JPWO2005071056A1 - Biochip and method for testing functionality of sample solution using the same - Google Patents

Abstract

Excellent data handling and reliability when making comprehensive measurements of enzyme activities such as blood and immunological properties and performing pathological diagnosis, easy chemical treatment, and increased detection density. A biochip capable of acquiring enzyme activity and its tendency even with the sample solution of is provided. A biochip for detecting an enzyme in a sample solution introduced into the chip substrate 11, and the sample solution channel 12 formed on the chip substrate in a channel pattern such as a spiral shape, a tree shape, or a radial shape as a whole And a plurality of enzyme activity detectors 13 arranged in the sample solution flow path 12 in a predetermined order and having different activities with respect to a predetermined enzyme.

Description

  The present invention relates to a biochip capable of comprehensively detecting activity and reaction behavior of a sample solution with respect to an enzyme and a method for testing the functionality of the sample solution using the biochip.

Conventionally, techniques for measuring enzyme activity by changing fluorescence in a solution using a single fluorescent enzyme substrate peptide have been established for many enzyme-fluorescent substrate pairs.
In addition, in order to perform such multiple measurements of enzyme activity in parallel, a technique has been developed in which a large number of reaction products obtained by hydrolyzing DNA or RNA are immobilized on a substrate to form a chip.
For example, an array obtained by immobilizing a large number of oligonucleotides on a substrate such as glass is called a DNA chip, and is often used for analysis of genes related in the fields of pathology, medicine, drug discovery, food, and the environment. ing.
In addition, development of a technique for exhaustively searching for proteins that are gene products using a protein chip (registered trademark of Cyphergen Co., Ltd.) in the same manner as a DNA chip is underway. Proteolytic enzymes (proteases) exist just as DNA exists where life phenomena are observed. Therefore, if proteases can be comprehensively detected, it becomes a technique necessary for personal health care, such as obtaining biological information related to homeostasis consisting of the secretion, production, and degradation processes of peptide hormones. In order to detect proteases comprehensively, there is a method for scientifically qualitatively determining the presence of one molecule as a protein.
However, since protein fragmentation results in loss of protein function, it is not easy to immobilize it on a substrate while maintaining the three-dimensional structure of the biopolymer, which is a technical bottleneck. Yes.

In recent years, with the development of medical fields such as pathological diagnosis and research fields such as proteome analysis, enzymes that do not have the disadvantages of fragmentation such as proteins and are easy to handle chemically and create a data library are used. As a result, there is a need to detect the activity against multiple enzymes, and there is a technology for measuring absorbed light and fluorescence in solution using a sensitive substance that is active against enzymes. Various studies have been conducted.
For example, in Patent Document 1, both ends of a substrate peptide that is specifically cleaved by caspase, which is a type of proteolytic enzyme, are modified with a fluorescent group, and the fluorescence wavelength and fluorescence of the fluorescent group generated by cleaving the substrate peptide are cleaved. A fluorescent probe is described which measures the intensity and detects the enzymatic activity of a solution containing caspase.
As a method for measuring such enzyme activity, there is a method in which a sample solution is injected into a cell formed on a microplate for fluorescence measurement, and the fluorescence of the cell is measured using a plate reader.
JP 2000-316598 A

However, the conventional techniques have the following problems.
(1) Since the technique described in Patent Document 1 only detects the enzyme activity of a sample solution with respect to a specific proteolytic enzyme, the enzyme is adapted to a complex system such as blood containing various enzymes and proteins. It is necessary to repeat complicated procedures to measure the activity and immunological characteristics comprehensively, and to locate the characteristics of the sample solution to be measured in many library data and perform pathological diagnosis. However, there was a problem that the measurement was not reliable or quick.
(2) Further, since it is necessary to use a substrate peptide whose specificity for a specific enzyme is known in advance as an enzyme sensor, a large number of the substrate peptides are specified and analyzed to analyze a complicated immune system or the like. However, there was a problem of requiring a huge accumulation of basic data.
(3) In the conventional method of measuring the fluorescence change of a fluorescent probe by injecting a specimen solution into a microplate having a millimeter-sized cell, a large amount of solution is required to examine a large number of enzyme activities, and handling is also necessary. It becomes complicated. Therefore, when performing fluorescence measurement, it took time to replace the microplate, and the measurement efficiency could not be improved, and it took time to measure.
(4) Further, there is a problem that it is difficult to acquire time series data when the sensor substance and the enzyme are bound or react with each other and lacks dynamic analysis.
(5) Since the enzyme substrate is bound in an array on the substrate, it is necessary to immerse the substrate surface in the sample solution containing the enzyme in order to perform simultaneous measurement, and the sample solution is required in milliliter units. There was a problem of requiring a large amount.
(6) The technology that uses a fluorescent enzyme substrate peptide to measure enzyme activity based on changes in fluorescence in the solution requires one fluorescence measurement well for a single enzyme measurement, and uses multiple fluorescence measurement devices. Even if the fluorescence measurement requires several minutes per 100 wells, it takes several hours or more to perform the time-varying measurement for each well in order to ensure precise quantitativeness. There was a problem.
(7) At the research level of enzyme activity detection biochip, the peptide of the enzyme substrate is bound to the surface of a substrate such as glass via trimethoxyaminopropylsilane, and the surface is immersed in the enzyme solution to emit fluorescence. Detected. However, since the binding amount of the fluorescent substrate peptide on the substrate is low, there is a problem that even if qualitative results can be obtained with or without enzyme activity, it is difficult to use for applications that require quantitativeness. It was.
(8) In addition, since the binding amount of the fluorescent substrate peptide on the substrate is not stable and it is difficult to quantify the binding amount, the results cannot be compared among a plurality of enzyme substrate peptides. It is difficult to quantify enzyme activity simultaneously on one chip.

The present invention was made in order to solve the above-mentioned conventional problems, and handling and reliability of data when performing pathological diagnosis by comprehensively measuring enzyme activity and immunological characteristics such as blood, An object of the present invention is to provide a biochip that is excellent in rapidity, easy in chemical treatment, and capable of increasing the degree of integration of the detection unit and acquiring enzyme activity and its tendency even with a small amount of sample solution.
In addition, the present invention does not require accumulation of basic data, is excellent in the analysis of time-series data, and has excellent operability and efficiency in which the enzyme activity of a sample solution can be measured in a short time using a small amount of sample solution. It aims at providing the functionality inspection method of a sample solution.

The biochip according to claim 1 of the present invention is a biochip for detecting an enzyme in a sample solution introduced into a chip substrate, and the entire chip has a flow path pattern such as a spiral shape, a tree shape, or a radial shape. A plurality of sample solution channels formed on the substrate, and enzyme activity detection units arranged in a predetermined order in the sample solution channels and having different activities with respect to a predetermined enzyme are configured.
With this configuration, the following effects can be obtained.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow path formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.

Here, as the sample solution, a body fluid or a culture solution containing blood or urine to be subjected to pathological diagnosis or the like can be applied. As the sample solution, a biological sample (blood or the like) can be used as it is, or a blood cell component or the like removed by a filter, centrifugation, or the like. Moreover, what performed the condition setting (pH adjustment, active agent introduction | transduction, etc.) that an enzyme expresses activity based on a biological sample can also be used. As a pH adjuster, a buffering agent such as Tris-HCl or Hepes-KOH can be added as a reaction buffer. In addition, salts and active protective agents necessary for the expression of enzyme activity can be added.
Examples of enzymes to be detected include serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, kallikrein, urokinase, elastase, aspartic proteases such as pepsin, cathepsin D, renin, chymosin, carboxypeptidase A, B, collagenase, thermolysin, etc. Endopeptidases that cleave internal peptide bonds such as cysteine proteases such as metalloproteases, cathepsins B, H, L, and calpain, blood coagulation proteases, capture proteases, hormone processing enzymes, and the like.
When the enzymes to be detected have an inactive state, they can be activated by adding trypsin or the like or removing the endogenous inhibitor.

  As the chip substrate, a substrate such as glass or synthetic resin having no enzyme specificity is used. Solvents used in condensation reactions to form peptide bonds (halogenated hydrocarbons such as chloroform and dichloromethane, esters such as ethyl acetate, polar organic solvents such as N, N-dimethylformamide and dimethyl sulfoxide, dioxane , Ethers such as tetrahydrofuran, alcohols such as methanol and ethanol, pyridine, etc.) made of insoluble synthetic resin (polystyrene, etc.) or glass, etc., formed into a curved surface such as a flat plate or spherical surface It is done. A commercially available carrier for solid phase organic synthesis such as Lantern series (registered trademark) manufactured by Mimotops can also be used.

The sample solution channel is a portion in which the vertical cross section of the chip substrate is formed in a rectangular shape, a U shape, a groove shape, or the like. The flow pattern of the sample solution flow path is continuous or branched so that the sample solution flows from one or a plurality of upstream openings toward the downstream openings, and in a plan view, a spiral shape, a tree shape, It can be formed in a radial shape, a zigzag shape, or the like. Thereby, the integration degree of a sample liquid flow path can be raised and a biochip can be reduced in size and size.
The sample solution channel can be formed on the chip substrate mechanically or chemically so that the volume, channel width, and depth of about 100 μL or less are about 1 mm or less. This is because the degree of integration of the sample solution flow path on the chip substrate decreases as the value becomes larger than these upper limit values, and it tends to be difficult to specify the fluorescent pattern to be formed on the chip substrate.
On one end side of the sample solution flow path, a recess-like liquid supply section for supplying the sample solution can be formed in a row with one or a plurality of upstream openings. Once the sample solution is supplied to the liquid supply part formed in the shape of a depression, the sample solution can be moved sequentially from the liquid supply part to the sample liquid flow path by capillary action etc. Because you can. The liquid supply part is provided with a filter such as a membrane film for removing red blood cells, white blood cells, lymphocytes, etc. in the supplied blood (sample solution) as necessary, so that the sample solution adheres to the channel wall. So that the flow is not obstructed.
On the other end side of the sample solution flow path, a liquid storage part for storing the sample solution can be formed continuously with one or a plurality of downstream openings. This liquid storage part is a recessed part formed in the center or peripheral part of the chip substrate. As a result, the sample solution flows out from the other end side of the sample solution flow path to the liquid storage section, so that a flow of the sample solution from the sample solution flow path to the liquid storage section can be formed. The oligopeptide cleaved from the enzyme activity detection part by the action of the enzyme in the sample solution can be reliably released into the sample solution without stagnation of the sample solution, and the detection sensitivity of the enzyme activity can be increased. .

The enzyme activity detection unit is configured by arranging a sensitive substrate such as a peptide chain having a selective and specific reaction behavior with respect to a specific enzyme. With such an enzyme-specific reaction or the like, the optical characteristics such as the fluorescence wavelength and fluorescence intensity of the sensitive substrate and the electrical characteristics change, and this can be detected.
A chip substrate when a plurality of enzyme activity detectors are arranged in the sample solution flow path, and an image sensor comprising a CCD or CMOS device integrated body is arranged at a position where the enzyme activity detector can receive light. By acquiring the upper two-dimensional fluorescence image, time-series fluorescence data of the enzyme activity detection unit or fluorescence data after a predetermined time can be obtained.
The enzyme activity detectors arranged adjacent to each other in the sample solution channel need not be arranged so as to be separated from the surroundings in a cell shape via a partition wall or the like, but along the bottom of the sample solution channel of the chip substrate. Alternatively, they may be arranged continuously in a planar shape.
In addition, the arrangement of each independent enzyme activity detection unit made of a peptide to which a fluorescent group is bound in the sample solution flow path is, for example, using a commercially available DNA chip plotter or using a syringe while looking at a microscope. Can be arranged. In addition, each predetermined region in the sample solution flow path formed in a spiral shape is filled with spherical particles such as polystyrene resin or flat plate with enzyme specificity on the surface thereof, and an independent enzyme activity detector is provided. It is also possible to arrange them.

The biochip according to claim 2 is the invention according to claim 1, wherein the enzyme activity detection unit is (a) a peptide chain whose base end is bound to the chip substrate and bound to the other end. The fluorescent group whose fluorescence characteristics such as the fluorescence frequency and the fluorescence intensity are changed depending on the chemical binding state, and (b) the enzyme-specific binding portion provided by the peptide chain of a specific amino acid sequence consisting of a combination of amino acids. And an oligopeptide bonded to a fluorescent group.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(1) Since it has a fluorescent group bound to the chip substrate and an oligopeptide bound to the fluorescent group by a peptide bond that is cleaved by an enzyme, the oligopeptide is released when the peptide bond is cleaved by reacting with the enzyme Is done. Since the fluorescence wavelength of the fluorescent group released from the oligopeptide or the fluorescence intensity at a predetermined wavelength is different from that before the release, the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
(2) Since the fluorescent group is bonded in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. In addition to detecting the activity, the biochip can be miniaturized by dramatically increasing the degree of integration of the enzyme activity detector that can detect the enzyme activity.
(3) Enzyme activity can be detected simply by using a sample solution that fills a very small volume of the sample solution flow path. Therefore, a large amount of sample solution is not required for measurement, and even a small amount of sample solution can be used for enzyme activity. Can be detected.
(4) By contacting the enzyme in the sample solution flowing through the sample solution flow path with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
(5) Since the enzyme specificity for a specific enzyme and the like is determined by the respective amino acid sequences constituting the oligopeptide, when possible combinations of these amino acid sequences are two-dimensionally arranged in the sample solution channel on the chip substrate in a certain order In the case of the same sample solution, images of the same fluorescent pattern arrangement are always obtained. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
(6) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
(7) Since knowledge of enzyme activity for a specific enzyme or protein is not required in advance for all such amino acid sequences, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.

  Here, the oligopeptide is a peptide composed of a peptide chain consisting of several amino acids, and has a substantially linear form unlike a protein having a large molecular weight and a mass. The amino acid sequence of the oligopeptide can characterize the enzyme specificity at the binding site with the fluorescent group to which the oligopeptide is bound.

As the fluorescent group, those in which the fluorescence wavelength and the fluorescence intensity change before and after the peptide bond with the oligopeptide is cleaved by the enzyme are used. In particular, a fluorescent group that emits fluorescence in the specific wavelength region when the peptide bond is bonded to the peptide chain and is a non-fluorescent substance in the specific wavelength region when the peptide bond is cleaved to release the peptide chain, for example, 4-Methylcoumaryl-7-amide (MCA), 7-amino-4-carboxymethylcoumarin (ACC), p-nitroanilide, α-naphthylamide, α-naphthyl ester and the like are preferably used.
As the amino acid residue located at the binding site, one in which the peptide bond on the C-terminal side is selectively cleaved by an enzyme is used. This is because it is possible to change the fluorescence intensity and the fluorescence frequency of the fluorescent group by releasing the binding portion that has been bonded to the fluorescent group.
In addition, when the oligopeptide is formed with a peptide chain having a predetermined length (for example, about 15 mm) or more, the substrate specificity for each amino acid is not high, but rather, elastase required for the action of cleaving a relatively long peptide chain. Such enzymes can be detected, and the types of enzymes that can be detected can be increased.

