KR20080007733A - Micro bio chip for immunoreaction and manufacture method thereof and immunoreaction detecting method using micro bio chip - Google Patents

Abstract

A micro bio-chip for immunoreaction is provided to maximize the reliability and the satisfaction index in use by reducing the amount of a sample used and a testing time and accurately and rapidly measuring a biochemical test. A micro bio-chip for immunoreaction(100) is characterized in that a glass chip(10) for detecting the immunoreaction using an electric signal through a microelectrode(11) and a PDMS chip(20) for injecting a sample for detecting the immunoreaction are placed on the top side of the glass chip and integrated thereinto, wherein the PDMS chip consists of a chamber(21), a microfilter(22) and a channel(23). A method for preparing the micro bio-chip for immunoreaction comprises the steps of: (a) forming the glass chip for detecting the immunoreaction using the electric signal; (b) forming the PDMS chip for injecting the sample such as a bio-chemical material for detecting the immunoreaction; and (c) after connecting the PDMS chip to the top side of the glass chip, treating it with oxygen plasma with constant power for a predetermined time to integrate the PDMS chip into the glass chip. A method for detecting the immunoreaction using the micro bio-chip comprises the steps of: (a) fixing an antigen and an antibody using the micro bio-chip and a micro-bead; and (b) performing to detect the immunoreaction through an electric signal using nano-gold and a silver solution.

Description

MICRO BIO CHIP FOR IMMUNOREACTION AND MANUFACTURE METHOD THEREOF AND IMMUNOREACTION DETECTING METHOD USING MICRO BIO CHIP}

1 is a schematic configuration diagram showing a glass chip according to the present invention;

2 is an exemplary view for schematically showing a configuration of a PDMS chip implemented by the present invention;

Figure 3 is an exemplary cross-sectional view for explaining the configuration of a micro biochip bonded glass chip and PDMS chip implemented according to the present invention,

4 is a perspective illustration showing an embodiment of a micro biochip implemented according to the present invention;

5 is an exemplary view schematically showing a step of forming a PDMS chip implemented by the present invention;

6 is an exemplary view schematically showing a step of forming a glass chip implemented by the present invention;

7 is an exemplary view schematically showing a process of detecting whether an immune response using the present invention,

Figure 8 is a graph measuring the electrical response of the immune response in real time using the present invention micro biochip.

<Description of the code | symbol about the principal part of drawing>

10 glass chip 11: micro electrode

20: PDMS chip 21: chamber

22: micro filter 100: micro biochip

The present invention relates to a micro biochip for immunoreaction for use in biochemical experiments such as an immune response experiment, a method for preparing the same, and a method for detecting an immune response using the same.

More specifically, the micro biochip for immunoreaction, a method of preparing the same, and an immune reaction using the same, which facilitates immobilization of a sample, which is a biochemical, and is detected by an electrical signal through an electrode for a biochemical reaction such as an immune response. It relates to a method of detecting whether or not.

Recently, due to the emergence of micro electro-mechanical systems (MEMS) and the development of biotechnology, research on biochips by BioMEMS based on this is being actively conducted.

The biochip is divided into two types, the first of which is a microarray type, which allows a specific biochemical contained in a sample to be searched by a capturing probe, and can serve as a capturing probe. After immobilizing the substance on the surface of the chip and reacting the biochemical to be analyzed there, information on the biochemical can be obtained by detecting and interpreting the presence or absence of such a reaction.

The second is a biochip using microfluidics, which implements microchannels, microchambers, and mixer valves on a chip to control microfluidics, immobilize biochemicals on the detection unit, and then use microfluidic flow for the biochemicals to be detected. This is a system that detects the reaction by reacting with the biochemicals immobilized on the detection unit, which is the field that is being actively researched in the long term and the recent miniaturization trend.

Essential technologies for such a biochip include immobilization of a probe that can react with a reactant, a detection technology that can detect the presence or absence of a reaction, and an information processing technology that can process detected information.

Conventional techniques for immobilizing the probes generally employ a method of immobilizing a probe capable of reacting with a reactant directly on a substrate and a method of immobilizing a detector using an external force.

However, the method of directly immobilizing on a substrate is useful in an array type, but in biochips using microfluidics, it is difficult to selectively immobilize on a substrate.

