JP2011013000A - Method for analyzing biochip, and system for automatic analysis of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing a biochip capable of rapidly obtaining a measuring result, by automating a reaction detection process of a specimen and an antigen after the specimen is sampled, using the biochip loaded with antigens of many kinds of allergens which are mutually independent, that is, isolated spots, and to provide a system for automatic analysis of biochip.SOLUTION: A consistent system for automatic analysis of biochip is provided with the biochip 2 loaded with the antigen as the spot 6; a prefilter for removing serum component from a sampled blood specimen and a reagent cassette 4a becoming a pair along with the biochip 2, can rapidly obtain an analysis and measurement result, by automating the respective processings from the reaction of the biochip 2, to which the specimen is added, with the antigen, the reaction with an antibody, cleaning and the addition of a detection reagent to the detection of chemoluminescence, shortens the analysis and measurement time and attains micronization of an installation area.

Description

  The present invention uses, for example, a biochip in which antigens of biomolecules such as proteins, nucleic acids, glycolipids, etc. are immobilized as independent spots, and analyzes a sample to be analyzed at a high speed, at a low cost, with easy handling, and over multiple items. The present invention relates to a biochip analysis method and an automatic analysis system thereof.

  In recent years, allergic diseases include various allergic diseases such as hay fever, atopic dermatitis, bronchial asthma, food allergies, drug allergies, etc. Of the 120 million people in Japan, 42 million have some relationship with allergies. It is said that there are people. The reason for the increase in allergic diseases is the increase in sensitizing substances (antigens) in the daily living environment and the heavy exposure to this. Since allergic diseases cannot be separated from the daily living environment, it is not enough to suppress the symptoms with drugs in the treatment of the disease, and it cannot escape the recurrence. It is necessary to identify and treat using the law. Accordingly, there are an increasing number of medical treatments related to allergic diseases and allergens that cause allergic symptoms in allergies, dermatology, internal medicine, etc. Tests that identify allergens usually require testing by specialized inspection agencies because of the complexity of the test and the need for large-scale test analyzers.

Allergic symptoms are antigen-antibody reactions in which an antigen acts on the body (living body) and an immune function works. As a result of this reaction, immunoglobulin (abbreviated as “Immunoglobulin”) is produced. Currently, five types of immunoglobulins, A, G, D, M, and E, are known. Among them, it is known that excessive reaction of immunoglobulin E (abbreviated as IgE) causes allergic symptoms. Currently, this is detected and used for diagnosis. This is called IgE antibody. An immunoglobulin is a protein and its structure is a chain of amino acids. IgE is as small as 200-240 ng / ml in normal individuals. Identification of allergens begins with a questionnaire, and the enzyme antibody method (enzyme-
Various examination methods such as linked immunosorbent assay (ELISA) are performed to identify the causative antigen.

  As an IgE calibration method, a fluorescent immunoassay method (for example, patent patent) detects the amount of IgE antibody bound to an allergen with a fluorescently labeled secondary antibody after allowing the target specimen to act on the solid surface on which the allergen is immobilized. Reference 1), or the amount of IgE antibody bound to the allergen with a chemiluminescent labeled secondary antibody, or the amount of IgE antibody bound to the allergen with a chemiluminescent labeled secondary antibody. , A dye immunoassay for colorimetric detection, for example, CAP-FEIA method (abbreviated as CAP method).

  There are a single item method and a multi-item method as a measuring method of an IgE analyzer which is put into practical use. In the single item method, one specimen sample is added to one well, and the presence or absence of IgE antibody against the allergen fixed in the well is determined. Since only one type of allergen can be determined per well, screening analysis to identify allergens requires the use of different types of wells. A typical example is the CAP method, which has become a global standard. Since the CAP method requires 40 microliters of specimen sample for each single item measurement, when measuring N items, the amount of blood sample increases and the apparatus becomes large. Will be installed. On the other hand, techniques for measuring multiple items at the same time have been developed for the purpose of improving analysis efficiency. In the multi-item method, a large number of allergens are locally arranged in one well, and the presence or absence of IgE antibody is determined from the immunity antibody determination at each location. For this purpose, a technique for locally fixing the allergen and a measurement technique for determining it are necessary.

