic polymer material;

b) manufacturing an upper substrate of a transparent organic polymer material by an injection method using a nickel plated plate having a pattern corresponding to the injection hole, the recovery hole, and the flow path; And

c) thermal bonding the lower substrate after step a) and the upper substrate of step b).

Disposable multi-channel SPR chip according to the present invention, both the upper substrate and the lower substrate is made of a transparent organic polymer material. The transparent organic polymer material may include polyethylene terephthalate (PET), polycarbonate (PC), and cyclic olefin copolymer (cyclic olefin copolymer; COC), and the like. The cyclic olefin copolymer has excellent light transmittance and is generally used. Compared to the polyethylene terephthalate (PET) and the polycarbonate (PC), which are transparent plastic substrates, the chemical resistance is excellent, and thus it is more suitable for use in place of the glass substrate used as a conventional lower substrate. In addition, the patterned nickel plated plate and the injection molding method can be easily produced a patterned upper substrate, there is an advantage that can be thermally bonded at a relatively low temperature of 70 to 120 ℃.

The cyclic olefin copolymer is formed by copolymerizing an alpha olefin and a cyclic alkene, and specifically, for example, the cyclic olefin copolymer represented by the following Chemical Formula 1 may be exemplified, and commercially available products include the trade name TOPAS series.

[Formula 1]

[In Formula 1, x and y are integers of 1 to 100000, R is selected from hydrogen, (C1-C7) alkyl group, (C6-C20) aryl group, or (C6-C20) ar (C1-C7) alkyl group. do.]

The material of the lower substrate according to the present invention preferably matches the refractive index of the base prism (BASE PRISM) used in SPR equipment for measuring the disposable multichannel SPR sensor chip. This can reduce the safety of the measured data and the loss of signal, and the same refractive index can satisfy the boundary condition that the optical path and the wave vector of light (light source) cause resonance. As an example, when the refractive index of the prism used in general SPR equipment is 1.53, it is even more preferable to use 5013 series materials in the TOPAS COC series as the cyclic olefin copolymer because the refractive index is consistent.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art. In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

1 is an exploded perspective view of a disposable multi-channel SPR chip according to a preferred embodiment of the present invention, FIG. 2 is a cross-sectional view taken along line aa 'when the metal thin film layer 20 of FIG. 1 is not patterned, and FIG. When the metal thin film layer 20 is patterned along a flow path, it is a cross-sectional view taken along aa ', and FIG. 4 is a front view of an upper substrate of a disposable multi-channel SPR chip according to an embodiment of the present invention, and FIG. 5 is an SPR according to the present invention. It is a schematic diagram for explaining the analysis method using a chip.

1 to 4, the disposable multi-channel SPR chip 100 includes a lower substrate 30, a metal thin film layer 20, and an upper substrate 10. The upper substrate is provided with a plurality of flow paths 12 and 13. When the SPR chip according to the present invention is inserted into the SPR analyzer during the SPR analysis, the prism of the SPR analyzer contacts the metal thin film layer with respect to the lower substrate. In this case, it is preferable to apply a matching oil such as the refractive index of the lower substrate and the base prism which are actually used to eliminate the change of the optical refractive index on the lower substrate surface in contact with the prism.

The upper substrate 10 is open to the biological sample and the buffer solution inlet 11 formed through the upper substrate, the biological sample and the buffer solution recovery port 14 formed through the upper substrate, and the lower side of the upper substrate 10. A plurality of independent flow paths 12 and 13 contacting the lower substrate 30 and the metal thin film layer 20 and connecting the injection hole 11 and the recovery port 14 via the metal thin film layer 20. It is provided. The metal thin film layer 20 may not be patterned as shown in FIG. 2, or may be patterned according to a flow path size as shown in FIG. 3. Two or more flow paths are formed independently. In FIG. 1, four flow paths are formed, but are not necessarily limited thereto. One of the flow paths may be used as a reference channel for measurement of a reference reagent during analysis.