In addition, there is a method of immobilizing by using an external force, such as a method of immobilizing a magnetic bead coated magnetic beads using a magnetic electrode formed on the substrate, but this requires a device that generates an additional force to immobilize have.

Meanwhile, detection methods for biochemical reactions, which are currently used mainly, include fluorescence detection using a laser and electrochemical detection using redox reactions. The fluorescence detection using a laser combines a fluorescent material with a sample and is immobilized on a substrate. It is the most widely used method to distinguish the reaction optically by using the fluorescent material combined with the reaction of the reaction with, but this requires an optical detection equipment to determine the reaction, which requires a lot of time In addition to this, there is a problem that such a detection method has some limitations in miniaturizing an analysis system based on a biochip.

In addition, the electrochemical detection method is a method of detecting whether the reaction using the electrochemical reaction of other chemicals on the electrode and the probe is coupled to the sample, but this has a problem that the detection ability is relatively lower than the fluorescence detection method.

Therefore, the immobilization method and detection method conventionally performed as described above have some limitations in their efficiency, and thus problems of minimizing the reliability and satisfaction of practical use of the biochemical experiments applied and applied are always included. .

The present invention has been made to solve the problems of the prior art as described above has the following object.

The present invention is easy to use without the need for an additional device for directly immobilizing the antigen or antibody in the biochemical experiments, such as immune response experiments, or by using external force by using a microbead structural form and microbeads It is an object of the present invention to provide a microbiochip for immune reaction, and a method of manufacturing the same and a method for detecting the immune response using the same.

Another object of the present invention is to provide a miniaturization at the same time to be measured with a multimeter that can be easily and cheaply purchased on the market even without a large detection system such as an optical detection device using a conventional laser, and a biochip also uses a polymer such as PDMS. The present invention provides a micro biochip for an immune reaction, a method for manufacturing the same, and a method for detecting an immune reaction using the same, which allow mass production to be minimized while minimizing the production cost through the molding method.

Another object of the present invention is to reduce the amount of use and the experiment time of the sample and at the same time to improve the efficiency, such as to enable accurate and rapid measurement of biochemical experiments to maximize the reliability and satisfaction of the use to apply it The present invention provides a micro biochip for immune response, a method for manufacturing the same, and a method for detecting the immune response using the same.

Hereinafter, the present invention will be described in detail.

In describing the present invention, when it is determined that the detailed description of the related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

Terms to be described later are terms set in consideration of functions in the present invention, and may be changed according to a user's intention or custom such as an experimenter and a measurer, and a definition thereof should be made based on the contents throughout the present specification.

First, the micro biochip for immune reaction to be carried out by the present invention is a glass chip for detecting whether the immune response as an electrical signal, and to inject a sample to detect the immune response on the upper surface of the glass chip PDMS (Polydimethylsiloxane) chip is located and integrated, but after processing for a certain time at a constant power with oxygen plasma in order to integrate with PDMS chip located on the upper surface of the glass chip so that the PDMS chip is bonded to the upper surface of the glass chip do.

In this case, the glass chip is provided with a micro electrode to detect the immune response as an electrical signal, the micro electrode is formed of a chromium and platinum thin film, interdigitated type (interdigitated type) to increase the detection efficiency The two electrodes by) become a pair.

In addition, the PDMS chip is composed of a chamber in which biochemicals are injected, a micro filter positioned at the center of the chamber, and a channel provided between the inlet and the outlet.

Accordingly, the micro filter located in the central portion of the chamber is caught so as to stably fix the microbeads injected into the inlet, and the microbeads are coated with an antigen that is a biochemical.

As described above, in the state in which the glass chip and the PDMS chip are integrated, the microelectrode provided in the glass chip uses a microfilter provided in the PDMS chip and a microbead injected into the PDMS chip and fixed by the microfilter. It is to be located at the front end of the micro filter so that the immune response can be easily detected by an electrical signal.

In another embodiment of the present invention, a method for manufacturing a micro biochip for an immune reaction may include forming a glass chip for detecting an immune response with an electrical signal, and a sample such as a biochemical to detect the immune response. Forming a PDMS chip to inject the same, and treating the PDMS chip on the upper surface of the glass chip formed thereon for a predetermined time at a constant power with an oxygen plasma to integrate the PDMS chip.