  The amino acid sequence is composed of, for example, peptide chains having amino acids of A (alanine), B (proline), C (lysine), and D (phenylalanine) as elements, and the enzyme activity detection unit has a peptide in the sample solution channel. When arranged in the order of the rule combination of each element of the chain, analysis and evaluation of test data can be performed easily and quickly based on basic data accumulated for basic amino acids. In addition, the biochip can be mechanically constructed using known peptide chain synthesis means so that oligopeptides composed of basic amino acids are regularly arranged in descending or ascending order such as the character code order of A to D. Excellent production efficiency.

The biochip according to claim 3 is the invention according to claim 1 or 2, wherein the fluorescent group is an aminomethylcoumarin fluorescent group and is chemically modified with a silane coupling agent or the like. Composed and configured.
With this configuration, in addition to the operation described in claim 1 or 2, the following operation is provided.
(1) Since the fluorescent group is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the fluorescent group to the chip substrate is an action of an enzyme or the like in the sample solution. Therefore, the biochip is not easily cut and the stability and durability of the biochip can be improved.
(2) Since an aminomethylcoumarin-based fluorescent group is used, it is possible to construct an enzyme activity detection unit having excellent selectivity by effectively utilizing its excellent fluorescence characteristics that differ depending on the binding state with each oligopeptide.

Here, as the silane coupling agent, for example, aminopropyltrimethoxysilane can be applied.
Examples of the aminomethylcoumarin-based fluorescent group include 4-methylcoumaryl-7-amide (MCA), 7-amino-4-carboxymethylcoumarin (ACC), and the like.

The biochip according to claim 4 is the image according to any one of claims 1 to 3, wherein the chip substrate is translucent, and a CCD, a CMOS element, or the like is arranged on the back side thereof. A sensor substrate is laminated.
With this configuration, in addition to the operation described in any one of claims 1 to 3, the following operation is provided.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide, the enzyme activity detectors are arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.

  As the image sensor substrate, it is possible to apply a CCD or CMOS element formed by providing a photosensitive portion in which photodiodes are two-dimensionally arranged on a silicon thin plate and a signal output portion thereof by a known semiconductor manufacturing technique.

The biochip according to claim 5 is the invention according to any one of claims 1 to 4, wherein the biochip is provided with a protective layer on a surface side of the chip substrate on which the sample solution flow path is formed. Yes.
With this configuration, in addition to the operation described in any one of claims 1 to 4, the following operation is provided.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, and measurement accuracy, reliability, and stability can be further enhanced.
(2) When the sample solution flow path is covered with a light-transmitting protective layer, excitation light can be irradiated from the protective layer side, and the fluorescence change of the fluorescent group can be measured from the outside. In addition, the biochip is excellent in handling and storage properties after the sample solution is introduced into the sample solution flow path.

For example, the protective layer can be configured by attaching a ceramic coating formed of silicon oxide, aluminum oxide, or the like on the chip substrate on which the sample solution flow path is formed.
A filter that passes the excitation wavelength of the fluorescent group can also be coated as a protective layer. Thereby, light is irradiated from the surface side where the sample solution flow path is formed, and the enzyme activity is detected by arranging an image sensor such as a CCD at a position where fluorescence of the enzyme activity detection unit can be detected. be able to.

The method for testing the functionality of the sample solution according to claim 6 supplies the sample solution to the liquid storage part of the sample solution flow path of the biochip according to any one of claims 1 to 5 under a predetermined condition. A sample solution supply step and a two-dimensional image on the chip substrate formed by a state change for each of the enzyme activity detection units arranged on the sample solution flow path are processed with a filter that removes the specific wavelength region, and A data acquisition step of acquiring series data or result data after a predetermined time, and comparing each of the time series data or result data with library data stored in advance under the same measurement conditions, by pattern recognition means and statistical data processing means And a data determination step for determining a pattern matching degree with the library data.
This configuration has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Since there is a data judgment step for comparing the time series data or result data obtained in this way with the library data and calculating the degree of pattern matching with each library data, a small amount of data can be obtained without requiring accumulation of basic data. The enzyme activity of the sample solution can be measured in a short time using this sample solution, and the operability and efficiency are excellent.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.

According to invention of Claim 1, the following effects are acquired.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow paths formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.

According to invention of Claim 2, in addition to the effect of Claim 1, it has the following effects.
(1) Since it has a fluorescent group bound to the chip substrate and an oligopeptide bound to the fluorescent group by a peptide bond that is cleaved by an enzyme, the oligopeptide is released when the peptide bond is cleaved by reacting with the enzyme Is done. Since the fluorescence wavelength of the fluorescent group released from the oligopeptide or the fluorescence intensity at a predetermined wavelength is different from that before the release, the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
(2) Since the fluorescent group is bonded in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. The activity can be detected, and the degree of integration of the enzyme activity detector that can detect the enzyme activity can be dramatically increased.
(3) Since the enzyme activity can be detected simply by using a sample solution sufficient to fill the sample solution flow path, a large amount of sample solution is not required for measurement, and the enzyme activity can be detected even with a small amount of sample solution. It can be carried out.
(4) By contacting the enzyme in the sample solution flowing through the sample solution flow path with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
(5) Since the enzyme specificity for a specific enzyme and the like is determined by the respective amino acid sequences constituting the oligopeptide, two possible combinations of these amino acid sequences are two-dimensionally arranged in a sample solution channel on the chip substrate in a certain order. As a result, the same fluorescent pattern array image can always be obtained for the same sample solution. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
(6) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
(7) Since knowledge of enzyme activity for a specific enzyme or protein is not required in advance for all such amino acid sequences, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.

According to invention of Claim 3, in addition to the effect of Claim 1 or 2, it has the following effects.
(1) Since the fluorescent group is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the fluorescent group to the chip substrate is an action of an enzyme or the like in the sample solution. Therefore, the biochip is not easily cut and the stability and durability of the biochip can be improved.
(2) Since an aminomethylcoumarin-based fluorescent group is used, it is possible to construct an enzyme activity detection unit having excellent selectivity by effectively utilizing its excellent fluorescence characteristics that differ depending on the binding state with each oligopeptide.

According to invention of Claim 4, in addition to the effect of any one of Claims 1 thru | or 3, it has the following effects.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide, the enzyme activity detectors are arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.

According to invention of Claim 5, in addition to the effect of any one of Claims 1 thru | or 4, it has the following effects.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, and measurement accuracy, reliability, and stability can be further enhanced.
(2) When the sample solution flow path is covered with a light-transmitting protective layer, excitation light can be irradiated from the protective layer side, and the fluorescence change of the fluorescent group can be measured from the outside. In addition, the biochip is excellent in handling and storage properties after the sample solution is introduced into the sample solution flow path.

According to invention of Claim 6, it has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Since there is a data judgment step for comparing the time series data or result data obtained in this way with the library data and calculating the degree of pattern matching with each library data, a small amount of data can be obtained without requiring accumulation of basic data. The enzyme activity of the sample solution can be measured in a short time using this sample solution, and the operability and efficiency are excellent.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.

Schematic diagram showing the enzyme activity detection principle of the enzyme activity detector Schematic diagram of enzyme activity detector with different arrangement Schematic perspective view of the biochip in the first embodiment Schematic diagram showing the state of the enzyme activity detectors arranged in the sample solution flow path Configuration diagram for measuring enzyme activity of sample solution introduced into biochip in embodiment 1 using image sensor Schematic diagram of biochip in embodiment 2 Schematic diagram showing a modification of the biochip in the second embodiment Graph plotting fluorescence intensity for each amino acid combination

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chip substrate 2 Fluorescent group 3 Oligopeptide 4 Enzyme 5 Fluorescent group in which the fluorescence wavelength was changed 6 Oligopeptide 7 First fluorescent group 7a First fluorescent group in which fluorescence is observed 8 Second oligopeptide 9 Second fluorescent group 10 Biochip 11 Chip substrate 12 Sample solution flow path 13 Enzyme activity detection unit 14 Liquid supply unit 15 Liquid storage unit 20 Image sensor 21 Lens 22, 23 Filter 24 Image sensor 30 Biochip 31 Chip substrate 32 Sample solution Flow path 32a Enzyme activity detection unit 33 Upper filter 34 Image sensor substrate 35 Lower filter 36 Silicon oxide layer

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic diagram showing an enzyme activity detection principle of an enzyme activity detection unit in a biochip according to an embodiment of the present invention.
In the figure, 1 is a chip substrate having a portion (sample solution flow path) that is mechanically or chemically formed into a flat plate shape with silicon oxide or glass, and 2 is directly bonded to the chip substrate 1 by a peptide bond or the like. Fluorescent group such as 4-methylcoumaryl-7-amide (MCA), 3 is an amino peptide bonded to fluorescent group 2 by peptide bond, oligopeptide such as peptide, 4 selectively binds fluorescent group 2 to oligopeptide 3 An enzyme having substrate specificity, such as serine protease to be cleaved, 5 is a fluorescent group such as 7-amino-methylcoumarin (AMC) whose fluorescence wavelength has been changed by selective release of oligopeptide 3 by enzyme 4. is there. The enzyme activity detection unit configured as described above is synthesized by using a known peptide synthesis technique and is bonded onto the chip substrate 1.

Hereinafter, the principle of detecting enzyme activity will be described with reference to FIG.
The fluorescent group 2 such as 4-methylcoumaryl-7-amide (MCA) shown in FIG. 1A is a non-fluorescent substance in a specific wavelength region and does not exhibit fluorescence. When the sample solution containing the enzyme 4 is brought into contact with the chip substrate 1 and reacted, the enzyme 4 having substrate specificity cleaves the peptide bond between the fluorescent group 2 and the oligopeptide 3 (see FIG. 1B). ).
The fluorescent group 5 from which the oligopeptide 3 is released becomes a fluorescent substance in the specific wavelength region such as 7-amino-methylcoumarin (AMC), and the fluorescence wavelength or the fluorescence intensity at a predetermined wavelength is a fluorescent group that is peptide-bonded to the oligopeptide 3 Therefore, the enzyme activity can be detected using a change in fluorescence intensity or the like as an index (see FIG. 1 (c)).

  Although the case where the fluorescent group 2 is directly bonded to the chip substrate 1 has been described here, the fluorescent group 2 may be bonded to the chip substrate 1 via an amino acid, a peptide, or the like. As a result, the distance between the chip substrate 1 and the enzyme action point can be optimized and the enzyme activity can be detected more accurately without being affected by the chip substrate 1. In this case, amino acids, peptides and the like between the fluorescent group 2 and the chip substrate 1 are synthesized with molecules or compounds having no enzyme specificity. This is to prevent the fluorescent group 2 from being released from the chip substrate 1 by the action of the enzyme.

FIG. 2 is a schematic diagram of an enzyme activity detection unit having an arrangement configuration different from that in FIG.
In FIG. 2, 6 is a first oligopeptide having one end immobilized on the chip substrate 1, 7 is a first fluorescent group introduced into the side chain of the oligopeptide 6, 8 is a peptide bond and the first oligopeptide A second oligopeptide bound to peptide 6, 9 is a second fluorescent group that binds to the second oligopeptide and causes fluorescence resonance energy transfer with the first fluorescent group 7, and 7a is originally observed by fluorescence resonance energy transfer. This is the first fluorescent group in which the fluorescence of the second fluorescent group 9 that should be attenuated is attenuated and fluorescence is observed instead.
Here, the fluorescence resonance energy transfer means that when two fluorescent compounds are present at positions close to each other, the fluorescence spectrum of one of the two fluorescent compounds (referred to as a donor) and the excitation of the other (referred to as an acceptor). When there is an overlap with the spectrum, this is a phenomenon in which the donor fluorescence that should be observed is attenuated when the excitation wavelength energy of the donor is applied, and the acceptor fluorescence is observed instead.
In this enzyme activity detection unit, the second oligopeptide is released by the enzyme 4 and the two fluorescent compounds are located far away from each other. Therefore, the second fluorescent group 9 that should originally be observed is detected. The fluorescence is attenuated, and the fluorescence of the first fluorescent group 7 is observed instead (7a), and the enzyme activity can be detected.

Here, as the first fluorescent group 7 and the second fluorescent group 9, a combination of a donor and an acceptor in which fluorescence resonance energy transfer occurs can be used. For example, the second fluorescent group 9 (or the first fluorescent group 7) which is an atomic group having an absorption band in a wavelength region overlapping with the fluorescent wavelength of the first fluorescent group 7 (or the second fluorescent group 9) is used. It is done. Specifically, the combination of (7-methoxycoumarin-4-yl) acetyl (MOAc), anthraniloylbenzyl (ABz), N-methylanthranilic acid (Nma) and the like and dinitrophenyl (Dnp), Dabsyl and EDANS ( 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid), tryptophan (Trp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns), carboxydichlorofluorescein (CDCF) and carboxymethyl A combination of rhodamine (CTMR), a combination of carboxydichlorofluorescein (CDCF) and carboxy X-rhodamine (CXR), a combination of lucifer yellow (LY) and carboxymethyl rhodamine (CTMR), and the like are used.
Any of these donors and acceptors may be the first fluorescent group 7 or the second fluorescent group 9. This is because if a spectral change occurs in the first fluorescent group 7, it can be used as a measurement index of enzyme activity.
In addition, between the coupling | bond part of the 1st fluorescence group 7 and the 2nd fluorescence group 9 which were each couple | bonded with the 1st fluorescence group 7 and the 2nd oligopeptide 8 which were introduce | transduced into the side chain of the 1st oligopeptide 6 The length is desirably 100 mm or less. This is because as the distance becomes longer, the fluorescence resonance energy transfer becomes smaller and the change in the fluorescence intensity or the like tends to become smaller.
By configuring the enzyme activity detection unit in this way, it is possible to easily detect an enzyme such as elastase which has a low substrate specificity for each amino acid and rather requires a relatively long peptide chain for its cleaving action. Detection sensitivity can be increased.