As shown in FIG. 5, a matching oil is disposed between the lower substrate and the prism of the SPR chip according to the present invention, and the flow path formed on the upper substrate is in contact with the metal thin film layer, so that the biological sample injected into the flow path is the metal thin film layer. Is adsorbed or entrapped. The incident light injected into the lower substrate through the prism is a total reflection condition in which light transmitted to the upper substrate is no longer removed when light is injected above the critical angle without any metal thin film layer and all incident light is reflected. However, when the metal thin film layer is present as shown in FIG. 5, the light incident beyond the total reflection angle is absorbed at the interface between the metal thin film and the dielectric material at a specific angle. In addition, the angle changes depending on the metal thin film and the adsorbed or entrapped material. It is said that surface plasmon is excited. Therefore, at certain angles above the total angle of reflection, the surface plasmon resonance causes the reflectivity to be minimized (in theory, the minimum value is zero).

Referring to the application of the SPR chip according to the present invention, the biological sample injected through the injection port 11 is a metal through the flow path (12, 13) by natural convection or capillary phenomenon, or external pressure (pump) to the aqueous solution phase The biological sample supplied on and supplied to the thin film layer is adsorbed on the surface of the metal thin film layer or is reacted with a monolayer or antibody of the biological sample formed on the surface of the metal thin film layer. When the biological sample is adsorbed or entrapped on the surface of the metal thin film layer, the biological sample, that is, the biological sample bound to the antibody, changes the plasmon resonance angle analyzed through the prism in contact with the lower substrate, and the component of the biological sample is changed by the change of the plasmon resonance angle. This analysis becomes possible. That is, when the biological sample is not adsorbed or entrapped in the metal thin film layer, the plasmon resonance angle of the light source incident and reflected through the prism and the plasmon resonance angle when the biological sample is adsorbed or entrapped have different values, and such plasmon resonance The change in angle is used to analyze the response of the biological sample.

Referring back to FIGS. 1 to 4, when a biological sample or a buffer solution is injected into the injection hole 11 provided in the upper substrate, it passes through the metal thin film layers through the channels 12 and 13 and is discharged to the recovery port. In addition, in the upper substrate, flow paths are separately provided for each biological sample inlet, so that biological samples can be supplied to the metal thin film layer through the injection path, and at the same time, the biological sample or the buffer solution can be washed through the injection path. In addition, different fluids can be selectively injected into the desired channel, allowing independent use of multiple channels to confirm the multi-material change response.

The metal thin film layer is formed at a position corresponding to the prism in contact with the lower substrate during the SPR analysis. The metal thin film material is not particularly limited as long as it can cause surface plasmon resonance. Gold, silver, copper, or aluminum may be used alone or in combination. In the case of the metal thin film, the thickness of the gold thin film may vary depending on the refractive index and the absorption coefficient. Must be different, generally 50 nm thickness is preferred for gold. Sputtering, evaporation, ion plating, electroplating, electroless plating, etc. may be used as a method for forming the metal thin film layer. However, in view of ease of manufacture, it is more preferable to form by sputtering or electron beam evaporation using a pattern mask. .

A single layer for detecting a biological sample may be fixed to the metal thin film layer. As a single layer for detecting a biological sample, a biomaterial such as an antibody may be used, and in order to coat a single layer on a metal thin film layer, a self assembly monolayer is fixed on a gold thin film. Method can be used. In addition, the method of forming a single layer may be a method using a polymer such as nylon, cellulose. The forming of the single layer may be performed before thermal bonding of the lower substrate and the upper substrate in the manufacturing process of the disposable multi-channel SPR chip according to the present invention, and after the bonding is completed, injecting a material for forming the single layer through the injection hole. It may be formed by reaching the metal thin film layer through the flow path.

Hereinafter, a manufacturing process of the multichannel SPR chip according to the present invention will be described in detail.

A method of manufacturing a multichannel SPR chip according to the present invention includes the following steps.

a) forming a metal thin film layer on a predetermined position of a lower substrate made of a transparent organic polymer material;

b) manufacturing an upper substrate by a method of injecting a transparent organic polymer material using a nickel plated plate having a pattern corresponding to the injection hole, the recovery hole, and the flow path; And

c) thermal bonding the lower substrate after step a) and the upper substrate of step b).