In addition, in another embodiment of the present invention, a method for detecting an immune reaction using a micro biochip for immune reaction is to immobilize an antigen and an antibody using a micro biochip and microbeads, and then nano gold and silver solution to a specific antigen or antibody. Using to detect whether the immune response as an electrical signal.

EXAMPLE

With reference to the accompanying drawings, a preferred embodiment for achieving the present invention will be described in detail.

First, the present invention is shown in the accompanying drawings, Figures 1 to 7, Figure 1 is a schematic configuration showing for explaining a glass chip according to the present invention, Figure 2 is a PDMS chip of the present invention 3 is an exemplary view for schematically illustrating a configuration, and FIG. 3 is an exemplary cross-sectional view for explaining a configuration of a micro biochip to which a glass chip and a PDMS chip are bonded according to the present invention, and FIG. FIG. 5 is a perspective view illustrating an embodiment of a micro biochip, and FIG. 5 is a schematic view illustrating a process of forming a PDMS chip according to the present invention, and FIG. 6 illustrates a glass chip implemented by the present invention. FIG. 7 is an exemplary view schematically showing a step of making a step, and FIG. 7 illustrates an exemplary embodiment schematically showing a process of detecting an immune response using the present invention. It is shown.

That is, the present invention is intended to detect the immune response, such as a biochemical reaction by using an electrode and then immobilize the sample of the biochemical as an electrical signal through the electrode.

To this end, the micro biochip 100 for immune reaction is largely composed of a glass chip 10 and a PDMS chip 20, the glass chip 10 is formed with an electrode to detect whether the immune response as an electrical signal In addition, the PDMS chip 20 is injected to the sample to detect whether the immune response and the injected sample is made to be easily fixed to the electrode.

Accordingly, the micro biochip 100 for the immune reaction is an oxygen plasma to bond the PDMS chip 20 to the upper surface of the glass chip 10 so as to detect the immune response as an electrical signal. The treatment is performed at a constant power for a predetermined time, wherein the bonding is preferably performed after treatment at 18 W for 1 minute and 30 seconds with oxygen plasma.

On the other hand, when bonding the PDMS chip 20 and the glass chip 10, it was confirmed that the treatment for 1 minute and 30 seconds at 18W was the optimal bonding condition in the course of performing various experiments, and in other conditions, the bonding was performed. This did not work properly.

The glass chip 10 includes a microelectrode 11 having a pair of two electrodes of an interdigitated type, and the microelectrode 11 is formed of a chromium and platinum thin film. This is to increase the detection efficiency in detecting whether an immune response is an electrical signal.

In addition, the PDMS chip 20 is easily injected with a sample of a biochemical material through the chamber 21, and because the micro filter 22 is located in the center of the chamber 21 is injected through the chamber The sample is caught by the micro filter 22, through which the sample can be stably fixed to detect whether the immune response.

That is, when the microbead coated with the antigen, which is a biochemical, is injected into the chamber, the microbead is caught by the microfilter 22 which is located at the center of the chamber. As a result, the microbeads are stably fixed and fixed to the microelectrode 11 positioned at the front end of the microfilter 22.

Accordingly, the immune response can be easily detected as an electrical signal using the microbeads which are stably fixed above.

In this case, the checking of the electrical signal detected through an external measuring device (for example, a multimeter) is performed by connecting to a channel 23 provided between the inlet part and the outlet part.

In addition, the size of the glass chip 10 having the microelectrode 11 formed therein should be larger than that of the PDMS chip 20 in the overall shape of the micro biochip 100, which is a microelectrode 11 for detecting an electrical signal. Since the size of the glass chip 10 is larger than the PDMS chip 20 because it must be connected to an external measuring device, the operation is easy and easy to control when bonding the two chips.

In addition, as shown in FIG. 3, it is preferable to set the space between the channel 23 and the glass chip 10 to 160 μm, since the size of the microbead is about 50 to 80 μm. If it is smaller, there is only one layer of microbeads on the electrode, so that the microbeads have two layers on the electrode part for a smoother electrical signal measurement. There is a limit of the height that can be implemented by the photosensitizer.