(Embodiment 1)
FIG. 3 is a schematic perspective view of the biochip according to the first embodiment of the present invention to which the above-described enzyme activity detection principle is applied, and FIG. 4 is a schematic diagram showing the state of the enzyme activity detection unit arranged in the sample solution flow path. FIG.
In FIG. 3, 10 is a biochip according to the first embodiment of the present invention, 11 is a chip substrate constituting the biochip 10 and formed in a substantially rectangular plate shape, and 12 is a chip that is spirally formed as a whole. A sample solution flow path 13 formed in the shape of a groove on the substrate 11 is formed in a cell shape and a spot shape in the sample solution flow path 12, and each has a predetermined enzyme activity and is arranged in a predetermined order. , 14 is a substantially rectangular liquid supply portion provided on the outer end side of the sample solution flow path 12 to which the sample solution is supplied, and 15 is substantially circular on the spiral center side which is the discharge side of the sample solution flow path 12 It is the liquid storage part provided.
The biochip 10 as a whole is formed on a chip substrate 11 made of glass or polystyrene resin having a substantially rectangular plate shape (length of about 20 to 30 mm on one side and thickness of about 2 mm), a flow path width of about 0.5 mm, and a depth. A sample solution channel 12 having a channel cross section of about 0.1 mm and an effective channel length of about 90 mm and formed in a spiral shape with a channel interval of 0.5 mm is provided. The liquid supply unit 14 is formed in a substantially square shape with a side of about 2.5 mm, and the effective volume of the sample solution channel 12 is about 4.5 μL.
A sample solution such as a blood sample collected in the liquid supply unit 14 on the chip substrate 11 is supplied, and sequentially moves through the sample solution flow path 12 by capillary action or the like to come into contact with the enzyme activity detection unit 13 to be in the sample solution. Characteristics such as the activity and antibody reactivity of each enzyme contained in are detected.

The enzyme activity detection unit 13 is formed as a cell-like, dent-like or flat spot-like site at the bottom of the sample solution flow path 12 formed in a spiral shape on the chip substrate 11 and has different enzyme activities. It is configured to be sequentially arranged at a predetermined interval.
As shown in FIGS. 3 and 4, the enzyme activity detector 13 includes a fluorescent group K whose one end is coupled to the bottom surface of the sample solution channel 12 formed in a substantially groove shape, and the other end of the fluorescent group K. It is comprised with the oligopeptide which consists of an amino acid sequence couple | bonded and arrange | positioned, for example at 4 sites of AD.
The fluorescent group K is, for example, an aminomethylcoumarin fluorescent group, and has different fluorescent characteristics depending on the binding state with each oligopeptide. If necessary, the chip substrate 11 is chemically modified with a silane coupling agent or the like so that the fluorescent group K is bonded into the sample solution flow path 12 of the chip substrate 11 so that the chip substrate 11 is bonded. Stability and durability can be increased.

For example, a peptide chain composed of any combination of ala: alanine, pro: proline, lys: lysine, and phe: phenylalanine is set at each site A to D of the oligopeptide conjugated to the fluorescent group K. I have to.
Examples of oligopeptides include (1) ala-pro-lys-phe, (2) pro-lys-phe-ala, (3) lys-phe-ala-pro, and (4) phe-ala-pro-lys. The amino acid sequence is set so that the enzyme specificity of the binding part of the fluorescent group to which the oligopeptide is bound is defined corresponding to each amino acid sequence.

Each amino acid sequence can be synthesized by a normal peptide synthesis method such as a solid phase method in which the peptide is extended from the C-terminus. Also, use a sequential extension method that sequentially extends from the C-terminal side to the N-terminal side of the target amino acid sequence, or a fragment condensation method that synthesizes a plurality of short peptide fragments and extends them by coupling between peptide fragments. Can do. Moreover, 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid, t-butyloxycarbonyl (Boc) amino acid, etc. can also be synthesize | combined using a well-known peptide synthesizer. Furthermore, a peptide bond can be generated using a protease, or it can be synthesized using a genetic engineering technique.
Furthermore, as a condensation method for forming a peptide bond, for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, DCC-additive method, active ester method, carbonyldiimidazole method , A redox method, a method using Woodward reagent K, and the like are used. In addition, before the condensation reaction, the carboxyl group and amino group which are not involved in the reaction in amino acids and peptides can be protected by known means, or the carboxyl group and amino group involved in the reaction can be activated. It can also be left.

Thus, as shown in FIG. 4, the S1-Sn enzyme activity detectors 13 each having a possible combination of amino acid sequences are arranged at the bottom of the sample solution channel 12 as follows, for example.
S1: Fluorescent group-oligopeptide (1) (ala-pro-lys-phe)
S2: fluorescent group-oligopeptide (2) (pro-lys-phe-ala)
S3: fluorescent group-oligopeptide (3) (lys-phe-ala-pro)
S4: fluorescent group-oligopeptide (4) (phe-ala-pro-lys)
・
・
Sn: fluorescent group-oligopeptide (n)
In this way, by flowing the sample solution through the sample solution flow path 12 in which the enzyme activity detection units of S1 to Sn are arranged and contacting with the enzyme activity detection unit 13, the enzymes and proteins in the sample solution have different enzyme specificities. Acts on the bond between the oligopeptide to which is attached and the fluorescent group, thereby cleaving this bond. By optically detecting the fluorescence characteristic change of the fluorescent group accompanying this cutting, the sample solution flow path 12 formed in a spiral shape can be expressed as a fluorescence pattern unique to the sample solution.
In addition, the amino acid sequence of the oligopeptide in S1 to Sn does not need to be known in terms of characteristics and behavior such as enzyme activity for each amino acid sequence, and may be of a specific unique sequence as the biochip 10. For this reason, S1-Sn includes, for example, a regular array or a random array including a periodic pattern.

The sample solution channel 12 is formed by etching or the like when the chip substrate 11 is made of silicon oxide or glass. Further, when the chip substrate 11 is made of synthetic resin or the like, it is formed by embossing, laser beam processing, grinding using a machining center, or the like. It can also be manufactured by injection molding or press molding.
The depth of the sample solution flow path 12 formed in a groove shape is preferably about 10 to 200 μm, and the width is preferably in the range of 10 to 1000 μm. If the depth and width of the flow path become smaller than the lower limit values, the flow path will be blocked due to the viscosity of the sample solution, or the measurement sensitivity will be insufficient. This is because when the size is larger, the degree of integration of the enzyme activity detection unit on the chip substrate becomes insufficient, and it becomes difficult to specify the fluorescent pattern to be formed on the chip substrate.

  The liquid supply unit 14 may be provided on the side of the chip substrate 11 and may be provided with a filter that removes red blood cells in blood such as a nonwoven fabric filter and a membrane film. As a result, red blood cells, white blood cells, and the like in the supplied sample solution can be filtered to adjust the viscosity of the sample solution, so that the flow path is blocked by adhering to the wall of the sample solution flow path 12. Can be effectively prevented.

Next, a method for testing the functionality of the sample solution applied to the biochip 10 configured as described above will be described with reference to a schematic operation diagram for a specific enzyme of the enzyme activity detector shown in FIG.
First, a biochip 10 is prepared in which enzyme activity detectors 13 each having a unique enzyme activity are arranged in a predetermined pattern at each site S1 to Sn of a sample solution flow path 12 formed in a spiral shape on a chip substrate 11. In the sample solution supply step, a sample solution such as blood is supplied to the liquid supply unit 14 of the biochip 10 at a predetermined flow rate and temperature. As a result, the blood component or the like supplied to the liquid supply unit 14 is supplied to the sample solution flow path 12 and sequentially brought into contact with the enzyme activity detection unit 13 at each of the sites S1 to Sn using capillary action or the like. it can.
Thus, as shown in the figure, the binding part between the oligopeptide having sensitivity to a specific enzyme in the blood component and the fluorescent group is cleaved, and the fluorescence characteristic of the fluorescent group is in a state before cleaving (here, α). ) To the state after cutting (here, β). As a result, the state of the fluorescent group at the sites S1 to Sn along the sample solution flow path 12 is changed from the initial state X (α, α, α, α, α,...), For example, to the detection state Y ( .alpha., .beta., .alpha., .alpha., .beta.

  In the next data acquisition step, a two-dimensional fluorescence image on the chip substrate 11 formed by the fluorescence change in each enzyme activity detection unit 13 on the sample solution flow path 12 is acquired by an image sensor or the like, and the specific wavelength range is obtained. The time series data or the result data after a predetermined time can be obtained by processing with a filter that removes.

  In the data determination step, the time series data or the result data after a predetermined time is compared with library data measured and accumulated for various samples in advance under the same measurement conditions, and each library is detected by a pattern recognition unit or a statistical determination unit. The degree of pattern matching with data can be determined, and various pathological findings can be obtained quickly and accurately.

As described above, in the sample solution functionality testing method using the biochip 10, the sample solution flow path arrayed with oligopeptides that impart enzyme specificity with the sample solution containing blood, urine, etc. as the test target Lead to react. The result of the enzyme reaction can be acquired by an image sensor that detects fluorescence intensity, and the acquired data can be compared with library data to comprehensively detect the activity of the protease mixture contained in the sample solution. Further, since the sample solution flow path 12 is formed in a spiral shape, the speed of the sample solution moving in the sample solution flow path 12 due to capillary action or the like can be made substantially constant, and the reproducibility of the acquired time series data is excellent. Further, since it continues without branching, the length of the path from the liquid supply unit 14 in the sample liquid channel 12 and the fluorescence intensity can be expressed in a one-to-one relationship, and comparison with the library data is facilitated. be able to.
In this way, comprehensive detection of proteases provides biological information on homeostasis, which is a process of secretion, production, and degradation of peptide hormones, which can be used efficiently for personal health care. .
In this way, the functional test method for sample solutions using biochips is applied to medical fields such as pathological diagnosis and research fields such as proteome analysis, so that the activity of multiple enzymes can be increased at high speed and in small quantities. Can be measured with a sample.

Next, a specific configuration for measuring the enzyme activity of the sample solution introduced into the biochip using an image sensor will be described.
FIG. 5 is a configuration diagram when the enzyme activity of the sample solution introduced into the biochip in Embodiment 1 is measured using an image sensor.
In FIG. 5, reference numeral 20 denotes an image sensor which is arranged on the biochip 10 and acquires two-dimensional data in the enzyme activity detection unit 13 on the chip substrate 11 via the lens 21. As described above, in the configuration in which the image sensor 20 is placed away from the chip substrate 11, the state change of the fluorescent group of the enzyme activity detection unit 13 forms an image on the image sensor 20 through the optical system such as the lens 21. Read.

As described above, since the biochip in the first embodiment is configured, only a very small amount of sample solution containing an enzyme is introduced into the sample solution channel 12 of the chip substrate 11 and the fluorescence intensity of the chip substrate 11 is measured. To detect enzyme activity. Furthermore, since it is not necessary to form a cell or the like for injecting the solution into the substrate for each individual sample, the enzyme activity detection unit 13 and the sample solution flow channel 12 can be miniaturized, so that the integration degree of the enzyme detection unit on the chip substrate 11 can be increased. It can be improved dramatically.
In addition, since enzyme activity can be detected simply by using a very small amount of sample solution containing an enzyme, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. Can do.

(Embodiment 2)
FIG. 6 is a schematic diagram of the biochip in Embodiment 2 of the present invention when the enzyme activity of the sample solution introduced into the biochip is measured by arranging the chip substrate directly on the image sensor.
In FIG. 6, reference numeral 22 denotes a filter as a light-transmitting protective layer disposed on the surface of the chip substrate 11 in which the enzyme activity detection unit 13 is arranged in the sample solution flow path 12. The selectivity is such that the excitation wavelength is allowed to pass but the fluorescence wavelength is blocked. Reference numeral 23 denotes a filter disposed on the back surface of the chip substrate 11 and has a selectivity that allows the fluorescence wavelength of the enzyme activity detector 13 to pass but blocks the excitation wavelength. Reference numeral 24 denotes an image sensor such as a CCD disposed on the back side of the chip substrate 11 via a filter 23.
In the present embodiment, the chip substrate 11 is made of translucent glass or the like.
As a result, the biochip in the second embodiment can be made compact without requiring an optical system such as a lens, because the fluorescence of the enzyme activity detector 13 can be read as it is with the detection element of the image sensor 24 arranged in close contact. be able to.

FIG. 7 is a schematic diagram showing a modification of the biochip in which the image sensors in Embodiment 2 are stacked.
In FIG. 7, 30 is a biochip according to a modification of the second embodiment, 31 is a translucent chip substrate made of silicon oxide, and 32 is a sample solution channel formed in the chip substrate 31 by etching or the like in a groove shape. 32a is an enzyme activity detector arranged in the sample solution flow path 32, and 33 is an upper filter made of glass or organic material that is disposed so as to cover the opening surface of the sample solution flow path 32 and transmits fluorescence of a predetermined wavelength. , 34 is an image sensor substrate in which CMOS elements and the like are stacked on the back side of the chip substrate 31 via a lower filter 35, and 36 is a silicon oxide layer that protects the surface of the image sensor substrate 34. When the CMOS element or the like of the sensor substrate 34 is formed, the insulating layer (protective layer) is formed by means such as CVD.
The upper filter 33 has such selectivity that the excitation wavelength of the enzyme activity detector 32a is allowed to pass but the fluorescence wavelength is blocked, and the lower filter 35 is excited to pass the fluorescence wavelength of the enzyme activity detector 32a. The wavelength has such selectivity that it blocks.
The chip substrate 31 is formed by CVD or the like, similar to a silicon oxide layer formed by means such as CVD as an insulating layer (protective layer) when forming a CMOS element or the like of the image sensor substrate 34.
In the biochip 30 according to the present embodiment configured as described above, the enzyme activity detection unit 32a of the chip substrate 31 is provided on the projection surface of the lower filter 35 disposed directly on the silicon oxide layer 36 of the image sensor substrate 34. Since the chip substrate 31 is manufactured by using the same semiconductor manufacturing technology as that for forming the CMOS element or the like of the image sensor substrate 34, the manufacturing process can be shortened. .

Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples.
For the amino acids, peptides, protecting groups, solvents, and the like described in the present Examples, abbreviations commonly used in the technical field or abbreviations adopted by the IUPAC-IUB naming committee are used.
For example, the following abbreviations are used. Ala: alanine, Pro: proline, Lys: lysine, Phe: phenylalanine, Aca: aminocaproic acid, Ac: acetyl, ACC: 7-amino-4-carboxymethylcoumarin, Boc: t-butyloxycarbonyl, Lys (Boc): Side chain t-butyloxycarbonyl protected lysine, DCC: dicyclohexylcarbodiimide, DCM: dichloromethane, DIEA: N, N-diisopropylethylamine, DMF: N, N-dimethylformamide, EtOH: ethanol, Fmoc: 9-fluorenylmethyloxy Carbonyl, HATU: o- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, HBTU: o- (benzotriazol-1-yl) -1,1 , 3,3-tetramethyluronium hexafluorophosphate, HOAt: 1-hydroxy-7-azoben Triazole, HOBt: 1-hydroxybenzotriazole, TFA: trifluoroacetic acid.