Step a) is a step of forming a metal thin film layer at a predetermined position on the lower substrate. As described above, the metal thin film layer is formed at a position corresponding to the prism contacting the lower substrate during SPR analysis.

Step b) is to prepare the upper substrate, the upper substrate is a plurality of injection holes for injecting the biological sample and the buffer solution, recovery ports for recovering the biological sample and the buffer solution, and the injection port and recovery port It connects and the flow path through a metal thin film layer is formed. Method of manufacturing a patterned nickel plated plate by plating nickel on a silicon wafer on which a pattern corresponding to the injection hole, the recovery port, and the flow path is formed, and then releasing the integrated organic polymer material using the same. It is preferable to manufacture the upper substrate. It is preferable to use a cyclic olefin copolymer (COC) as the transparent organic polymer material, and the injection temperature is sequentially applied at 150 to 300 ° C. In this case, it is preferable that the flow path formed has a width of a flow path of a portion via the metal thin film layer and a portion connected to the recovery port to be 1.5 to 5 times the width of the flow path of the portion connected to the injection hole. The flow path formed on the portion where the metal thin film layer is formed is further reduced in height during the thermal bonding process, which is a subsequent process, and the biological sample or buffer solution injected through the flow path gradually compensates for the metal thin film layer. To pass through. Therefore, when the width of the flow path 13 in the portion passing through the metal thin film layer is less than 1.5 times, the passage of the biological sample is not easy, and when it exceeds 5 times, it is difficult to form several channels on the metal thin film layer. It is not easy.

More specifically, the height of the flow path of the upper substrate before thermal bonding is 100 to 400 um, the width of the flow path 12 of the portion connected to the injection hole is 100 to 300 um, and the flow path of the portion connected to the portion passing through the metal thin film layer and the recovery port. (13) The width is preferably 200 to 600 um. This takes into account the capillary phenomenon and the size of the SPR chip according to the thickness SPR analyzer of the metal thin film layer. If the size is out of the size, it may be difficult to properly implement the performance of the multi-channel SPR chip.

The step c) is a step of thermally bonding the lower substrate and the upper substrate on which the metal thin film layer is formed. More specifically, the surface of the lower substrate and the upper substrate on which the metal thin film layer is formed is argon (Ar) gas and oxygen (O ₂ ). Plasma treatment using a gas, and the step of contact bonding under the conditions of 70 to 120 ℃, 40 to 1000 N / cm ² after contacting the upper surface of the lower substrate and the lower surface of the upper substrate. If it is less than the temperature range, the bonding is not made properly, and if the temperature is exceeded, the width or height of the fine flow path may be changed, which may be detrimental to the formation of a uniform flow path. If the pressure range is exceeded, the shape of the fine flow path pattern of the upper substrate may be changed, which may be disadvantageous for forming a uniform flow path.

After the step a), or after the step c) may further comprise fixing a single layer for detecting a biological sample on the metal thin film layer. The monolayer may be a biomaterial such as an antibody and the like, and in order to coat the monolayer on the metal thin film layer, a method of fixing a self assembly monolayer on the gold thin film may be used. . In addition, a method using a polymer such as nylon and cellulose may be used.

As described above, since the multi-channel SPR chip according to the present invention includes a plurality of biological sample / buffer solution inlets and a recovery port, multiple biological samples may be simultaneously introduced or discharged. Therefore, two or more unknown biological samples can be compared and analyzed, and after the reaction with the detection antibody formed on the metal thin film, the remaining amount of the biological sample can be recovered without loss through the recovery port. In addition, by applying a transparent organic polymer material having excellent optical properties and chemical resistance to the upper substrate and the lower substrate it can be easily fastened by thermal bonding, and mass production is possible to manufacture a low-cost disposable multi-channel SPR chip.

Hereinafter, a method of manufacturing a disposable multi-channel SPR chip according to the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following Examples.