  The multi-item method currently in practical use is a simultaneous multi-item allergen-specific IgE antibody measurement method (Multiple Antigen Simultaneous Test: MAST method) (for example, see Non-Patent Document 1). In this method, the long wells are divided into smaller wells, different types of antigens are fixed to each well, and the sample sample is applied to the whole well, and then the individual well signal intensity is analyzed, The amount of IgE antibody against the antigen is analyzed in the whole well. This method currently allows simultaneous analysis of up to 33 antigens, but the sample volume is only 200 microliters. However, the time required for the measurement is 5 hours.

  Another multi-item method is the imfast check method using thin layer chromatography. This uses a thin layer method in which the sample liquid is automatically added without using wells. Since only the step of injecting the sample solution and the color developing solution is required, it is a point of care test (POCT) type that can be easily used as a cassette and can be used in the field. However, the number of allergens that can be used is limited to about three.

  A protein chip method (abbreviated as microarray chip method) for the purpose of high-speed on-site analysis is known as one of the multi-item methods (see, for example, Non-Patent Document 2). The allergen is fixed in the spot shape of a slide substrate coated with a special surface, and a flow path for the sample serum is provided there. After the reaction, washing, secondary antibody reaction, washing, a chemiluminescence reagent is allowed to act, The emission intensity is measured with a CCD camera, and the IgE amount is determined from the intensity. The process of injecting the sample specimen into the chip container and the subsequent process are automated. The time required for the measurement is 30 minutes or more. However, the size of the device is large and requires a large installation area.

JP 2005-49276 A

Yamashita Kohei, Simultaneous multi-item allergen-specific IgE assay reagent (MASTII-S), Clinical Allergy, 28 (4), 314-321 (2008). Y. Ito et al, Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigenmicroarray, Biotechnol.Prog. 24, American Institute of Chemical Engineers, 1384-1392 (2008).

  Regarding the analysis method of biochip and its analysis system, the conventional technology is capable of simultaneous measurement of multiple items, but is a problem as a point-of-care test (POCT) system, that is, a compact and inexpensive device that does not take up installation space. Satisfying that it is a simple operation system that can be used by anyone without requiring proficiency, that results can be obtained quickly on the spot, and that more than 10 types of allergen screening can be performed with a single operation using a small amount of sample. There was nothing.

  Accordingly, an object of the present invention is to solve the above-mentioned problems, and after collecting a sample using a biochip on which antigens of various allergens are mounted as spots that are independent from each other, that is, separated from each other, It is an object of the present invention to provide a biochip analysis method and an automatic analysis system thereof that can automate the reaction detection process and obtain measurement results quickly.

  The present invention provides a substrate on which a biochip in which different types of antigens, which are biomolecules such as proteins, nucleic acids, and cytokines, are spaced apart from each other and fixed in a plurality of spots, and a retaining wall surrounding the biochip are fixed. Installed on a moving table at an installation station, supplies a specimen to be analyzed to the biochip area, and includes a cleaning liquid nozzle, an antibody nozzle, a reagent nozzle, and a suction nozzle for the biochip in the retaining wall on the substrate. And moving the antibody in the specimen to the antigen in the spot by causing the antibody in the specimen to react with the antigen in the spot, and then capturing the antibody in the specimen in the spot. A secondary antibody reagent is injected into the area from the antibody nozzle to react the secondary antibody with the antibody captured in the spot. In order to analyze the amount of the secondary antigen antibody reactively bound to the antibody, a luminescent reagent is injected into the biochip from the reagent nozzle, and chemiluminescence is produced by reacting the luminescent reagent with the secondary antigen antibody. Inducing and moving the biochip to an observation area, observing the emission intensity of the spot with a CCD camera, and analyzing the amount of antibody in the specimen corresponding to the spot of each antigen The present invention relates to a chip analysis method.

  In this biochip analysis method, prior to the injection of the secondary antibody reagent into the area of the biochip, the specimen is aspirated and removed from the area with the suction nozzle, and the cleaning liquid is supplied from the cleaning liquid nozzle to the area. Supplying and washing each of the biochips, and then suctioning and removing the cleaning solution from the area with the suction nozzle; and prior to the injection of the luminescent reagent, the secondary antibody reagent is removed from the area with the suction nozzle. Then, the cleaning liquid is supplied to the area from the cleaning liquid nozzle to clean the biochip, and then the cleaning liquid is suctioned and removed from the area by the suction nozzle.