In Example 1, as a preliminary experiment for forming an enzyme activity detection unit, a peptidyl fluorescent group-bound spherical substrate was synthesized as an enzyme activity detection substrate, and the activity on enzymes (trypsin and chymotrypsin) was measured. The method will be described below.
In addition, a biochip can be configured by filling and arranging such a spherical substrate in each region to be the enzyme activity detection units S1 to Sn of the sample solution flow path formed in a spiral shape or the like.
<Synthesis of peptidyl fluorescent group-bonded spherical substrate>
As a substrate, spherical NH ₂ -PEGA-resin (manufactured by Watanabe Chemical Industry) was used. The vessel for solid phase synthesis was fixed vertically, NH ₂ -PEGA-resin (0.05 mmol / g, 0.5 g) was added, DMF (10 ml) was allowed to flow with the vessel cock open, and the solvent was replaced. Then close the vessel cock and add Fmoc-Aca-OH (0.13 mmol, 44 mg), HBTU (0.13 mmol, 48 mg), DIEA (0.13 mmol, 0.022 ml) dissolved in DMF (2 ml), Reacted overnight. After the reaction, the cock was opened and washed with DMF (10 ml) and methanol (10 ml) to obtain Fmoc-Aca-PEGA resin.

Next, DMF (10 ml) was allowed to flow through the Fmoc-Aca-PEGA resin (0.05 mmol / g, 0.5 g) in the vessel with the cock open, and solvent substitution and washing were performed. The cock was closed and 20% piperidine / DMF was added and reacted for 30 minutes to remove Fmoc. The cock was then opened to remove 20% piperidine / DMF and then washed with DMF (10 ml). Next, with the cock closed, Fmoc-Aca-OH (3 eq), HATU (3 eq), HOAt (3 eq), and DIEA (5 eq) were dissolved in DMF (2 ml) and allowed to react for 3 hours. It was. The cock was then opened and washed with DMF (10 ml) and methanol (10 ml) to obtain Fmoc-Aca-Aca-PEGA resin in which the binding part (Aca-Aca-) was bound to the substrate (PEGA resin). .
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 mL). After washing three times with DCM (1 ml) and once with DMF (1 ml), Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol), DCC (11 mg , 54 mmol), HOBt · H 2 O (8.3 mg, 54 mmol) was added and allowed to react for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give a fluorescent group. Fmoc-Lys (Boc) -ACC-Aca-Aca-PEGA resin in which (ACC) was bound to the binding site (Aca-Aca-) was obtained.
Then, it was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml) and stirred for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to acetylate the unreacted product.

Subsequently, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Pro-OH (18 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin.
Hereinafter, Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin was used to repeat the same procedure using Fmoc-Ala-OH, and the peptide was extended to attach the binding moiety (Ala-Ala -Pro-Lys) bound to Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin. Then, the cock was closed and DIEA (2.5 mmol, 0.435 ml) and acetic anhydride (1.25 mmol, 0.117 ml) were diluted in DMF (2 ml) and reacted for 1 hour to acetylate the terminal group of the bond. Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin was obtained.
Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin (0.05 mmol, 0.5 mg) was placed in the vessel, and DCM (10 ml) was flowed with the cock open to replace the solvent. Then, close the cock and add 25% TFA / DCM (2 ml), react for 30 minutes, remove Boc, wash with DCM (10 ml), H2O (10 ml) The peptidyl fluorescent group-bonded spherical substrate (Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin) of Example 1 was obtained.

<Measurement of enzyme activity>
The peptidyl fluorescent group-bound spherical substrate Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin as the enzyme activity detection substrate of Example 1 was added to wells A to D of a 96-well fluorescence measurement microplate. The following solutions were added, and the difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes was measured. The fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVO ™ SX 1420 multilabel counter (manufactured by PerkinElmer).
A: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 240 μl, trypsin (1 mg / 1 ml) to 10 μl
B: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 240 μl, chymotrypsin (1 mg / 1 ml) to 10 μl
C: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 250 μl
D: 250 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2),
The difference between well A and well B is the type of enzyme, and the difference between well A (or well B) and well C is the presence or absence of an enzyme. Well A (or well B, C) and well The difference from D is the presence or absence of a substrate for enzyme activity detection.
The difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes is shown in Table 1 for each well.

Comparing the fluorescence values of wells A and B and well C in Table 1, the enzyme activity detection substrate of Example 1 was about 315 times in well A where trypsin was present, and about 315 times in well B where chymotrypsin was present. It was confirmed that the difference was 10 times. Thus, it was shown that the enzyme activity corresponding to the amount of enzyme, the type of enzyme, and the like can be detected by applying the enzyme activity detection substrate of Example 1. Further, comparing the fluorescence values of well A and well B, the enzyme activity detection substrate of Example 1 has a fluorescence value in the case of trypsin that is approximately 30 times or more larger than that in the case of chymotrypsin. confirmed. This is because the amino acid of the binding part that binds to the fluorescent group of the enzyme activity detection substrate of Example 1 is lysine, and trypsin has the specificity of selectively cleaving the peptide bond mainly on the C-terminal side of lysine. It is guessed that it was expressed from On the other hand, chymotrypsin mainly has the specificity of selectively cleaving the peptide bond on the C-terminal side of the aromatic amino acid residue, so it is presumed that the change in fluorescence value before and after the reaction was small.
Accordingly, it was shown that the enzyme activity detection substrate of Example 1 has specificity depending on the enzyme, so that qualitative analysis of the enzyme having activity is possible. In addition, if a sample solution containing the same type of enzyme is brought into contact with the same type of enzyme activity detection substrate and the fluorescence value is measured after a predetermined time, the change in the fluorescence value is affected by the action of the enzyme. It was speculated that quantitative analysis of the enzyme was possible.

  In Example 2, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded planar substrate as a substrate for enzyme activity detection. The method will be described below. The enzyme activity detector can be formed by applying the synthesis method described below, and enzyme activity detectors having different enzyme specificities can be arranged in a predetermined pattern in each region of the sample solution flow path.

<Synthesis of a peptidyl fluorescent group-bonded planar substrate>
As the substrate, a synthetic resin carrier made by separating only one plate from a multi-plate synthetic resin carrier for peptide synthesis (Lantern series (registered trademark) manufactured by Mimotops Co., Ltd.) was used.
Put one synthetic resin carrier lantern (Mimotops D-series, introduction rate 18 mmol / piece) as a substrate in the screw tube and stir for 30 minutes with 20% piperidine / DCM (1 mL) to remove the Fmoc group. Went. After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Aca-OH (19 mg 54 mmol), DCC (17 mg 81 mmol), HOBt · H2O (8 mg 54 mmol) were DMF. It was dissolved in (1 ml) and added to react for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give Fmoc- Obtained Aca-lantern.
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Aca-OH (19 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Aca-Aca-lantern with one end of the binding site (Aca-Aca) immobilized on the substrate (lantern) It was.

Subsequently, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 mL). Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol), washed with DCM (1 ml) three times and DMF (1 ml) once to swell lantern and dissolved in DMF 1 ml, DCC (11 mg, 54 mmol) and HOBt · H2O (8.3 mg, 54 mmol) were added and reacted for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give a binding site. Fmoc-Lys (Boc) -ACC-Aca-Aca-lantern in which a fluorescent group (ACC) was bound to (Aca-Aca) was obtained.
Then, it was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml) and stirred for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to acetylate the unreacted product.
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Pro-OH (18 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern.

Hereinafter, Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern was repeated twice using Fmoc-Ala-OH to elongate the peptide, and bound to the fluorescent group (ACC) (Ala Fmoc-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-lantern to which -Ala-Pro-Lys) was bound was obtained.
Thereafter, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing with DMF (1 ml) 3 times with DCM (1 ml), DMF (1 mL), DIEA 32 ml (183 mmol) and acetic anhydride 8.5 ml (90 mmol) were added to DMF (1 mL). Diluted and added for 1 hour. Thereafter, the lantern was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then, wash 3 times with DCM (1 ml), 5 times with H2O (1 ml), 3 times with DCM (1 ml), dry under reduced pressure, and end group of the binding site (Ala-Ala-Pro-Lys) Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-lantern was obtained in which was acetylated.
The Boc group of Lys side chain was removed using 25% TFA / DCM (1 ml), and the target enzyme activity detection substrate of Example 2 (Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca- lantern).

<Measurement of enzyme activity>
100 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2), 100 μl of methanol, and 50 μl of trypsin (1 mg / 1 ml) were added to the enzyme activity detection substrate of Example 2 and reacted. The fluorescence values before and after the reaction were measured. The fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVO ™ SX 1420 multilabel counter (manufactured by PerkinElmer).
As a result, the initial fluorescence value was 39000, the fluorescence value after reaction for 18 hours was 145000, and it was confirmed that the fluorescence value changed about 4 times. Thereby, it was shown that the enzyme having activity can be detected even in the enzyme activity detection substrate of Example 2 using lantern as the substrate.

In Example 3, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for enzyme activity detection. The method will be described below.
<Synthesis of a peptidyl fluorescent group-bonded planar substrate>
As the substrate, a synthetic resin carrier made flat by separating only one plate from a multi-plate synthetic resin carrier for peptide synthesis (Lantern series (registered trademark) manufactured by Mimotopus Co., Ltd.) was used.
Put 1 lantern of synthetic resin carrier lantern (Mimotops D-series, introduction rate 18 mmol / piece) into the screw tube and stir for 30 minutes with 20% piperidine / DCM (1 mL) to cut out the Fmoc group. It was. Fmoc-Aca-OH 20.0 mg (56.6 mmol), DCC 17.5 mg (84.5 mmol), HOBt in which lantern was swollen with DMF (1 ml × 1) and dissolved in 1 ml of DMF after DCM washing (1 ml × 3) -H2O 8.8 mg (57.5 mmol) was added and it stirred for 23 hours. DMF (1 ml x 2), DCM (1 ml x 2), DCM / EtOH = 1: 1 (1 ml x 2), EtOH (1 ml x 2), DCM (1 ml x 1), diethyl ether (1 The resin was washed with ml × 1) and dried to obtain Fmoc-Aca-lantern.

Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to excise the Fmoc group, washed with DCM (1 ml × 3), and then 23.0 mg of Fmoc-Aca-OH dissolved in 1 ml of DMF ( 65.1 mmol), HATU 21.6 mg (56.8 mmol), HOAt 8.1 mg (59.5 mmol), and DIEA 15.7 ml (90.2 mmol) were added and stirred for 1 hour. The plate was washed with DMF (1 ml × 3) and DCM (1 ml × 3) to obtain Fmoc-Aca-Aca-lantern in which one end of the binding portion (Aca-Aca) was immobilized on the substrate (lantern). Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 ml × 3) to obtain H-Aca-Aca-lantern.
Subsequently, Fmoc-Phe-ACC-OH (33.4 mg 56.7 mmol), DCC 12.5 mg (60.6 mmol), HOBt * H2O 8.9 mg (58.0 mmol) dissolved in 1 ml of DMF were added and stirred for 22 hours. DMF (1 ml x 2), DCM (1 ml x 2), DCM / EtOH = 1: 1 (1 ml x 2), EtOH (1 ml x 2), DCM (1 ml x 1), diethyl ether (1 After washing with ml × 1), drying was performed to obtain Fmoc-Phe-ACC-Aca-Aca-Lantern in which the fluorescent group (ACC) was bound to the binding portion (Aca-Aca).

Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 ml × 3). Next, Fmoc-Pro-OH 24.7 mg (73.2 mmol), HATU 21.4 mg (56.2 mmol), HOAt 8.2 mg (60.2 mmol), DIEA 15.7 ml (90.2 mmol) dissolved in DMF 1 ml were added and stirred for 1 hour. After washing with DMF (1 ml × 3) and DCM (1 ml × 3), Fmoc-Pro-Phe-ACC-Aca-Aca-lantern was obtained.
Thereafter, Fmoc-Ala-OH was introduced twice by introducing Fmoc-Ala-Pro-Phe with the binding moiety (Ala-Ala-Pro-Phe) bound to the fluorescent group (ACC). -Obtained ACC-Aca-Aca-lantern.
Then, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, washed with DCM (1 ml × 3), DMF (1 mL), DIEA 32 ml (183 mmol) and anhydrous Acetic acid 8.5 ml (90 mmol) was added and allowed to stir for 1 hour. Thereafter, washing with DMF (1 ml × 3) and DCM (1 ml × 3) is performed, and the terminal group of the binding site (Ala-Ala-Pro-Phe) is acetylated to achieve the target enzyme activity of Example 3 A peptidyl fluorescent group-bonded planar substrate Ac-Ala-Ala-Pro-Phe-ACC-Aca-Aca-lantern was obtained as a detection substrate.

<Measurement of enzyme activity>
The substrate for enzyme activity detection of Example 2 (introduction rate 2 μmol / substrate) in wells A and C of the microplate for fluorescence measurement, and the substrate for enzyme activity detection in Example 3 (introduction rate 2 μmol / substrate) in wells B and D Each was added with 100 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl 2) and 100 μl of methanol, and further 50 μg of the following enzyme was added and reacted.
A: Chymotrypsin B: Chymotrypsin C: Trypsin D: Trypsin The measurement was performed using a WALLAC ARVOTM SX 1420 multilabel counter (manufactured by PerkinElmer) at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm. The fluorescence value after 30 minutes was measured and the difference was obtained.
The difference in the fluorescence value calculated in each well is shown in (Table 2).

  According to Table 2, for chymotrypsin, a large change in the fluorescence value was confirmed on the enzyme activity detection substrate of Example 3 in which the amino acid at the binding portion that binds to the fluorescent group was one kind of phenylalanine of the aromatic amino acid. It was. In addition, for trypsin, a large change in fluorescence value was confirmed on the enzyme activity detection substrate of Example 2 in which the amino acid at the binding site that binds to the fluorescent group was lysine. The difference between these changes is that chymotrypsin has the specificity of selectively cleaving peptide bonds mainly on the C-terminal side of aromatic amino acid residues, and trypsin mainly reduces peptide bonds on the C-terminal side of lysine. It is presumed that it was expressed because of its specificity for selective cleavage. As a result, it became clear that the activity detection ability for the type of enzyme can be changed by changing the type of amino acid at the binding site that binds to the fluorescent group.