The method for manufacturing the micro biochip 100 for immune reaction described above forms a glass chip 10 and a PDMS chip 20 and then integrates the PDMS chip 20 on the upper surface of the glass chip 10. This can be done after treatment for 1 minute and 30 seconds at 18W with oxygen plasma.

At this time, in order to form the PDMS chip 20, the 4-inch silicon wafer is first immersed in a SPM (sulfuric acid and hydrogen peroxide mixture) solution to remove foreign substances, and then a spin coater is used to apply a negative photosensitive agent SU-8 to a constant thickness. The thickness of applying SU-8, which is the negative photosensitive agent, is preferably 150 μm.

After the application, the soft bake is performed to remove the organic solvent contained in the negative photosensitive agent SU-8, and then a resin bond of the negative photosensitive agent is formed and developed through an exposure process and a post bake.

Thus, after forming and developing the resin bond of the negative photosensitive agent, a hard baking is performed to manufacture the PDMS chip 20 to complete the SU-8 mold.

When the SU-8 mold is completed, it is left in a vacuum oven for a certain time to remove bubbles, and then mixed with PDMS and a curing agent in a constant ratio (for example, a 10: 1 ratio of PDMS and curing agent) and then the SU-8 mold. Pour over and cure at a certain temperature for a certain time, preferably at 65 ℃ and 3 hours 30 minutes.

Accordingly, the ratio of the PDMS and the curing agent, and the temperature and time for curing are conditions that can be performed under optimum conditions when the experiment is performed based on various conditions.

The PDMS is separated from the mold that has completed the above process. At this time, the inlet part and the outlet part are formed by using a tool such as a punch to separate the PDMS. This is completed and formed.

In addition, in order to form the glass chip 10, the 4-inch Pyrex glass wafer is first immersed in a SPM (sulfuric acid and hydrogen peroxide mixture) solution to remove foreign substances, and then coated with a positive coating agent AZ1512 using a spin coater.

When the coating is completed in the above, the electrode shape is patterned through a photographic process. At this time, after depositing chromium and platinum at 100 kPa and 700 kPa on the patterned part, the positive photoresist remaining under the metal layer is removed.

The chromium and platinum are deposited at 100 kPa and 700 kPa, respectively, which usually raises an adhesive layer when forming metal wires or the like on a glass wafer or a silicon wafer, and the chromium is used as the adhesive layer.

If only platinum is deposited on the wafer, it may not be easily deposited on the wafer, which may cause a problem of falling off the wafer during the process, and to deposit an adhesive layer to prevent such a problem.

It is preferable to raise the adhesive layer by about 50 kPa to 100 kPa depending on the thickness, and in general, the ratio of the thickness of the adhesive layer and the metal layer to be raised is preferably about 1:10, but in the case of the present invention, the platinum is set to 700 kPa in consideration of the cost. It can be raised above 700Å, but there is a limit that metal deposition equipment can raise (usually ~ thousands).

When the positive photosensitive agent is removed, the glass chip 10 having the final electrode is completed.

On the other hand, the method for detecting the immune response using the micro-biochip for immune reaction to be carried out by the present invention is to immobilize the antigen and the antibody using the micro-biochip 100 and the microbeads, at this time to the specific antigen or antibody nano Gold and silver solutions are used to detect the immune response as an electrical signal.

To this end, as shown in Figure 7, the micro-biochip (100) is injected into a microbead coated with a biochemical material such as an antigen is located in the central portion of the chamber 21 formed on the PDMS chip 20 The microbeads are caught and fixed by the filter 22, and then the primary antibody is injected into the chamber 21 in which the microbeads are fixed.

In this case, when the primary antibody is injected into the chamber 21 in which the microbeads are fixed, in the case of reacting with the antigen, the primary antibody binds to the antigen on the surface of the microbead and remains on the surface of the microbead. Primary antibodies that react nonspecifically with the antigen will not bind to the antigen and will not remain on the surface of the microbead.

Thus, in the present invention, when the primary antibody injected above reacts specifically with the antigen, the primary antibody immobilized on the surface of the microbead is recombined with the secondary antibody to which nanogold particles are attached.