  In addition, the biochip includes a chip in which 20 or more different allergen antigens are spaced apart from each other and fixed as a plurality of the spots, and 8 or more different autoimmune antigens are spaced from each other to obtain a plurality of spots. A chip fixed as a spot and / or a chip fixed as a plurality of spots by separating the cytokine or antibody protein from each other.

  The present invention also provides a biochip in which different types of antigens, which are biomolecules such as proteins, nucleic acids, glycolipids, etc., are spaced apart from each other and fixed as a plurality of spots, and a retaining wall surrounding the biochip area A fixed table, a moving table in which the substrate is installed and detachable at an installation station, a cleaning liquid nozzle for discharging a cleaning liquid to the area in the retaining wall, and a secondary antibody reagent in the area in the retaining wall. Antibody nozzle for injection, reagent nozzle for injecting luminescent reagent into the area in the retaining wall, specimen to be analyzed supplied to the area in the retaining wall, the washing liquid, the secondary antibody reagent, and the luminescent reagent A nozzle station equipped with a suction nozzle for sucking out the gas, and a CCD camera provided in the observation station for observing the light emission state of the spot on the biochip. A first driving device for moving the moving table to sequentially move the biochip to the installation station, the nozzle station and the observation station, and the cleaning liquid nozzle, the antibody nozzle, the reagent nozzle, and the suction nozzle And a biochip automatic analysis system comprising a second driving device for vertically moving the CCD camera relative to the biochip.

  In this biochip automatic analysis system, the biochip loaded with the allergen antigen is formed as an open chip surrounded by wells.

  This biochip automatic analysis system is capable of appropriately stirring the reaction solution on the biochip by the first driving device that moves the biochip to the reaction position in the nozzle station and the observation position by the CCD camera. is there.

In this biochip automatic analysis system, the CCD camera hood that covers the CCD camera is lowered to the biochip moved to the observation station, and the biochip is hermetically housed in a dark box shape in the CCD camera hood. The light emission state of the spot of the biochip is observed.
Ochip automatic analysis system.

  This biochip automatic analysis system is unitized so that the amount of analysis can be easily increased or decreased according to the number of specimens, and control, data collection and analysis can be performed for each unit connected via LAN. is there.

  The biochip analysis method according to the present invention includes a so-called one biochip in which various different allergens are mounted as spots spaced apart from each other, a prefilter for extracting serum components from a blood sample to be analyzed, and a biochip. The reagent cassette is provided and reacted with the antigen of the biochip to which the sample has been added. The reaction, washing, detection reagent addition, and chemiluminescence detection are automated, and measurement results can be obtained quickly. In addition, the above analysis method is to build a consistent automatic analysis system consisting of devices that can achieve a reduction in installation surface with a short measurement time. This biochip analysis method and system uses a photo-immobilized substrate in which non-specific adsorption is suppressed for point-of-care test (POCT) analysis for use in clinical settings. By preparing a biochip in which the sample is immobilized, injecting the specimen to be analyzed into the biochip, and then automating the reaction detection process, it is possible to obtain an analytical measurement result quickly and accurately with a very small amount of specimen sample.

It is a flowchart which shows one Example of the analysis method of the biochip which concerns on embodiment of this invention. It is explanatory drawing which shows one Example of the biochip automatic analysis system which concerns on embodiment of this invention. A biochip used in this biochip analysis method is shown, (A) is a perspective view showing an appearance image of the biochip, (B) is a configuration outline of the biochip, the biochip and a cleaning liquid nozzle, an antibody nozzle, a luminescent reagent Sectional drawing which shows a nozzle and a suction nozzle, (C) is a top view which shows a biochip external appearance upper surface. An example of an observation spot by a CCD camera for each specimen to be analyzed is shown, and since the emission spot is different for each specimen, it is an observation photograph that can discriminate the antibody of the specimen. It is an observation photograph by a CCD camera showing an example of the result of observation with a CCD camera by arranging an autoantibody as an allergen antigen on a biochip, and is an explanatory diagram showing the name of the autoantibody corresponding to the luminescent spot . It is a graph which shows an example of the correlation with the data obtained by the automatic analysis system by this invention, and the analysis result by ELISA, (a) is SSA, (b) is UI-RNP68kDa, (c) is DNA, (d ) Is SSB, and (e) is each antibody of Sm.