0.3 g of Aca was introduced into 1 g of ordinary resin for peptide synthesis as in Example 1 and divided into 361 groups. ACC was introduced into each of these resins using a peptide synthesizer, then 19 kinds of amino acid residues except cysteine were introduced twice, and finally capping with an acetyl group was performed after deprotection. Thereby, 361 kinds of peptides were synthesized on the resin.
Peptides were cut out from each resin to obtain 361 types of C-terminal unprotected peptides.
On a glass substrate or a silicon oxide layer (chip substrate) constructed as a protective layer (insulating layer) on an image sensor, a spiral sample solution flow path having a flow path width of 500 μm and a flow path depth of 100 μm is formed by etching. Formed. In addition, as a sample supply portion, a concave portion having a depth of 100 μm and a vertical and horizontal length of 2.5 mm was continuously formed at one end portion of the sample solution flow channel by etching.
The entire surface of the sample solution flow channel was coated with amino groups, and 361 types of peptides as the enzyme activity detection unit were introduced into the sample solution flow channel using a condensing agent (HATU). For the introduction, an automatic spotter (model: 222XL) manufactured by Gilson was used, and one kind of peptide was introduced into a circle of about 200 μm at the center of a flow path width of 500 μm, and another kind of peptide was spaced at an interval of about 100 μm. Peptides were introduced one after the other.
A filter (which cuts off wavelengths longer than 350 ± 10 nm) that presses the cover (protective layer) and transmits fluorescence excitation light was pressure-bonded to the upper surface of the sample solution flow channel into which the peptide was introduced. A fluorescent chip (which cuts off wavelengths shorter than 450 ± 10 nm) was adhered to the image sensor substrate, and a chip substrate was further disposed thereon to manufacture a biochip similar to that described in the second embodiment.

As a test sample, a solution of enzyme group 1 centered on trypsin and enzyme group 2 centered on chymotrypsin (20 nM, 5 mM HEPES pH 7.4) was prepared. This was introduced into each of the two biochips using a microsyringe, and the output of the image sensor from the start of introduction was recorded.
The fluorescence intensity of each spot (enzyme activity detection unit) was calculated every 10 msec from the output of the image sensor, and the enzyme activity (fluorescence intensity) for each spot was calculated based on that.

FIG. 8 is a graph in which the fluorescence intensity is plotted for each amino acid constituting each spot. In FIG. 8, the x-axis and the y-axis indicate the combination sequences of the respective amino acids, and the z-axis indicates the fluorescence intensity corresponding to the combination of the amino acids.
From FIG. 8, it was confirmed that the fluorescence intensity patterns output from the biochips having the same enzyme activity detection unit array differ between enzyme group 1 and enzyme group 2. Thus, for example, by using human body fluid as a sample solution and detecting the enzyme activity as image data periodically using the biochip of the present invention, it is determined whether or not the enzyme activity in the body fluid has changed. It was confirmed that comprehensive monitoring was possible and pathological diagnosis and the like could be performed accurately and promptly.

  The biochip of the present invention is applied to the analysis of sample solutions containing enzymes, proteins, and genes related in the fields of pathology, medical care, drug discovery, food, and environment, and comprehensively includes characteristic data such as enzyme activity. Can be evaluated and specified, and can contribute to medical fields such as pathological diagnosis and research such as proteome analysis.

[0004]
[0006]
The biochip according to claim 1 of the present invention is a biochip for detecting an enzyme in a sample solution introduced into a chip substrate, and the entire chip has a flow path pattern such as a spiral shape, a tree shape, or a radial shape. A plurality of sample solution channels formed on the substrate, and a plurality of enzyme activity detectors arranged in a predetermined order in the sample solution channels and having different activities with respect to a predetermined enzyme, and the enzyme activity detector However, (a) the fluorescence characteristics of the fluorescence frequency and the fluorescence intensity vary depending on the state of chemical bonding with the peptide chain bonded to the chip substrate directly or via a molecule or compound having no enzyme specificity. And (b) a specific amino acid sequence consisting of a combination of amino acids, which is attached by the peptide chain and cleaved by the enzyme, and has an enzyme-specific binding site that binds to the fluorescent group. Configured to include an oligopeptide that is, a.
With this configuration, the following effects can be obtained.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow path formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.
(5) Since it has a fluorescent group bound to the chip substrate and an oligopeptide bound to the fluorescent group with a peptide bond that is cleaved by an enzyme, the oligopeptide is released when the peptide bond is cleaved by reacting with the enzyme. Is done. Since the fluorescence wavelength of the fluorescent group released from the oligopeptide or the fluorescence intensity at a predetermined wavelength is different from that before the release, the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
(6) Since the fluorescent group is bonded in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. The activity can be detected, and the degree of integration of the enzyme activity detection unit capable of detecting the enzyme activity can be dramatically increased, and the biochip can be downsized.
(7) Enzyme activity can be detected simply by using a sample solution that fills a very small volume of sample solution flow path, so that a large amount of sample solution is not required for measurement, and even a small amount of sample solution can be used for enzyme activity. Can be detected.
(8) By contacting the enzyme in the sample solution flowing through the sample solution flow path with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
(9) Since the enzyme specificity for a specific enzyme and the like is determined by the respective amino acid sequences constituting the oligopeptide, when possible combinations of these amino acid sequences are two-dimensionally arranged in the sample solution flow path on the chip substrate In the case of the same sample solution, images of the same fluorescent pattern arrangement are always obtained. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
(10) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
(11) Since knowledge of enzyme activity for a specific enzyme or protein for all such amino acid sequences is not required in advance, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.
[0007]
Here, as the sample solution, a body fluid or a culture solution containing blood or urine to be subjected to pathological diagnosis or the like can be applied. As the sample solution, a biological sample (blood or the like) can be used as it is, or a blood cell component or the like removed by a filter, centrifugation, or the like. In addition, those that have been subjected to condition settings (pH adjustment, activator introduction, etc.) that cause the enzyme to exhibit activity based on biological samples


4/2

[0007]
You may make it continuously arrange | position in a planar form along the bottom part.
In addition, the arrangement of each independent enzyme activity detection unit made of a peptide to which a fluorescent group is bound in the sample solution flow path is, for example, using a commercially available DNA chip plotter or using a syringe while looking at a microscope. Can be arranged. In addition, each predetermined region in the sample solution flow path formed in a spiral shape is filled with spherical particles such as polystyrene resin or flat plate with enzyme specificity on the surface thereof, and an independent enzyme activity detector is provided. It is also possible to arrange them.
[0011]


7

[0008]
[0012]
Here, an oligopeptide is a peptide composed of a peptide chain consisting of several amino acids, and has a substantially linear form unlike a protein such as a lump having a large molecular weight. The amino acid sequence of the oligopeptide can characterize the enzyme specificity in the binding moiety with the fluorescent group to which the oligopeptide is bound.
[0013]
As the fluorescent group, those in which the fluorescence wavelength and the fluorescence intensity change before and after the peptide bond with the oligopeptide is cleaved by the enzyme are used. In particular, a fluorescent group that is a non-fluorescent substance in a specific wavelength region when a peptide bond is formed with a peptide chain bond, and emits fluorescence in the specific wavelength region when the peptide bond is cleaved and the peptide chain is released, for example, 4-Methylcoumalyl-7-amide (MCA), 7-amino-4-cal


8

[0009]
Boxymethylcoumarin (ACC), p-nitroanilide, α-naphthylamide, α-naphthyl ester and the like are preferably used.
As the amino acid residue located at the binding site, one in which the peptide bond on the C-terminal side is selectively cleaved by an enzyme is used. This is because it is possible to change the fluorescence intensity and the fluorescence frequency of the fluorescent group by releasing the binding portion that has been bonded to the fluorescent group.
In addition, when the oligopeptide is formed with a peptide chain having a predetermined length (for example, about 15 mm) or more, the substrate specificity for each amino acid is not high, but rather, elastase required for the action of cleaving a relatively long peptide chain. Such enzymes can be detected, and the types of enzymes that can be detected can be increased.
[0014]
The amino acid sequence is composed of, for example, peptide chains having amino acids of A (alanine), B (proline), C (lysine), and D (phenylalanine) as elements, and the enzyme activity detection unit has a peptide in the sample solution channel. When arranged in the order of the rule combination of each element of the chain, analysis and evaluation of test data can be performed easily and quickly based on the basic data accumulated for basic amino acids. In addition, the biochip can be mechanically constructed using known peptide chain synthesis means so that oligopeptides composed of basic amino acids are regularly arranged in descending or ascending order such as the character code order of A to D. Excellent production efficiency.
[0015]
A biochip according to a second aspect is the biochip according to the first aspect, wherein the fluorescent group is an aminomethylcoumarin-based fluorescent group and is bonded onto the chip substrate chemically modified with a silane coupling agent or the like. Configured.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(1) Since the fluorescent group is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the fluorescent group to the chip substrate is an action of an enzyme or the like in the sample solution. Therefore, the biochip is not easily cut and the stability and durability of the biochip can be improved.
(2) Since an aminomethylcoumarin-based fluorescent group is used, it is possible to construct an enzyme activity detection unit having excellent selectivity by effectively utilizing its excellent fluorescence characteristics that differ depending on the binding state with each oligopeptide.
[0016]
Here, examples of the silane coupling agent include aminopropyltrimethoxysilane.


9/1

[0010]
Applicable.
Examples of the aminomethylcoumarin-based fluorescent group include 4-methylcoumaryl-7-amide (MCA), 7-amino-4-carboxymethylcoumarin (ACC), and the like.
[0017]
The biochip according to claim 3 is the invention according to claim 1 or 2, wherein the chip substrate is translucent, and an image sensor substrate in which CCDs, CMOS elements, and the like are arranged on the back side is laminated. It is configured.
With this configuration, in addition to the operation described in claim 1 or 2, the following operation is provided.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide soot, the enzyme activity detection unit is arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.
[0018]
As the image sensor substrate, it is possible to apply a CCD or CMOS element formed by providing a photosensitive portion in which photodiodes are two-dimensionally arranged on a silicon thin plate and a signal output portion thereof by a known semiconductor manufacturing technique.
[0019]
According to a fourth aspect of the present invention, there is provided the biochip according to any one of the first to third aspects, further comprising a protective layer on the surface of the chip substrate on which the sample solution flow path is formed. Yes.
With this configuration, in addition to the operation described in any one of claims 1 to 3, the following operation is provided.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, and measurement accuracy, reliability, and stability can be further enhanced.
(2) When the sample solution channel is covered with a translucent protective layer, excitation is performed from the protective layer side.


10

[0011]
In addition to being able to irradiate light and measuring the fluorescence change of the fluorescent group from the outside, it is excellent in handling and storage of the biochip after the sample solution is introduced into the sample solution flow path.
[0020]
For example, the protective layer can be configured by attaching a ceramic coating formed of silicon oxide, aluminum oxide, or the like on the chip substrate on which the sample solution flow path is formed.
A filter that passes the excitation wavelength of the fluorescent group can also be coated as a protective layer. Thereby, light is irradiated from the surface side where the sample solution flow path is formed, and the enzyme activity is detected by arranging an image sensor such as a CCD at a position where fluorescence of the enzyme activity detection unit can be detected. be able to.
[0021]
The sample solution functionality inspection method according to claim 5 supplies the sample solution to the liquid storage part of the sample solution flow path of the biochip according to any one of claims 1 to 4 under a predetermined condition. A sample solution supply step and a two-dimensional image on the chip substrate formed by a state change for each of the enzyme activity detection units arranged on the sample solution flow path are processed with a filter that removes the specific wavelength region, and A data acquisition step of acquiring series data or result data after a predetermined time, and comparing each of the time series data or result data with library data stored in advance under the same measurement conditions, by pattern recognition means and statistical data processing means And a data determination step for determining a pattern matching degree with the library data.
This configuration has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Compare the time series data or result data obtained in this way with library data.


11

[0012]
Since it has a data judgment process that calculates the degree of pattern matching with each library data, the enzyme activity of the sample solution can be measured in a short time using a small amount of sample solution without the need to accumulate basic data, and operability and efficiency Excellent in properties.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.
【The invention's effect】
[0022]
According to invention of Claim 1, the following effects are acquired.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow paths formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.
(5) Since it has a fluorescent group bound to the chip substrate and an oligopeptide bound to the fluorescent group with a peptide bond that is cleaved by an enzyme, the oligopeptide is released when the peptide bond is cleaved by reacting with the enzyme. Is done. Since the fluorescence wavelength of the fluorescent group released from the oligopeptide or the fluorescence intensity at a predetermined wavelength is different from that before the release, the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
(6) Since the fluorescent group is bonded in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. The activity can be detected, and the degree of integration of the enzyme activity detector that can detect the enzyme activity can be dramatically increased.
(7) Since the enzyme activity can be detected simply by using a sample solution sufficient to fill the sample solution flow path, a large amount of sample solution is not required for measurement, and the enzyme activity can be detected even with a small amount of sample solution. It can be carried out.
(8) By contacting the enzyme in the sample solution flowing through the sample solution flow path with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
(9) Since the enzyme specificity for a specific enzyme or the like is determined by the respective amino acid sequences constituting the oligopeptide, two possible combinations of these amino acid sequences are two-dimensionally arranged in a sample solution channel on the chip substrate in a certain order. As a result, the same fluorescent pattern array image can always be obtained for the same sample solution. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
(10) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
(11) Since knowledge of enzyme activity for a specific enzyme or protein for all such amino acid sequences is not required in advance, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.
[0023]


12/2

[0014]
[0024]
According to invention of Claim 2, in addition to the effect of Claim 1, it has the following effects.
(1) Since the fluorescent group is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the fluorescent group to the chip substrate is an action of an enzyme or the like in the sample solution. Therefore, the biochip is not easily cut and the stability and durability of the biochip can be improved.
(2) Since an aminomethylcoumarin-based fluorescent group is used, it is possible to construct an enzyme activity detection unit having excellent selectivity by effectively utilizing its excellent fluorescence characteristics that differ depending on the binding state with each oligopeptide.
[0025]
According to invention of Claim 3, in addition to the effect of Claim 1 or 2, it has the following effects.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide, the enzyme activity detectors are arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.
[0026]
According to invention of Claim 4, in addition to the effect of any one of Claims 1 thru | or 3, it has the following effects.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, ensuring measurement accuracy, reliability, and stability.


14

[0015]
It can be further increased.
(2) When the sample solution flow path is covered with a light-transmitting protective layer, excitation light can be irradiated from the protective layer side, and the fluorescence change of the fluorescent group can be measured from the outside. In addition, the biochip is excellent in handling and storage properties after the sample solution is introduced into the sample solution flow path.
[0027]
According to invention of Claim 5, it has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Since there is a data judgment step for comparing the time series data or result data obtained in this way with the library data and calculating the degree of pattern matching with each library data, a small amount of data can be obtained without requiring accumulation of basic data. The enzyme activity of the sample solution can be measured in a short time using this sample solution, and the operability and efficiency are excellent.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.
[Brief description of the drawings]
[0028]
[FIG. 1] Schematic diagram showing the principle of enzyme activity detection of the enzyme activity detector [FIG. 2] Schematic diagram of the enzyme activity detector having a different arrangement configuration [FIG. 3] A schematic perspective view of the biochip in the first embodiment [FIG. FIG. 4 is a schematic diagram showing the state of the enzyme activity detectors arranged in the sample solution flow path. FIG. 5 shows the enzyme activity of the sample solution introduced into the biochip in the first embodiment.