EXAMPLE

Disposable multichannel SPR chips as shown in FIGS. 1 and 2 were prepared.

Fabrication of the Upper Substrate

The photosensitive resin PR is formed on the silicon wafer to form a pattern in which the injection hole 11, the recovery hole 14, and the flow paths 12 and 13 are formed, and then fastened to a plating jig. The fastened jig is washed with an aqueous sulfuric acid solution and then an aqueous hydrofluoric acid solution, and then immersed in a nickel sulfamate plating solution maintained at 53 degrees. The current applied during plating was applied at 500 mA 90 min, 1000 mA 60 min, 2000 mA 60 min, 4000 mA 60 min, and 7000 mA 60 min, and then plated at 15 A for 240 minutes to prepare a nickel plated plate having a thickness of 0.5 mm. Insert the nickel plated plate into the mold and maintain the mold temperature at 110 ℃. Using COC (TOPAS 5013, refractive index 1.53), the injection temperature is sequentially applied in five steps from 150 to 300 ° C., and the injection pressure is injected in five steps in steps of up to 20 MPa, and the ejection cooling time is At 30 seconds, a patterned upper substrate of COC material was prepared. The thickness of the manufactured upper substrate is 1 mm, the flow path width of the injection hole is 200 um, the width of the flow path connected to the metal thin film layer forming portion and the recovery port is 500 um, and the depth of the flow path is 200 um.

Manufacturing of Disposable SPR Chips

After placing a pattern mask where only a portion of the metal thin film layer is to be formed on a lower substrate composed of a COC (TOPAS 5013, a refractive index of 1.53) having a thickness of 1 mm, gold having an average thickness of 50 nm was formed by a shadow masking method using a metal mask. The thin film layer (Au) was formed using the electron beam evaporation method.

The lower substrate on which the gold thin film layer was formed and the upper substrate prepared above were subjected to ultrasonic cleaning for 5 minutes in acetone, ultrasonic cleaning for 5 minutes in methanol, finally ultrasonic cleaning for 5 minutes in distilled water, and drying using nitrogen gas. After the process, the upper surface of the lower substrate on which the gold thin film layer was formed and the lower surface of the upper substrate on which the flow path was formed were treated with a plasma mixed with argon and oxygen gas.

After contacting the upper surface of the lower substrate and the lower surface of the upper substrate, pressure bonding was carried out under conditions of 100 ° C. and 200 N / cm ² to prepare a disposable SPR chip according to the present invention.

[Test Example 1]

The multi-channel SPR chip prepared in the above example was mounted on an SPR analyzer (μ-SPR, K-MAC Co., Ltd.), and deionized water and ethanol were injected to measure the change in resonance angle according to the solvent. 6 is shown.

Referring to FIG. 6, it can be seen that the SPR resonance angle changes due to the change of the refractive index according to the type of buffer solution passing on the Au thin film layer.

[Test Example 2]

The multi-channel SPR chip prepared in Example was mounted on an SPR analyzer (μ-SPR, K-MAC Co., Ltd.) to measure the change in resonance angle according to the concentration of ethanol, and the results are shown in FIG. 7. As a comparative example, a bulk-type SPR chip without a channel was manufactured by continuously depositing Cr 1 nm and Au 50 nm by electron beam evaporation using a cover glass (manufacturer: MATSUNAMI, product name: Micro cover glass No. 2). , Mounted on the same SPR analyzer and injected with changing the concentration of ethanol through the sample inlet of the SPR analyzer to measure the change in the resonance angle according to the concentration and the results are shown in FIG.

Referring to FIG. 7, as the concentration of ethanol passing through the Au thin film layer was changed from 0 to 80 wt%, the resonance angle was changed in the range of 62 to 70 degrees. Referring to FIG. 8, the result measured by a conventional measuring method As the concentration of ethanol was changed from 0 to 80 wt%, the resonance angle was changed in the range of 52 to 58 degrees. It can be seen from the results of FIGS. 7 and 8 that the measurement method according to the present invention has a wider resonance angle change range than the conventional method, so that the change in refractive index according to the sample can be more sensitively analyzed.