  Hereinafter, a biochip analysis method and an automatic analysis system thereof according to the present invention will be described with reference to the drawings. FIG. 2 is a conceptual diagram of the biochip automatic analysis system 1 according to the present invention. The biochip automatic analysis system 1 includes a biochip 2, a prefilter (not shown), a reagent supply system or reagent cassette 4a, a reaction / measurement integrated automation device, a control / analysis / display device, and a notebook personal computer 6. Has been.

  The biochip automatic analysis system 1 is controlled by a notebook personal computer 6 to collect and analyze measurement data. The control / analysis / display device 6 performs overall control of the automation device, determines the position of each allergen spot 2d from the image data, and estimates the IgE antibody concentration corresponding to the allergen from the optical signal intensity. It is an automated device that performs allergen screening and displays the measurement results. Further, the biochip automatic analysis system 1 unitizes the system so that the amount of analysis can be easily increased or decreased according to the number of samples. When the analysis amount of the biochip 2 is increased, it is increased for each unit, and control, data collection, analysis, and the like are performed for each unit connected via the LAN. Also, processing such as data collection and analysis of the entire unit connected to the LAN can be performed.

-About Biochip 2-
The biochip 2 used in the automatic biochip analysis system 1 is one in which 10 or more types of allergen antigens are fixed as a large number of spots 2d that are independent, ie, spaced apart from each other. In the biochip 2, a plurality of allergen extract or recombinant allergen antibody are arranged as spots 2d on the immobilization substrate 2a. The substrate 2a of the biochip 2 is preferably a glass substrate, a silicon substrate, or a plastic substrate that has been surface-treated for protein immobilization. Among these substrates, in particular, an allergen-extracted extract or a recombinant allergen antibody is spot-shaped on a metal substrate coated with a light-fixing polymer (see Japanese Patent Application No. 2008-080292 developed by the present inventor and previously applied for a patent). A plurality of biochips 2 arranged and fixed to each other are suitable for use in an automated process in a biochip analysis method because of less non-specific adsorption and good drainage.

-Prefilter-
A prefilter (not shown) used in the biochip automatic analysis system 1 removes red blood cells from a blood sample collected from a subject and adds a reagent for adjusting viscosity and the like so as to be suitable for reaction processing and measurement conditions. This is a cassette filter to be adjusted.

-Reagent supply system or reagent cassette 4a-
The reagent supply system or the reagent cassette 4a is a cassette on which a reagent that is easy to denature and difficult to store is used up once, and the convenience is improved by using up the reagent. For example, an antibody nozzle 9b for discharging reagent solution A from a reagent solution bottle for labeling of anti-IgE antibody added with horseradish peroxidase (HRP), a reagent solution bottle containing hydrogen peroxide, and a reagent solution containing luminol The reagent nozzle 9c that mixes and discharges the reagent solution B from the bottle and can be dropped on the biochip 2 independently by the reagent nozzle 9c, or can be adapted to cope with one biochip 2. A reagent cassette composed of an IgE antibody labeling reagent solution ampoule and an ampoule having a mechanism capable of mixing the reagent solution A and the reagent solution B immediately before dropping them onto the biochip 2 can be used. It ’s good.

-About the reaction / measurement integrated automation device-
The reaction / measurement integrated automation device uses the CCD camera 10c and the function to perform the reaction of the biochip 2 to which the sample to be analyzed has been added, the washing, the labeled antibody reaction, the washing, and the color reagent reaction by automation. This is an automated device that has a function of measuring a color signal image from the biochip 2 into which a color reagent for injection is injected. A mechanism for setting the biochip 2 and the reagent cassette 4a, that is, an installation station 8 for setting the biochip 1 on the moving table 12, a driving device 8a that is a mechanism for moving both to the reaction table, that is, a reaction position, a specimen, After a certain reaction time with the antigen, the suction nozzle 9d for sucking and draining the reagent on the biochip 2 and independent nozzles that can drop the cleaning liquid, the antibody reagent or the luminescent reagent without delay after the waste liquid, that is, the cleaning liquid nozzle 9a and the antibody nozzle 9b A reaction device having a luminescent reagent nozzle 9c, that is, a nozzle station 9 and a driving device 9e, and a mechanism capable of moving to the CCD camera observation system by the CCD camera 10c after dropping the luminescent reagent, that is, the CCD camera station 10 and the camera observation position; After the measurement of the sample is completed, the biochip 2 is returned to the reaction table, and the reagent is aspirated and drained. The kinematic device 8a, an automated reactor having a mechanism, which can be returned biochips 2 and the reagent cassette 4a to the initial set state.