15

  The present invention relates to a biochip capable of comprehensively detecting activity and reaction behavior of a sample solution with respect to an enzyme and a method for testing the functionality of the sample solution using the biochip.

Conventionally, techniques for measuring enzyme activity by changing fluorescence in a solution using a single fluorescent enzyme substrate peptide have been established for many enzyme-fluorescent substrate pairs.
In addition, in order to perform such multiple measurements of enzyme activity in parallel, a technique has been developed in which a large number of reaction products obtained by hydrolyzing DNA or RNA are immobilized on a substrate to form a chip.
For example, an array obtained by immobilizing a large number of oligonucleotides on a substrate such as glass is called a DNA chip, and is often used for analysis of genes related in the fields of pathology, medicine, drug discovery, food, and the environment. ing.
In addition, development of a technique for exhaustively searching for proteins that are gene products using a protein chip (registered trademark of Cyphergen Co., Ltd.) in the same manner as a DNA chip is underway. Proteolytic enzymes (proteases) exist just as DNA exists where life phenomena are observed. Therefore, if proteases can be comprehensively detected, it becomes a technique necessary for personal health care, such as obtaining biological information related to homeostasis consisting of the secretion, production, and degradation processes of peptide hormones. In order to detect proteases comprehensively, there is a method for scientifically qualitatively determining the presence of one molecule as a protein.
However, since protein fragmentation results in loss of protein function, it is not easy to immobilize it on a substrate while maintaining the three-dimensional structure of the biopolymer, which is a technical bottleneck. Yes.

In recent years, with the development of medical fields such as pathological diagnosis and research fields such as proteome analysis, enzymes that do not have the disadvantages of fragmentation such as proteins and are easy to handle chemically and create a data library are used. As a result, there is a need to detect the activity against multiple enzymes, and there is a technology for measuring absorbed light and fluorescence in solution using a sensitive substance that is active against enzymes. Various studies have been conducted.
For example, in Patent Document 1, both ends of a substrate peptide that is specifically cleaved by caspase, which is a type of proteolytic enzyme, are modified with a fluorescent group, and the fluorescence wavelength and fluorescence of the fluorescent group generated by cleaving the substrate peptide are cleaved. A fluorescent probe is described which measures the intensity and detects the enzymatic activity of a solution containing caspase.
As a method for measuring such enzyme activity, there is a method in which a sample solution is injected into a cell formed on a microplate for fluorescence measurement, and the fluorescence of the cell is measured using a plate reader.
JP 2000-316598 A

However, the conventional techniques have the following problems.
(1) Since the technique described in Patent Document 1 only detects the enzyme activity of a sample solution with respect to a specific proteolytic enzyme, the enzyme is adapted to a complex system such as blood containing various enzymes and proteins. It is necessary to repeat complicated procedures to measure the activity and immunological characteristics comprehensively, and to locate the characteristics of the sample solution to be measured in many library data and perform pathological diagnosis. However, there was a problem that the measurement was not reliable or quick.
(2) Further, since it is necessary to use a substrate peptide whose specificity for a specific enzyme is known in advance as an enzyme sensor, a large number of the substrate peptides are specified and analyzed to analyze a complicated immune system or the like. However, there was a problem of requiring a huge accumulation of basic data.
(3) In the conventional method of measuring the fluorescence change of a fluorescent probe by injecting a specimen solution into a microplate having a millimeter-sized cell, a large amount of solution is required to examine a large number of enzyme activities, and handling is also necessary. It becomes complicated. Therefore, when performing fluorescence measurement, it took time to replace the microplate, and the measurement efficiency could not be improved, and it took time to measure.
(4) Further, there is a problem that it is difficult to acquire time series data when the sensor substance and the enzyme are bound or react with each other and lacks dynamic analysis.
(5) Since the enzyme substrate is bound in an array on the substrate, it is necessary to immerse the substrate surface in the sample solution containing the enzyme in order to perform simultaneous measurement, and the sample solution is required in milliliter units. There was a problem of requiring a large amount.
(6) The technology that uses a fluorescent enzyme substrate peptide to measure enzyme activity based on changes in fluorescence in the solution requires one fluorescence measurement well for a single enzyme measurement, and uses multiple fluorescence measurement devices. Even if the fluorescence measurement requires several minutes per 100 wells, it takes several hours or more to perform the time-varying measurement for each well in order to ensure precise quantitativeness. There was a problem.
(7) At the research level of enzyme activity detection biochip, the peptide of the enzyme substrate is bound to the surface of a substrate such as glass via trimethoxyaminopropylsilane, and the surface is immersed in the enzyme solution to emit fluorescence. Detected. However, since the binding amount of the fluorescent substrate peptide on the substrate is low, there is a problem that even if qualitative results can be obtained with or without enzyme activity, it is difficult to use for applications that require quantitativeness. It was.
(8) In addition, since the binding amount of the fluorescent substrate peptide on the substrate is not stable and it is difficult to quantify the binding amount, the results cannot be compared among a plurality of enzyme substrate peptides. It is difficult to quantify enzyme activity simultaneously on one chip.

The present invention was made in order to solve the above-mentioned conventional problems, and handling and reliability of data when performing pathological diagnosis by comprehensively measuring enzyme activity and immunological characteristics such as blood, An object of the present invention is to provide a biochip that is excellent in rapidity, easy in chemical treatment, and capable of increasing the degree of integration of the detection unit and acquiring enzyme activity and its tendency even with a small amount of sample solution.
In addition, the present invention does not require accumulation of basic data, is excellent in the analysis of time-series data, and has excellent operability and efficiency in which the enzyme activity of a sample solution can be measured in a short time using a small amount of sample solution. It aims at providing the functionality inspection method of a sample solution.

The biochip of claim 1 of the present invention is a biochip for detecting an enzyme in a sample solution to be introduced into the chip substrate, and a sample solution flow path formed in the chip substrate, the sample solution A plurality of enzyme activity detection units arranged in a predetermined order in a flow path and having different activities with respect to a predetermined enzyme, wherein the enzyme activity detection unit is not directly on the chip substrate or has no enzyme specificity. Alternatively , the first oligopeptide bound via a compound, the first fluorescent group introduced into the side chain of the first oligopeptide, and the second oligopeptide bound to the first oligopeptide via a peptide bond An oligopeptide and a second fluorescence that causes fluorescence resonance energy transfer with the first fluorescent group by binding to the second oligopeptide and releasing the second oligopeptide by an enzyme When configured with a.
With this configuration, the following effects can be obtained.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow path formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.
(5) The first oligopeptide bound to the chip substrate, the first fluorescent group introduced into the side chain of the first oligopeptide, and the second oligopeptide bound to the first oligopeptide by peptide bonds And the second fluorescent peptide that binds to the second oligopeptide and is released by the enzyme to cause the first fluorescent group and the second fluorescent group to cause fluorescence resonance energy transfer . When the second oligopeptide is released and the two fluorescent compounds are present at a distance, the fluorescence of the second fluorescent group that should be observed is attenuated. Instead, the first fluorescent group The enzyme activity can be detected using a change in fluorescence intensity or the like as an index.
(6) The substrate specificity with respect to each amino acid is not high, and rather, an enzyme such as elastase that requires a relatively long peptide chain for cleaving can be easily detected, and the detection sensitivity can be increased.
( 7 ) Since the fluorescent group is bound in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. The activity can be detected, and the degree of integration of the enzyme activity detection unit capable of detecting the enzyme activity can be dramatically increased, and the biochip can be downsized.
( 8 ) Since the enzyme activity can be detected simply by using a sample solution that fills the small volume sample solution flow path, a large amount of sample solution is not required for measurement, and even a small amount of sample solution does not require enzyme activity. Can be detected.
( 9 ) By contacting the enzyme in the sample solution flowing through the sample solution channel with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
( 10 ) Since the enzyme specificity for a specific enzyme and the like is determined by the respective amino acid sequences constituting the oligopeptide, when possible combinations of these amino acid sequences are two-dimensionally arranged in the sample solution flow path on the chip substrate In the case of the same sample solution, images of the same fluorescent pattern arrangement are always obtained. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
( 11 ) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
( 12 ) Since knowledge of enzyme activity for a specific enzyme or protein is not required in advance for all such amino acid sequences, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.

Here, as the sample solution, a body fluid or a culture solution containing blood or urine to be subjected to pathological diagnosis or the like can be applied. As the sample solution, a biological sample (blood or the like) can be used as it is, or a blood cell component or the like removed by a filter, centrifugation, or the like. Moreover, what performed the condition setting (pH adjustment, active agent introduction | transduction, etc.) that an enzyme expresses activity based on a biological sample can also be used. As a pH adjuster, a buffering agent such as Tris-HCl or Hepes-KOH can be added as a reaction buffer. In addition, salts and active protective agents necessary for the expression of enzyme activity can be added.
Examples of enzymes to be detected include serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, kallikrein, urokinase, elastase, aspartic proteases such as pepsin, cathepsin D, renin, chymosin, carboxypeptidase A, B, collagenase, thermolysin, etc. Endopeptidases that cleave internal peptide bonds such as cysteine proteases such as metalloproteases, cathepsins B, H, L, and calpain, blood coagulation proteases, capture proteases, hormone processing enzymes, and the like.
When the enzymes to be detected have an inactive state, they can be activated by adding trypsin or the like or removing the endogenous inhibitor.

  As the chip substrate, a substrate such as glass or synthetic resin having no enzyme specificity is used. Solvents used in condensation reactions to form peptide bonds (halogenated hydrocarbons such as chloroform and dichloromethane, esters such as ethyl acetate, polar organic solvents such as N, N-dimethylformamide and dimethyl sulfoxide, dioxane , Ethers such as tetrahydrofuran, alcohols such as methanol and ethanol, pyridine, etc.) made of insoluble synthetic resin (polystyrene, etc.) or glass, etc., formed into a curved surface such as a flat plate or spherical surface It is done. A commercially available carrier for solid phase organic synthesis such as Lantern series (registered trademark) manufactured by Mimotops can also be used.

The sample solution channel is a portion in which the vertical cross section of the chip substrate is formed in a rectangular shape, a U shape, a groove shape, or the like. The flow pattern of the sample solution flow path is continuous or branched so that the sample solution flows from one or a plurality of upstream openings toward the downstream openings, and in a plan view, a spiral shape, a tree shape, It can be formed in a radial shape, a zigzag shape, or the like. Thereby, the integration degree of a sample liquid flow path can be raised and a biochip can be reduced in size and size.
The sample solution channel can be formed on the chip substrate mechanically or chemically so that the volume, channel width, and depth of about 100 μL or less are about 1 mm or less. This is because the degree of integration of the sample solution flow path on the chip substrate decreases as the value becomes larger than these upper limit values, and it tends to be difficult to specify the fluorescent pattern to be formed on the chip substrate.
On one end side of the sample solution flow path, a recess-like liquid supply section for supplying the sample solution can be formed in a row with one or a plurality of upstream openings. Once the sample solution is supplied to the liquid supply part formed in the shape of a depression, the sample solution can be moved sequentially from the liquid supply part to the sample liquid flow path by capillary action etc. Because you can. The liquid supply part is provided with a filter such as a membrane film for removing red blood cells, white blood cells, lymphocytes, etc. in the supplied blood (sample solution) as necessary, so that the sample solution adheres to the channel wall. So that the flow is not obstructed.
On the other end side of the sample solution flow path, a liquid storage part for storing the sample solution can be formed continuously with one or a plurality of downstream openings. This liquid storage part is a recessed part formed in the center or peripheral part of the chip substrate. As a result, the sample solution flows out from the other end side of the sample solution flow path to the liquid storage section, so that a flow of the sample solution from the sample solution flow path to the liquid storage section can be formed. The oligopeptide cleaved from the enzyme activity detection part by the action of the enzyme in the sample solution can be reliably released into the sample solution without stagnation of the sample solution, and the detection sensitivity of the enzyme activity can be increased. .

The enzyme activity detection unit is configured by arranging a sensitive substrate such as a peptide chain having a selective and specific reaction behavior with respect to a specific enzyme. With such an enzyme-specific reaction or the like, the optical characteristics such as the fluorescence wavelength and fluorescence intensity of the sensitive substrate and the electrical characteristics change, and this can be detected.
A chip substrate when a plurality of enzyme activity detectors are arranged in the sample solution flow path, and an image sensor comprising a CCD or CMOS device integrated body is arranged at a position where the enzyme activity detector can receive light. By acquiring the upper two-dimensional fluorescence image, time-series fluorescence data of the enzyme activity detection unit or fluorescence data after a predetermined time can be obtained.
The enzyme activity detectors arranged adjacent to each other in the sample solution channel need not be arranged so as to be separated from the surroundings in a cell shape via a partition wall or the like, but along the bottom of the sample solution channel of the chip substrate. Alternatively, they may be arranged continuously in a planar shape.
In addition, the arrangement of each independent enzyme activity detection unit made of a peptide to which a fluorescent group is bound in the sample solution flow path is, for example, using a commercially available DNA chip plotter or using a syringe while looking at a microscope. Can be arranged. In addition, each predetermined region in the sample solution flow path formed in a spiral shape is filled with spherical particles such as polystyrene resin or flat plate with enzyme specificity on the surface thereof, and an independent enzyme activity detector is provided. It is also possible to arrange them.

  Here, the oligopeptide is a peptide composed of a peptide chain consisting of several amino acids, and has a substantially linear form unlike a protein having a large molecular weight and a mass. The amino acid sequence of the oligopeptide can characterize the enzyme specificity at the binding site with the fluorescent group to which the oligopeptide is bound.

As the fluorescent group, those in which the fluorescence wavelength and the fluorescence intensity change before and after the peptide bond with the oligopeptide is cleaved by the enzyme are used. In particular, a fluorescent group that emits fluorescence in the specific wavelength region when the peptide bond is bonded to the peptide chain and is a non-fluorescent substance in the specific wavelength region when the peptide bond is cleaved to release the peptide chain, for example, 4-Methylcoumaryl-7-amide (MCA), 7-amino-4-carboxymethylcoumarin (ACC), p-nitroanilide, α-naphthylamide, α-naphthyl ester and the like are preferably used.
As the amino acid residue located at the binding site, one in which the peptide bond on the C-terminal side is selectively cleaved by an enzyme is used. This is because it is possible to change the fluorescence intensity and the fluorescence frequency of the fluorescent group by releasing the binding portion that has been bonded to the fluorescent group.
In addition, when the oligopeptide is formed with a peptide chain having a predetermined length (for example, about 15 mm) or more, the substrate specificity for each amino acid is not high, but rather, elastase required for the action of cleaving a relatively long peptide chain. Such enzymes can be detected, and the types of enzymes that can be detected can be increased.