Accordingly, a silver enhancer is injected to amplify the size of the nano gold particles, and when the silver solution is injected, the nano gold particles adhered to the secondary antibody bound to the primary antibody on the surface of the microbead. The diameter is increased to be connected to each other, and finally the diameters of the microbeads and microbeads are amplified to form an electrical bridge connecting the electrodes to the electrodes by the nanogold particles.

This serves to lower the resistance measured at both electrodes by the electrical legs formed above, and to determine whether the immune response by measuring the resistance by the electrical connection.

Accompanying drawings Figure 8 is a graph measuring the electrical response of the immune response in real time using the micro-biochip of the present invention, which shows the results of measuring the specific and non-specific cases of the antigen antibody response.

As described above, the present invention has been limited to only detecting whether the immune response as an electrical signal, but when applied to determine whether the reaction to the biochemicals causing the general specific reaction as well as the experiment of the immune response. Even if it belongs to the technical scope of the present invention in advance.

Finally, in the implementation configuration for the micro-biochip for the immune reaction and the method of manufacturing the same and the method for detecting the immune response using the same can be variously modified and can take various forms.

It is to be understood, however, that the present invention is not limited to the specific forms referred to in the above description, but rather includes all modifications, equivalents, and substitutions within the spirit and scope of the invention as defined by the appended claims. It should be understood to include.

As described above, the present invention not only consumes less amount of sample than the conventional immunoassay, but also has the effect of measuring faster and more accurately than the conventional experiment.

In addition, since the present invention utilizes a microbead structural form and microbeads, an additional device for directly immobilizing antigens or antibodies or immobilization using external forces in biochemical experiments such as immune response experiments. There is an effect that can be easily and simply immobilized antigen or antibody without.

In addition, the present invention can be measured with a multimeter that can be easily and cheaply purchased on the market even without a large detection system such as an optical detection device using a conventional laser, and at the same time, it can be miniaturized, and a biochip such as PDMS Through the molding method using a polymer, mass production is possible and the production cost is minimized.

In addition, the present invention as a whole, the reliability and satisfaction of the application to be implemented by implementing the effect due to the effect of improving the efficiency, such as reducing the amount of use of the sample and the experiment time, enabling accurate and rapid measurement of biochemical experiments There are several effects that are maximized.

Claims (15)