  -Control, analysis, and display device-The above-mentioned automated reaction device is controlled by a program installed in the notebook personal computer 6, reads the information acquired from the CCD camera 10c into the personal computer 6, and reads the information read into the personal computer 6 It is possible to automatically analyze and display the abundance of allergen antibodies from image data.

  Further, the above reaction apparatus has a mechanism capable of optically shielding and shielding the lens set on the CCD camera 10c and the biochip 2 to which the luminescent reagent is added at the time of chemiluminescence measurement, that is, the camera hood 10a and the driving device 10b. It is characterized by that. Thereby, size reduction and weight reduction of an apparatus can be achieved. In the above reaction apparatus, the cleaning reagent is supplied to the nozzles 9b and 9c via the reagent supply tube 9f by a pump from a separately provided bottle. Further, in the above reaction apparatus, the reagent on the biochip 2 is discharged into the waste liquid processing tray 8b by the suction pump 4a through the suction nozzle 9d. In the above reaction apparatus, the cleaning liquid nozzle 9a, the suction nozzle 9d, the antibody nozzle 9b, and the luminescent reagent nozzle 9c on the biochip 2 are set simultaneously.

  FIG. 3A shows an external appearance image of the biochip 2. FIG. 3B shows a configuration outline of the biochip 2 and an arrangement state of the biochip 2 and various nozzles 9a to 9d. FIG. 3C shows a plan view of the appearance of the biochip 2. The cleaning liquid nozzle 9a, the antibody nozzle 9b, and the luminescent reagent nozzle 9c are formed to have the same length, and the suction nozzle 9d is formed to be longer than the nozzles 9a to 9c. The reason why the suction nozzle 9d is formed long is that all of the cleaning liquid residue, the specimen, the antibody, and the luminescent reagent present at the bottom of the area 2b of the biochip 2 are sucked. The four types of nozzles 9a to 9d are configured to simultaneously move up and down on the biochip 2 at the nozzle station 9 by the driving device 9e, rather than moving the nozzles 9a to 9d up and down individually. It has a practical structure that can shorten the processing time, that is, the measurement time.

Next, a method for producing the biochip 2 used in the biochip analysis method according to the present invention will be described.
The photo-immobilization technology for immobilizing the allergen antigen developed in this invention on the biochip 2 can stably immobilize any biomolecule by covalent bond, has a high effective concentration of the immobilized product, and suppresses nonspecific adsorption. , High signal-to-noise (S / N) ratio can be realized. These features make it possible to detect more types more quickly with a smaller amount of sample.

The biochip 2 used in the present invention was produced as follows.
(1) A methacrylic polymer having a phenyl azide group and a polyethylene glycol group having a molecular weight of about 500 in the side chain was prepared as a photoreactive matrix so as to have a final concentration of 1%. (2) The prepared polymer was pre-coated on a polystyrene or metal-deposited glass chip by spin coating.
(3) The biochip 2 was dried under reduced pressure at 0.09 MPa for 15 minutes.
(4) The biochip 2 was irradiated with UV light for 7 minutes using a black light.
(5) Bisazide was mixed at 10% with respect to antigens of biomolecules such as proteins, nucleic acids and cytokines to prepare antigen samples.
(6) The prepared antigen sample was spotted on the precoated biochip 2 using a DNA arrayer.
(7) The biochip 2 was dried at room temperature and normal pressure for 7 minutes, and then irradiated with UV light for 7 minutes using a black light.