  The amino acid sequence is composed of, for example, peptide chains having amino acids of A (alanine), B (proline), C (lysine), and D (phenylalanine) as elements, and the enzyme activity detection unit has a peptide in the sample solution channel. When arranged in the order of the rule combination of each element of the chain, analysis and evaluation of test data can be performed easily and quickly based on basic data accumulated for basic amino acids. In addition, the biochip can be mechanically constructed using known peptide chain synthesis means so that oligopeptides composed of basic amino acids are regularly arranged in descending or ascending order such as the character code order of A to D. Excellent production efficiency.

The biochip according to claim 2 is configured by binding the first oligopeptide onto the chemically modified chip substrate in the invention according to claim 1.
With this configuration, in addition to the operation of the first aspect, the following operation is provided.
(1) Since the first oligopeptide is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the first oligopeptide to the chip substrate can be improved. It is not easily cleaved by the action of the enzyme in the inside, and the stability and durability of the biochip can be improved.

  Here, as the silane coupling agent, for example, aminopropyltrimethoxysilane can be applied.

A biochip according to a third aspect is the invention according to the first or second aspect, wherein the chip substrate is translucent and an image sensor substrate is laminated on the back side thereof.
With this configuration, in addition to the operation described in claim 1 or 2, the following operation is provided.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide, the enzyme activity detectors are arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.

  As the image sensor substrate, it is possible to apply a CCD or CMOS element formed by providing a photosensitive portion in which photodiodes are two-dimensionally arranged on a silicon thin plate and a signal output portion thereof by a known semiconductor manufacturing technique.

According to a fourth aspect of the present invention, there is provided the biochip according to any one of the first to third aspects, further comprising a protective layer on the surface of the chip substrate on which the sample solution flow path is formed. Yes.
With this configuration, in addition to the operation described in any one of claims 1 to 3, the following operation is provided.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, and measurement accuracy, reliability, and stability can be further enhanced.
(2) When the sample solution flow path is covered with a light-transmitting protective layer, excitation light can be irradiated from the protective layer side, and the fluorescence change of the fluorescent group can be measured from the outside. In addition, the biochip is excellent in handling and storage properties after the sample solution is introduced into the sample solution flow path.

For example, the protective layer can be configured by attaching a ceramic coating formed of silicon oxide, aluminum oxide, or the like on the chip substrate on which the sample solution flow path is formed.
A filter that passes the excitation wavelength of the fluorescent group can also be coated as a protective layer. Thereby, light is irradiated from the surface side where the sample solution flow path is formed, and the enzyme activity is detected by arranging an image sensor such as a CCD at a position where fluorescence of the enzyme activity detection unit can be detected. be able to.

The sample solution functionality inspection method according to claim 5 supplies the sample solution to the liquid storage part of the sample solution flow path of the biochip according to any one of claims 1 to 4 under a predetermined condition. A sample solution supply step and a two-dimensional image on the chip substrate formed by a state change for each of the enzyme activity detection units arranged on the sample solution flow path are processed with a filter that removes the specific wavelength region, and A data acquisition step of acquiring series data or result data after a predetermined time, and comparing each of the time series data or result data with library data stored in advance under the same measurement conditions, by pattern recognition means and statistical data processing means And a data determination step for determining a pattern matching degree with the library data.
This configuration has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Since there is a data judgment step for comparing the time series data or result data obtained in this way with the library data and calculating the degree of pattern matching with each library data, a small amount of data can be obtained without requiring accumulation of basic data. The enzyme activity of the sample solution can be measured in a short time using this sample solution, and the operability and efficiency are excellent.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.

According to invention of Claim 1, the following effects are acquired.
(1) By supplying a small amount of sample solution, a flow of the sample solution can be formed in the sample solution flow path, and the enzymes in the sample solution can be reacted with enzyme activity detection units having different enzyme activities. In this way, by detecting optically or electrically the state change associated with the enzyme reaction in each enzyme activity detector, the activity behavior specific to the sample solution is comprehensively acquired as a two-dimensional image displayed on the chip substrate. can do.
(2) The two-dimensional image data is identified and grouped by comparing with reference to known library data obtained in the same way, and the enzyme activity and immune characteristics of the sample solution are comprehensively understood. Pathological diagnosis can be performed accurately and promptly.
(3) Since the enzyme activity detectors having different activities are arranged in the sample solution flow paths formed in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, the acquired two-dimensional image data It is also possible to directly perform pathological diagnosis based on visual observation without using a pattern recognition process of data by a computer. In addition, by forming the sample solution flow path with a specific geometric arrangement, it is possible to facilitate the scanning operation at the time of data acquisition and to omit the data processing.
(4) A sample solution detection cell can be constructed by binding a fluorescent group to a chip substrate using a DNA chip plotter, printing means, etc., and the degree of integration of the detector can be dramatically increased. Thus, data such as enzyme activity can be efficiently obtained using the sample solution.
(5) The first oligopeptide bound to the chip substrate, the first fluorescent group introduced into the side chain of the first oligopeptide, and the second oligopeptide bound to the first oligopeptide by peptide bonds And the second fluorescent peptide that binds to the second oligopeptide and is released by the enzyme to cause the first fluorescent group and the second fluorescent group to cause fluorescence resonance energy transfer . When the second oligopeptide is released and the two fluorescent compounds are present at a distance, the fluorescence of the second fluorescent group that should be observed is attenuated. Instead, the first fluorescent group The enzyme activity can be detected using a change in fluorescence intensity or the like as an index.
(6) The substrate specificity with respect to each amino acid is not high, and rather, an enzyme such as elastase that requires a relatively long peptide chain for cleaving can be easily detected, and the detection sensitivity can be increased.
( 7 ) Since the fluorescent group is bound in the sample solution flow path on the chip substrate, the enzyme can be obtained simply by filling the sample solution flow path with a very small amount of sample solution containing the enzyme and measuring the fluorescence intensity of the chip substrate. The activity can be detected, and the degree of integration of the enzyme activity detector that can detect the enzyme activity can be dramatically increased.
( 8 ) Since the enzyme activity can be detected simply by using a sample solution that fills the sample solution flow path, a large amount of sample solution is not required for measurement, and the enzyme activity can be detected even with a small amount of sample solution. It can be carried out.
( 9 ) By contacting the enzyme in the sample solution flowing through the sample solution channel with the specific binding site of the enzyme activity detection unit, the binding unit between the oligopeptide corresponding to this enzyme and the fluorescent group is cleaved, The fluorescence emission frequency of the fluorescent group can be shifted or its fluorescence intensity can be changed with changes in fluorescence resonance energy or the like. This change can be observed through a filter or the like under excitation light irradiation, or can be converted into data by measuring a fluorescence spectrum.
( 10 ) Since the enzyme specificity for a specific enzyme and the like is determined by each amino acid sequence constituting the oligopeptide, possible combinations of these amino acid sequences are two-dimensionally arranged in a sample solution channel on the chip substrate in a certain order. As a result, the same fluorescent pattern array image can always be obtained for the same sample solution. Therefore, by comparing this image data with library data including data of known components, pathological characteristics can be comprehensively determined from the tendency of the enzyme specificity.
( 11 ) As the amino acids constituting the amino acid sequence, 20 or more types included in the protein can be set. When the amino acid sequence is composed of four types of amino acids, a part or all of peptide chains comprising 20 ⁴ = 160000 or more permutations can be arranged in each enzyme activity detection part of the sample solution flow path.
( 12 ) Since knowledge of enzyme activity for a specific enzyme or protein is not required in advance for all such amino acid sequences, the regular sequence can be mechanically formed by applying computer control means, Peptide chains consisting of a certain amino acid sequence can be sequentially arranged in the sample solution flow path, and a biochip configured with a predetermined enzyme activity for a known or unknown enzyme can be easily produced and provided.

According to invention of Claim 2, in addition to the effect of Claim 1, it has the following effects.
(1) Since the first oligopeptide is bound to the chip substrate chemically modified with the silane coupling agent, the binding can be strengthened more reliably, and the binding of the first oligopeptide to the chip substrate can be improved. It is not easily cleaved by the action of the enzyme in the inside, and the stability and durability of the biochip can be improved.

According to invention of Claim 3, in addition to the effect of Claim 1 or 2, it has the following effects.
(1) Since the image sensor substrate is laminated on the back side of the chip substrate made of a light-transmitting material such as silicon oxide or aluminum oxide, the enzyme activity detectors are arranged from the back side of the chip substrate arranged in a predetermined pattern. The fluorescence change can be acquired directly and as a two-dimensional image.
(2) Since the enzyme component in the sample solution can be analyzed in a state where the enzyme activity detector is integrated on the chip substrate at a high density, a biochip having a compact size and excellent reliability can be provided.

According to invention of Claim 4, in addition to the effect of any one of Claims 1 thru | or 3, it has the following effects.
(1) Since the opening of the sample solution channel formed in the shape of a groove on the chip substrate is covered with a light-transmitting or non-light-transmitting protective layer such as a silicon oxide film, the sample solution to be introduced Is protected without contact with air in the sample solution flow path, and measurement accuracy, reliability, and stability can be further enhanced.
(2) When the sample solution flow path is covered with a light-transmitting protective layer, excitation light can be irradiated from the protective layer side, and the fluorescence change of the fluorescent group can be measured from the outside. In addition, the biochip is excellent in handling and storage properties after the sample solution is introduced into the sample solution flow path.

According to invention of Claim 5, it has the following effects.
(1) Since the sample solution supply step of supplying the sample solution to the sample solution channel formed entirely in a spiral shape, tree shape, radial shape or the like is provided, the enzyme activity detection unit of the sample solution channel and the enzyme in the sample solution And the bond between the fluorescent peptide and the oligopeptide that specifically cleaves the enzyme can be cleaved.
(2) Next, since there is a data acquisition step for acquiring a two-dimensional image by changing the fluorescence characteristics of the fluorescent group by enzyme cleavage for each enzyme activity detection unit, many kinds of oligopeptides are performed without performing complicated calculation processing. The fluorescence behavior corresponding to can be comprehensively captured as a two-dimensional image.
(3) Since there is a data judgment step for comparing the time series data or result data obtained in this way with the library data and calculating the degree of pattern matching with each library data, a small amount of data can be obtained without requiring accumulation of basic data. The enzyme activity of the sample solution can be measured in a short time using this sample solution, and the operability and efficiency are excellent.
(4) Since proteases in blood and the like exist as a mixture in vivo and in vivo, only the enzyme activity is comprehensively detected in the mixture, and this is compared with the data of the peptide library, and the peptide secretion is secreted. Biometric information on homeostasis, which is a process of generation and decomposition, can be obtained, which can contribute to personal health care.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a schematic diagram showing an enzyme activity detection principle of an enzyme activity detector in a biochip according to a reference example of the present invention.
In the figure, 1 is a chip substrate having a portion (sample solution flow path) that is mechanically or chemically formed into a flat plate shape with silicon oxide or glass, and 2 is directly bonded to the chip substrate 1 by a peptide bond or the like. Fluorescent group such as 4-methylcoumaryl-7-amide (MCA), 3 is an amino peptide bonded to fluorescent group 2 by peptide bond, oligopeptide such as peptide, 4 selectively binds fluorescent group 2 to oligopeptide 3 An enzyme having substrate specificity, such as serine protease to be cleaved, 5 is a fluorescent group such as 7-amino-methylcoumarin (AMC) whose fluorescence wavelength has been changed by selective release of oligopeptide 3 by enzyme 4. is there. The enzyme activity detection unit configured as described above is synthesized by using a known peptide synthesis technique and is bonded onto the chip substrate 1.

Hereinafter, the principle of detecting enzyme activity will be described with reference to FIG.
The fluorescent group 2 such as 4-methylcoumaryl-7-amide (MCA) shown in FIG. 1A is a non-fluorescent substance in a specific wavelength region and does not exhibit fluorescence. When the sample solution containing the enzyme 4 is brought into contact with the chip substrate 1 and reacted, the enzyme 4 having substrate specificity cleaves the peptide bond between the fluorescent group 2 and the oligopeptide 3 (see FIG. 1B). ).
The fluorescent group 5 from which the oligopeptide 3 is released becomes a fluorescent substance in the specific wavelength region such as 7-amino-methylcoumarin (AMC), and the fluorescence wavelength or the fluorescence intensity at a predetermined wavelength is a fluorescent group that is peptide-bonded to the oligopeptide 3 Therefore, the enzyme activity can be detected using a change in fluorescence intensity or the like as an index (see FIG. 1 (c)).

  Although the case where the fluorescent group 2 is directly bonded to the chip substrate 1 has been described here, the fluorescent group 2 may be bonded to the chip substrate 1 via an amino acid, a peptide, or the like. As a result, the distance between the chip substrate 1 and the enzyme action point can be optimized and the enzyme activity can be detected more accurately without being affected by the chip substrate 1. In this case, amino acids, peptides and the like between the fluorescent group 2 and the chip substrate 1 are synthesized with molecules or compounds having no enzyme specificity. This is to prevent the fluorescent group 2 from being released from the chip substrate 1 by the action of the enzyme.

FIG. 2 is a schematic diagram of an enzyme activity detection unit according to an embodiment of the present invention having an arrangement configuration different from that in FIG.
In FIG. 2, 6 is a first oligopeptide having one end immobilized on the chip substrate 1, 7 is a first fluorescent group introduced into the side chain of the oligopeptide 6, 8 is a peptide bond and the first oligopeptide A second oligopeptide bound to peptide 6, 9 is a second fluorescent group that binds to the second oligopeptide and causes fluorescence resonance energy transfer with the first fluorescent group 7, and 7a is originally observed by fluorescence resonance energy transfer. This is the first fluorescent group in which the fluorescence of the second fluorescent group 9 that should be attenuated is attenuated and fluorescence is observed instead.
Here, the fluorescence resonance energy transfer means that when two fluorescent compounds are present at positions close to each other, the fluorescence spectrum of one of the two fluorescent compounds (referred to as a donor) and the excitation of the other (referred to as an acceptor). When there is an overlap with the spectrum, this is a phenomenon in which the donor fluorescence that should be observed is attenuated when the excitation wavelength energy of the donor is applied, and the acceptor fluorescence is observed instead.
In this enzyme activity detection unit, the second oligopeptide is released by the enzyme 4 and the two fluorescent compounds are located far away from each other. Therefore, the second fluorescent group 9 that should originally be observed is detected. The fluorescence is attenuated, and the fluorescence of the first fluorescent group 7 is observed instead (7a), and the enzyme activity can be detected.