In micro biochip for immune reaction, A glass chip for detecting an immune response with an electrical signal and a PDMS chip for injecting a sample to detect an immune response on an upper surface of the glass chip are located and integrated. Biochip. The method of claim 1, Integrating with the PDMS chip located on the upper surface of the glass chip is a micro biochip for immune reaction, characterized in that the PDMS chip is bonded to the upper surface of the glass chip after a certain time at a constant power with oxygen plasma. The method of claim 1, The glass chip is provided with a micro electrode to detect whether the immune response as an electrical signal, the micro electrode is formed of a chromium and platinum thin film, in an interdigitated type (interdigitated type) to increase the detection efficiency The micro-biochip for immune reaction, characterized in that the two electrodes consisting of a pair. The method of claim 1, The PDMS chip is a micro biochip for immune reaction, comprising a chamber in which a biochemical is injected, a micro filter positioned in the center of the chamber, and a channel provided between the inlet and the outlet. The method of claim 4, wherein The micro-filter located in the center of the chamber is a micro biochip for immune response, characterized in that to catch the microbead injected into the inlet to stably be fixed. The method of claim 5, The microbeads microbiochips for immune reaction, characterized in that the antigen is coated with a biochemical material. The method according to any one of claims 1 to 6, The glass chip and the PDMS chip are integrated, and the microfilter included in the PDMS chip and the microbeads injected into the PDMS chip are fixed by the microfilter so that the immune response can be easily detected as an electrical signal. Micro biochip for immune reaction, characterized in that the microelectrode provided on the glass chip is located at the front end of the micro filter. In micro biochip for immune reaction, A glass chip for detecting an immune response with an electrical signal and a PDMS chip for injecting a sample to detect an immune response on the upper surface of the glass chip are positioned at a constant time at a constant power with an oxygen plasma. After processing for a PDMS chip is bonded to the upper surface of the glass chip, The glass chip is provided with a microelectrode, and the microelectrode is formed of a chromium and a platinum thin film, and in order to increase the detection efficiency, two electrodes of an interdigitated type are formed in pairs. The PDMS chip is located in the center of the chamber to stably fix the chamber into which the biochemical is injected, the channel provided between the inlet and the outlet, and the microbead coated with the antigen injected into the inlet. Consisting of a micro filter, The microelectrode provided in the glass chip uses the microfilter provided in the PDMS chip and the microbeads injected into the PDMS chip and fixed by the microfilter so that the microreaction can be easily detected as an electrical signal. Micro biochip for immune reaction, characterized in that located in the front end of the. In the method for producing a micro biochip for immune reaction, Forming a glass chip for detecting an immune response with an electrical signal; Forming a PDMS chip to inject a sample, such as a biochemical, to detect the immune response; Treating the PDMS chip on the upper surface of the glass chip formed above by bonding the PDMS chip with an oxygen plasma for a predetermined time at a constant power; Method of producing a micro-biochip for immune reaction, characterized in that for executing. The method of claim 9, In the forming of the PDMS chip, Dipping the 4-inch silicon wafer in a SPM (sulfuric acid and hydrogen peroxide mixture) solution to remove debris; After removing the foreign matter and applying a negative photosensitive agent SU-8 to a constant thickness using a spin coater, Performing a soft bake to remove the organic solvent contained in the SU-8, which is a negative photosensitizer, after the coating; Forming and developing a resin bond of a negative photosensitive agent through an exposure process and a post bake after the soft bake is finished; Forming a resin bond of the negative photosensitive agent and developing the resin bond, and then performing hard baking to complete a SU-8 mold for manufacturing a PDMS chip; After performing the hard baking (hard baking) in the SU-8 mold is completed, the step of leaving the vacuum oven for a predetermined time to remove bubbles; After performing the above bubble removal, mixing PDMS and a curing agent at a constant ratio, pouring it onto a SU-8 mold, curing the mixture for a predetermined time at a constant temperature, and then separating the PDMS from the mold, The method of manufacturing a micro-biochip for immune reaction, characterized in that the PDMS separated in the step of forming an inlet (inlet) and an outlet (outlet) by a tool such as a punch to complete the final PDMS chip. The method of claim 10, The thickness is 150㎛, the ratio of the PDMS and the curing agent is 10: 1, the temperature and time to cure in the process of producing a micro biochip for immune reaction, characterized in that carried out to 65 ℃ and 3 hours 30 minutes. . The method of claim 9, In the step of forming the glass chip, Dipping a 4-inch Pyrex glass wafer in a SPM (sulfuric acid and hydrogen peroxide mixture) solution to remove debris; After removing the foreign matter in the above step of applying a positive photosensitive agent AZ1512 using a spin coater, Performing a photographic process to pattern the electrode shape after the application is completed; Depositing chromium and platinum in a thin constant thickness on the patterned part through the photo process, respectively, Removing the positive photoresist remaining under the metal layer after the deposition in the method of producing a micro-chip for immune reaction, characterized in that to perform the step of completing the glass chip on which the final electrode is formed. The method of claim 12, The thickness of each of the chromium and platinum to be deposited is formed by 100 Å and 700 Å, the method of producing a micro biochip for immune reaction, characterized in that the execution. Immobilization of antigens and antibodies using microbiochips and microbeads, and then micro-chips for immune reactions characterized in that the detection of the immune response with an electrical signal using nanogold and silver solutions to specific antigens or antibodies Method for detecting whether the immune response using. The method of claim 14, In order to detect whether the immune response is an electrical signal, Injecting microbeads coated with biochemicals such as antigens into the microbiochips so as to catch and fix the microbeads by a microfilter positioned at the center of a chamber formed in the PDMS chip; Injecting a primary antibody into the chamber to which the microbead is fixed; The primary antibody immobilized on the surface of the microbeads by specific reaction with the antigen is recombined with the secondary antibody to which nanogold particles are attached; Injecting a silver enhancer to amplify the size of the nanogold particles; When the silver solution is injected, the diameters of the nanogold particles attached to the secondary antibody bound to the primary agent on the surface of the microbead become larger and connected to each other. Forming an electrical bridge connecting the electrode with the electrode; Acting to lower the resistance measured at both electrodes by the formed electrical legs, and determining the immune response by measuring resistance by electrical connection; Method for detecting whether the immune response using the micro-biochip for immune response, characterized in that to carry out.

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