  Next, in particular, the biochip analysis method will be described with reference to FIGS. FIG. 1 is a flowchart showing an embodiment of the biochip analysis method according to the present invention. First, the biochip 2 in which 20 or more types of allergen antigens are fixed in advance as independent spots 2d is fixed to the substrate 2a (step S1). The substrate 2a to which the biochip 2 is fixed is loaded at the installation station 8 of the biochip automatic analysis system 1 and fixed by the fixing means such as the clamp 19 and the analysis of the biochip 2 is started (step S2). In the installation station 8, the temperature is controlled to a predetermined temperature by the temperature controller 7, and the biochip 2 is also adjusted and maintained at the predetermined temperature. Next, blood serum, which is a sample to be analyzed, is discharged onto the biochip 2 with a manual pipette or the like (step S3). By supplying the specimen to the biochip 2, the specimen to be analyzed reacts with the antigen of any spot 2d.

  Next, the drive device 8a (first drive device) such as a motor is driven by a command from the multi-axis motor controller 5, and the moving table 12 is operated via the pulley 13 and the belt conveyor 14 provided on the drive shaft, thereby the biochip 2 Is moved from the installation station 8 to the position of the nozzle station 9. Here, it takes a predetermined time for the specimen to react with any antigen, for example, a reaction time of 8 minutes or less (step S4). Next, it is determined whether or not a predetermined reaction time has passed between any spot 2d antigen and the sample (step S5). After a predetermined time sufficient for the specimen to react with the antigen of any of the spots 2d, a driving device 9e (second driving device) such as a motor is driven by a command from the multi-axis motor controller 5. Various nozzles 9 a to 9 d and a camera hood 10 a are supported on the support frame 20, and a rack 17 for moving the support frame 20 up and down is fixed. By driving the driving device 9e, the support frame 20 is lowered together with the rack 17 as the pinion 16 is rotated, and the lowering suction nozzle 9d is brought closer to the biochip 2. Next, the suction pump 4b is operated through the I / O control 11 in accordance with a command from the control computer 3, and the specimen liquid injected into the area 2b in which the antigen is immobilized in a spot shape is sucked out through the suction tube 9g by the suction nozzle 9d (step S6). After the sample liquid existing on the biochip 2 is sucked out, the cleaning liquid from the cleaning liquid tank 21 is discharged to the area 2b through the cleaning liquid nozzle 9a to wash the biochip 2 (step S7). Again, the suction pump 4b is operated to suck out the cleaning liquid that has cleaned the biochip 2 existing in the area 2b by the suction nozzle 9d (step S8).

  Next, a predetermined secondary antibody reagent is injected from the reagent cassette or reagent supply system into the area 2b of the biochip 2 through the reagent supply tube 9f in accordance with a command from the multi-axis motor controller 5, where A predetermined time, for example, 8 minutes or less, is required for the reaction between the reaction spot 2d reacted with the sample and the secondary antibody reagent (step S9). It is determined whether or not a sufficient predetermined time for the reaction between the secondary antibody reagent and the reaction spot 2d has elapsed, and the reaction ends when the reaction time has elapsed (step S10). Next, the suction pump 4b is operated through the I / O control 11 in accordance with a command from the control computer 3, and the secondary antibody reagent injected into the area 2b is sucked out by the suction nozzle 9d (step S11). Next, after sucking out the secondary antibody reagent present on the biochip 2, the cleaning liquid from the cleaning liquid tank 21 is discharged to the area 2b through the cleaning liquid nozzle 9a to clean the biochip 2. Again, the suction pump 4b is operated to suck out the cleaning liquid that has cleaned the biochip 2 existing in the area 2b by the suction nozzle 9d (step S12). Therefore, a predetermined luminescent reagent is injected from the reagent cassette or reagent supply system into the area 2b of the biochip 2 through the reagent supply tube 9f in accordance with a command from the multi-axis motor controller 5 (step S13). The luminescent reagent reacts with the reaction spot reacted with the antibody reagent, and the reaction spot on the biochip 2 emits light, and the reagent injection process on the biochip 2 is completed (step S14).