1 is an exploded perspective view of a multi-channel SPR chip according to a preferred embodiment of the present invention,

2 is a cross-sectional view taken along aa ′ when the metal thin film layer 20 of FIG. 1 is not patterned.

3 is a cross-sectional view taken along aa ′ when the metal thin film layer 20 of FIG. 1 is patterned along a flow path,

4 is a front view of the upper substrate of a disposable multi-channel SPR chip according to an embodiment of the present invention,

5 is a schematic diagram for explaining an analysis method using an SPR chip according to the present invention;

6 is a graph measuring the change of the SPR resonance angle according to the buffer using the SPR chip according to the present invention,

7 is a graph measuring the change of the SPR resonance angle according to the ethanol concentration using the SPR chip according to the present invention,

Figure 8 is a graph measuring the change in the SPR resonance angle according to the ethanol concentration using a bulk type SPR chip prepared on a glass substrate as a comparative example.

<Explanation of symbols for the main parts of the drawings>

100: multi-channel SPR chip 10: upper substrate

20: metal thin film layer 30: lower substrate

11: biological sample and buffer inlet

12, 13: Euro 14: recovery port

Claims (10)

In the SPR chip consisting of a lower substrate, an upper substrate and a metal thin film layer interposed at a predetermined position between the lower substrate and the upper substrate, The upper substrate may include a plurality of injection holes through which the biological sample and the buffer solution are injected and penetrate the upper substrate; A recovery port for recovering the biological sample and the buffer solution; And a plurality of independent flow paths which open to the lower side of the upper substrate to contact the lower substrate and the metal thin film layer and connect the injection hole and the recovery port via the metal thin film layer. The multi-channel SPR chip having a lower substrate and the upper substrate made of a transparent organic polymer material. The method of claim 1, The upper substrate and the lower substrate is a multi-channel SPR chip, characterized in that consisting of a cyclic olefin copolymer (COC). The method of claim 2, The upper substrate and the lower substrate is a multi-channel SPR chip, characterized in that bonded by thermal bonding. The method of claim 3, wherein The thermal bonding is performed by plasma forming a lower substrate and an upper substrate surface on which a metal thin film layer is formed. And a step of performing pressure bonding under conditions of 70 to 120 ° C. and 40 to 1000 N / cm ² after contacting the lower substrate upper surface and the upper substrate lower surface. The method of claim 3, wherein The flow path formed in the upper substrate is a multi-channel SPR chip, characterized in that the flow path width of the portion via the metal thin film layer and the portion connected to the recovery port is 1.5 to 5 times the width of the flow path of the portion connected to the injection hole. The method of claim 3, wherein The height of the flow path of the upper substrate before the thermal bonding is 100 to 400um, the flow path width of the portion connected to the injection hole is 100 to 300um, the flow path width of the portion via the metal thin film layer and the portion connected to the recovery port is 200 to 600um. Multi-channel SPR chip, characterized in that. The method of claim 6, The upper substrate is manufactured by injecting a cyclic olefin copolymer using a patterned nickel plated plate manufactured by plating nickel on a silicon wafer on which a pattern corresponding to the injection hole, the recovery port, and the flow path is formed. Channel SPR chip made with. The method of claim 1, The metal thin film layer is a multi-channel SPR chip, characterized in that to form a metal material selected from gold, silver, copper, or aluminum by a sputtering or deposition method using a pattern mask at a predetermined position on the upper surface of the lower substrate. The method of claim 8, The metal thin film layer is a multi-channel SPR chip, characterized in that the gold (Au) thin film of 10 to 100 nm thickness. a) forming a metal thin film layer at a predetermined position on a lower substrate made of a cyclic olefin copolymer (COC); b) manufacturing an upper substrate by an injection method of a cyclic olefin copolymer (COC) using a nickel plated plate having a pattern corresponding to the injection hole, the recovery hole and the flow path; And c) thermal bonding the lower substrate after step a) and the upper substrate of step b); Method for producing a disposable multi-channel SPR chip comprising a.

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