  When the reagent injection process is completed, the driving device 9e is driven by a command from the multi-axis motor controller 5. By driving the drive device 9e, the pinion 16 is rotated to raise the support frame 20 together with the rack 17, thereby raising the various nozzles 9a to 9d to their original standby positions. Next, the driving device 8a is driven by a command from the multi-axis motor controller 5, the moving table 12 is operated via the pulley 13 and the belt conveyor 14 provided on the driving shaft, and the biochip 2 is moved from the nozzle station 9 to the CCD camera station. The position is moved to the position 10 (step S15). Again, by driving the driving device 10b (second driving device) according to the command of the multi-axis motor controller 5, the pinion 16 is rotated, the support frame 20 is lowered together with the rack 17, and the camera frame provided on the support frame 20 is moved. 10a is lowered so as to cover the area 2b of the biochip 2 in a sealed manner, and the light emission state at the spot 2d of the biochip 2 in the dark box state is observed by the camera 10a with the CCD camera 10c. Information on observation of the light emission state of the spot 2d is taken into the personal computer 6 (step S16). Observation information from the CCD camera 10c in the light emission state of the spot 2d is taken into the personal computer 6 and the observation is completed (step S17).

  When the observation of the biochip 2 by the CCD camera 10c is completed, the driving device 10b (second driving device) is driven by the command of the multi-axis motor controller 5, the pinion 16 is rotated, and the support frame 20 is raised together with the rack 17, The camera hood 10a is raised to the original position so as to be separated from the area 2b of the biochip 2, and then the driving device 8a is driven by the command of the multi-axis motor controller 5 to operate the moving table 12 and Is moved from the CCD camera station 10 to the position of the nozzle station 9, and the driving device 10 b is driven by a command from the multi-axis motor controller 5 to lower the support frame 20. The nozzles 9 a to 9 d are lowered to the area 2 b of the biochip 2. Next, the suction pump 4b is actuated through the I / O control 11 in response to a command from the control computer 3, and the luminescent reagent injected into the area 2b is sucked out by the suction nozzle 9d (step S18). Therefore, the driving device 10b is driven to raise the support frame 20, and the various nozzles 9a to 9d are returned to their original positions. Next, the driving device 8a is driven to operate the moving table 12, and the biochip 2 is moved from the nozzle station 9 to the initial position of the installation station 8 (step S19). Finally, the clamp 19 is released and the biochip 2 is released. The substrate 2a on which the chip 2 is mounted is removed from the moving table 12, and the analysis of the biochip 2 is completed (step S20).

  FIG. 4 shows an example of the light emission state of the spot 2d of the biochip 2 observed by the CCD camera 10c. Since it can be seen that the luminescence state is different for each antibody, the antibody species can be discriminated.

  FIG. 5 shows the light emission state of the spot 2d of the biochip 2 observed by the CCD camera 10c and the antibody corresponding to each spot 2d. The mark indicates the standard emission intensity, and the intensity of the actual observation spot is compared to determine which spot 2d has the antigen-antibody reaction on the personal computer 6.

  In (a) to (e) of FIG. 6, the results of analyzing the five types of antibodies SSA, U1-RNP68 kDa, dsDNA, SSB, and Sm by the analysis system that performs the biochip analysis method according to the present invention, The correlation of the result analyzed by ELISA used as a standard machine in the industry is shown.

  Table 1 shows Example 1 and Comparative Examples 1 and 2 according to the present invention. Comparative Examples 1 and 2 show the specifications described in Non-Patent Documents 1 and 2.

  Table 2 shows an example of the correlation between the data of the above-mentioned five types of antibodies obtained by the biochip automatic analysis system according to the present invention and the analysis results by the widely used ELISA. This was obtained by plotting the data of FIG. 6 to obtain the correlation coefficient. High correlation coefficients were obtained for all antibodies, indicating that the biochip automatic analysis system according to the present invention is highly reliable.

  The biochip analysis method and its automatic analysis system according to the present invention, for example, use a biochip in which antigens of different types of biomolecules are fixed as a plurality of spots, and handle a sample to be analyzed at high speed and at low cost. It can easily analyze multiple items at the same time, and can be applied to point of care test (POCT) analysis for use in clinical settings.

DESCRIPTION OF SYMBOLS 1 Biochip automatic analysis system 2 Biochip 2b Area 2c Retaining wall 2d Spot 4a Reagent supply system or reagent cassette 4b Suction pump 6 PC 8 Installation station
8a Drive device (first drive device)
9 Nozzle station 9a Cleaning liquid nozzle 9b Antibody nozzle 9c Reagent nozzle 9d Suction nozzle 9e, 10b Driving device (second driving device)
9f Reagent supply tube 9g Suction tube
10 CCD camera station 10a Camera hood 10c CCD camera 12 Moving table 20 Support frame

Claims (8)

A biochip in which different types of antigens that are biomolecules are spaced apart from each other and fixed as a plurality of spots and a substrate on which a retaining wall surrounding the biochip is fixed are placed on a moving table at an installation station,
Supplying the specimen to be analyzed to the biochip area;
By moving the biochip in the retaining wall on the substrate to a nozzle station having a cleaning liquid nozzle, an antibody nozzle, a reagent nozzle and a suction nozzle, and reacting the antibody in the specimen with the antigen in the spot Allowing the antigen in any of the spots to capture the antibody in the specimen;
Next, a secondary antibody reagent is injected from the antibody nozzle into the area of the biochip to cause a secondary antibody to react and bind to the antibody captured in the spot,
In order to analyze the amount of the secondary antigen antibody reactively bound to the antibody, a chemiluminescence is induced by injecting a luminescent reagent from the reagent nozzle into the biochip and reacting the luminescent reagent with the secondary antigen antibody. ,
Analyzing the biochip, wherein the biochip is moved to an observation area, the emission intensity of the spot is observed with a CCD camera, and the amount of antibody in the specimen corresponding to the spot of each antigen is analyzed Method.   Prior to the injection of the secondary antibody reagent into the area of the biochip, the specimen is aspirated and removed from the area with the aspiration nozzle, and the cleaning liquid is supplied from the cleaning liquid nozzle to the area, thereby supplying the biochip to the area. Washing, and then sucking and removing the washing liquid from the area with the suction nozzle; and prior to the injection of the luminescent reagent, the secondary antibody reagent is sucked and removed from the area with the suction nozzle; The biochip analysis method according to claim 1, wherein the cleaning liquid is supplied to the area to clean the biochip, and then the cleaning liquid is sucked and removed from the area with the suction nozzle.   The biochip includes a chip in which 20 or more different allergen antigens are spaced apart from each other and fixed as a plurality of spots, and a plurality of spots in which 8 or more different autoimmune antigens are spaced from each other. 3. The biochip analysis method according to claim 1, wherein the chip is fixed as a plurality of spots by separating the cytokines and antibody proteins from each other. . A biochip in which different types of antigens, which are biomolecules, are separated from each other and fixed in a plurality of spots, and a retaining wall surrounding the biochip area,
A moving table on which the substrate is installed and detachable at an installation station;
A cleaning liquid nozzle for discharging a cleaning liquid into the area in the retaining wall, an antibody nozzle for injecting a secondary antibody reagent into the area in the retaining wall, a reagent nozzle for injecting a luminescent reagent into the area in the retaining wall, and A nozzle station comprising a specimen to be analyzed supplied to the area in the retaining wall, the washing liquid, the secondary antibody reagent, and a suction nozzle for sucking out the luminescent reagent;
A CCD camera provided in an observation station for observing the light emission state of the spot on the biochip;
A first driving device for moving the moving table to sequentially move the biochip to the installation station, the nozzle station and the observation station; and the cleaning liquid nozzle, the antibody nozzle, the reagent nozzle, the suction nozzle, and the An automatic biochip analysis system comprising a second driving device for moving a CCD camera up and down relative to the biochip.   5. The biochip automatic analysis system according to claim 4, wherein the biochip loaded with an allergen antigen is formed in an open chip surrounded by wells.   The reaction liquid on the biochip is appropriately stirred by the first driving device that moves the biochip to a reaction position in the nozzle station and an observation position by the CCD camera. Biochip automatic analysis system.   The CCD camera hood that covers the CCD camera is lowered to the biochip moved to the observation station, and the biochip is hermetically housed in a dark box shape in the CCD camera hood, thereby emitting light from the spots on the biochip. A state is observed, The biochip automatic analysis system of any one of Claims 4-6 characterized by the above-mentioned.   The unit according to claim 4, wherein the amount of analysis can be easily increased / decreased according to the number of samples, and control, data collection and analysis are performed for each unit connected via a LAN. Biochip automatic analysis system.

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