Here, as the first fluorescent group 7 and the second fluorescent group 9, a combination of a donor and an acceptor in which fluorescence resonance energy transfer occurs can be used. For example, the second fluorescent group 9 (or the first fluorescent group 7) which is an atomic group having an absorption band in a wavelength region overlapping with the fluorescent wavelength of the first fluorescent group 7 (or the second fluorescent group 9) is used. It is done. Specifically, the combination of (7-methoxycoumarin-4-yl) acetyl (MOAc), anthraniloylbenzyl (ABz), N-methylanthranilic acid (Nma) and the like and dinitrophenyl (Dnp), Dabsyl and EDANS ( 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid), tryptophan (Trp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns), carboxydichlorofluorescein (CDCF) and carboxymethyl A combination of rhodamine (CTMR), a combination of carboxydichlorofluorescein (CDCF) and carboxy X-rhodamine (CXR), a combination of lucifer yellow (LY) and carboxymethyl rhodamine (CTMR), and the like are used.
Any of these donors and acceptors may be the first fluorescent group 7 or the second fluorescent group 9. This is because if a spectral change occurs in the first fluorescent group 7, it can be used as a measurement index of enzyme activity.
In addition, between the coupling | bond part of the 1st fluorescence group 7 and the 2nd fluorescence group 9 which were each couple | bonded with the 1st fluorescence group 7 and the 2nd oligopeptide 8 which were introduce | transduced into the side chain of the 1st oligopeptide 6 The length is desirably 100 mm or less. This is because as the distance becomes longer, the fluorescence resonance energy transfer becomes smaller and the change in the fluorescence intensity or the like tends to become smaller.
By configuring the enzyme activity detection unit in this way, it is possible to easily detect an enzyme such as elastase which has a low substrate specificity for each amino acid and rather requires a relatively long peptide chain for its cleaving action. Detection sensitivity can be increased.

(Embodiment 1)
FIG. 3 is a schematic perspective view of the biochip according to the first embodiment of the present invention to which the above-described enzyme activity detection principle is applied, and FIG. 4 is a schematic diagram showing the state of the enzyme activity detection unit arranged in the sample solution flow path. FIG.
In FIG. 3, 10 is a biochip according to the first embodiment of the present invention, 11 is a chip substrate constituting the biochip 10 and formed in a substantially rectangular plate shape, and 12 is a chip that is spirally formed as a whole. A sample solution flow path 13 formed in the shape of a groove on the substrate 11 is formed in a cell shape and a spot shape in the sample solution flow path 12, and each has a predetermined enzyme activity and is arranged in a predetermined order. , 14 is a substantially rectangular liquid supply portion provided on the outer end side of the sample solution flow path 12 to which the sample solution is supplied, and 15 is substantially circular on the spiral center side which is the discharge side of the sample solution flow path 12 It is the liquid storage part provided.
The biochip 10 as a whole is formed on a chip substrate 11 made of glass or polystyrene resin having a substantially rectangular plate shape (length of about 20 to 30 mm on one side and thickness of about 2 mm), a flow path width of about 0.5 mm, and a depth. A sample solution channel 12 having a channel cross section of about 0.1 mm and an effective channel length of about 90 mm and formed in a spiral shape with a channel interval of 0.5 mm is provided. The liquid supply unit 14 is formed in a substantially square shape with a side of about 2.5 mm, and the effective volume of the sample solution channel 12 is about 4.5 μL.
A sample solution such as a blood sample collected in the liquid supply unit 14 on the chip substrate 11 is supplied, and sequentially moves through the sample solution flow path 12 by capillary action or the like to come into contact with the enzyme activity detection unit 13 to be in the sample solution. Characteristics such as the activity and antibody reactivity of each enzyme contained in are detected.

The enzyme activity detection unit 13 is formed as a cell-like, dent-like or flat spot-like site at the bottom of the sample solution flow path 12 formed in a spiral shape on the chip substrate 11 and has different enzyme activities. It is configured to be sequentially arranged at a predetermined interval.
As shown in FIGS. 3 and 4, the enzyme activity detector 13 includes a fluorescent group K whose one end is coupled to the bottom surface of the sample solution channel 12 formed in a substantially groove shape, and the other end of the fluorescent group K. It is comprised with the oligopeptide which consists of an amino acid sequence couple | bonded and arrange | positioned, for example at 4 sites of AD.
The fluorescent group K is, for example, an aminomethylcoumarin fluorescent group, and has different fluorescent characteristics depending on the binding state with each oligopeptide. If necessary, the chip substrate 11 is chemically modified with a silane coupling agent or the like so that the fluorescent group K is bonded into the sample solution flow path 12 of the chip substrate 11 so that the chip substrate 11 is bonded. Stability and durability can be increased.

For example, a peptide chain composed of any combination of ala: alanine, pro: proline, lys: lysine, and phe: phenylalanine is set at each site A to D of the oligopeptide conjugated to the fluorescent group K. I have to.
Examples of oligopeptides include (1) ala-pro-lys-phe, (2) pro-lys-phe-ala, (3) lys-phe-ala-pro, and (4) phe-ala-pro-lys. The amino acid sequence is set so that the enzyme specificity of the binding part of the fluorescent group to which the oligopeptide is bound is defined corresponding to each amino acid sequence.

Each amino acid sequence can be synthesized by a normal peptide synthesis method such as a solid phase method in which the peptide is extended from the C-terminus. Also, use a sequential extension method that sequentially extends from the C-terminal side to the N-terminal side of the target amino acid sequence, or a fragment condensation method that synthesizes a plurality of short peptide fragments and extends them by coupling between peptide fragments. Can do. Moreover, 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid, t-butyloxycarbonyl (Boc) amino acid, etc. can also be synthesize | combined using a well-known peptide synthesizer. Furthermore, a peptide bond can be generated using a protease, or it can be synthesized using a genetic engineering technique.
Furthermore, as a condensation method for forming a peptide bond, for example, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, DCC-additive method, active ester method, carbonyldiimidazole method , A redox method, a method using Woodward reagent K, and the like are used. In addition, before the condensation reaction, the carboxyl group and amino group which are not involved in the reaction in amino acids and peptides can be protected by known means, or the carboxyl group and amino group involved in the reaction can be activated. It can also be left.

Thus, as shown in FIG. 4, the S1-Sn enzyme activity detectors 13 each having a possible combination of amino acid sequences are arranged at the bottom of the sample solution channel 12 as follows, for example.
S1: Fluorescent group-oligopeptide (1) (ala-pro-lys-phe)
S2: fluorescent group-oligopeptide (2) (pro-lys-phe-ala)
S3: fluorescent group-oligopeptide (3) (lys-phe-ala-pro)
S4: fluorescent group-oligopeptide (4) (phe-ala-pro-lys)
・
・
Sn: fluorescent group-oligopeptide (n)
In this way, by flowing the sample solution through the sample solution flow path 12 in which the enzyme activity detection units of S1 to Sn are arranged and contacting with the enzyme activity detection unit 13, the enzymes and proteins in the sample solution have different enzyme specificities. Acts on the bond between the oligopeptide to which is attached and the fluorescent group, thereby cleaving this bond. By optically detecting the fluorescence characteristic change of the fluorescent group accompanying this cutting, the sample solution flow path 12 formed in a spiral shape can be expressed as a fluorescence pattern unique to the sample solution.
In addition, the amino acid sequence of the oligopeptide in S1 to Sn does not need to be known in terms of characteristics and behavior such as enzyme activity for each amino acid sequence, and may be of a specific unique sequence as the biochip 10. For this reason, S1-Sn includes, for example, a regular array or a random array including a periodic pattern.

The sample solution channel 12 is formed by etching or the like when the chip substrate 11 is made of silicon oxide or glass. Further, when the chip substrate 11 is made of synthetic resin or the like, it is formed by embossing, laser beam processing, grinding using a machining center, or the like. It can also be manufactured by injection molding or press molding.
The depth of the sample solution flow path 12 formed in a groove shape is preferably about 10 to 200 μm, and the width is preferably in the range of 10 to 1000 μm. If the depth and width of the flow path become smaller than the lower limit values, the flow path will be blocked due to the viscosity of the sample solution, or the measurement sensitivity will be insufficient. This is because when the size is larger, the degree of integration of the enzyme activity detection unit on the chip substrate becomes insufficient, and it becomes difficult to specify the fluorescent pattern to be formed on the chip substrate.

  The liquid supply unit 14 may be provided on the side of the chip substrate 11 and may be provided with a filter that removes red blood cells in blood such as a nonwoven fabric filter and a membrane film. As a result, red blood cells, white blood cells, and the like in the supplied sample solution can be filtered to adjust the viscosity of the sample solution, so that the flow path is blocked by adhering to the wall of the sample solution flow path 12. Can be effectively prevented.

Next, a method for testing the functionality of the sample solution applied to the biochip 10 configured as described above will be described with reference to a schematic operation diagram for a specific enzyme of the enzyme activity detector shown in FIG.
First, a biochip 10 is prepared in which enzyme activity detectors 13 each having a unique enzyme activity are arranged in a predetermined pattern at each site S1 to Sn of a sample solution flow path 12 formed in a spiral shape on a chip substrate 11. In the sample solution supply step, a sample solution such as blood is supplied to the liquid supply unit 14 of the biochip 10 at a predetermined flow rate and temperature. As a result, the blood component or the like supplied to the liquid supply unit 14 is supplied to the sample solution flow path 12 and sequentially brought into contact with the enzyme activity detection unit 13 at each of the sites S1 to Sn using capillary action or the like. it can.
Thus, as shown in the figure, the binding part between the oligopeptide having sensitivity to a specific enzyme in the blood component and the fluorescent group is cleaved, and the fluorescence characteristic of the fluorescent group is in a state before cleaving (here, α). ) To the state after cutting (here, β). As a result, the state of the fluorescent group at the sites S1 to Sn along the sample solution flow path 12 is changed from the initial state X (α, α, α, α, α,...), For example, to the detection state Y ( .alpha., .beta., .alpha., .alpha., .beta.

  In the next data acquisition step, a two-dimensional fluorescence image on the chip substrate 11 formed by the fluorescence change in each enzyme activity detection unit 13 on the sample solution flow path 12 is acquired by an image sensor or the like, and the specific wavelength range is obtained. The time series data or the result data after a predetermined time can be obtained by processing with a filter that removes.

  In the data determination step, the time series data or the result data after a predetermined time is compared with library data measured and accumulated for various samples in advance under the same measurement conditions, and each library is detected by a pattern recognition unit or a statistical determination unit. The degree of pattern matching with data can be determined, and various pathological findings can be obtained quickly and accurately.

As described above, in the sample solution functionality testing method using the biochip 10, the sample solution flow path arrayed with oligopeptides that impart enzyme specificity with the sample solution containing blood, urine, etc. as the test target Lead to react. The result of the enzyme reaction can be acquired by an image sensor that detects fluorescence intensity, and the acquired data can be compared with library data to comprehensively detect the activity of the protease mixture contained in the sample solution. Further, since the sample solution flow path 12 is formed in a spiral shape, the speed of the sample solution moving in the sample solution flow path 12 due to capillary action or the like can be made substantially constant, and the reproducibility of the acquired time series data is excellent. Further, since it continues without branching, the length of the path from the liquid supply unit 14 in the sample liquid channel 12 and the fluorescence intensity can be expressed in a one-to-one relationship, and comparison with the library data is facilitated. be able to.
In this way, comprehensive detection of proteases provides biological information on homeostasis, which is a process of secretion, production, and degradation of peptide hormones, which can be used efficiently for personal health care. .
In this way, the functional test method for sample solutions using biochips is applied to medical fields such as pathological diagnosis and research fields such as proteome analysis, so that the activity of multiple enzymes can be increased at high speed and in small quantities. Can be measured with a sample.

Next, a specific configuration for measuring the enzyme activity of the sample solution introduced into the biochip using an image sensor will be described.
FIG. 5 is a configuration diagram when the enzyme activity of the sample solution introduced into the biochip in Embodiment 1 is measured using an image sensor.
In FIG. 5, reference numeral 20 denotes an image sensor which is arranged on the biochip 10 and acquires two-dimensional data in the enzyme activity detection unit 13 on the chip substrate 11 via the lens 21. As described above, in the configuration in which the image sensor 20 is placed away from the chip substrate 11, the state change of the fluorescent group of the enzyme activity detection unit 13 forms an image on the image sensor 20 through the optical system such as the lens 21. Read.

As described above, since the biochip in the first embodiment is configured, only a very small amount of sample solution containing an enzyme is introduced into the sample solution channel 12 of the chip substrate 11 and the fluorescence intensity of the chip substrate 11 is measured. To detect enzyme activity. Furthermore, since it is not necessary to form a cell or the like for injecting the solution into the substrate for each individual sample, the enzyme activity detection unit 13 and the sample solution flow channel 12 can be miniaturized, so that the integration degree of the enzyme detection unit on the chip substrate 11 can be increased. It can be improved dramatically.
In addition, since enzyme activity can be detected simply by using a very small amount of sample solution containing an enzyme, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. Can do.

(Embodiment 2)
FIG. 6 is a schematic diagram of the biochip in Embodiment 2 of the present invention when the enzyme activity of the sample solution introduced into the biochip is measured by arranging the chip substrate directly on the image sensor.
In FIG. 6, reference numeral 22 denotes a filter as a light-transmitting protective layer disposed on the surface of the chip substrate 11 in which the enzyme activity detection unit 13 is arranged in the sample solution flow path 12. The selectivity is such that the excitation wavelength is allowed to pass but the fluorescence wavelength is blocked. Reference numeral 23 denotes a filter disposed on the back surface of the chip substrate 11 and has a selectivity that allows the fluorescence wavelength of the enzyme activity detector 13 to pass but blocks the excitation wavelength. Reference numeral 24 denotes an image sensor such as a CCD disposed on the back side of the chip substrate 11 via a filter 23.
In the present embodiment, the chip substrate 11 is made of translucent glass or the like.
As a result, the biochip in the second embodiment can be made compact without requiring an optical system such as a lens, because the fluorescence of the enzyme activity detector 13 can be read as it is with the detection element of the image sensor 24 arranged in close contact. be able to.

FIG. 7 is a schematic diagram showing a modification of the biochip in which the image sensors in Embodiment 2 are stacked.
In FIG. 7, 30 is a biochip according to a modification of the second embodiment, 31 is a translucent chip substrate made of silicon oxide, and 32 is a sample solution channel formed in the chip substrate 31 by etching or the like in a groove shape. 32a is an enzyme activity detector arranged in the sample solution flow path 32, and 33 is an upper filter made of glass or organic material that is disposed so as to cover the opening surface of the sample solution flow path 32 and transmits fluorescence of a predetermined wavelength. , 34 is an image sensor substrate in which CMOS elements and the like are stacked on the back side of the chip substrate 31 via a lower filter 35, and 36 is a silicon oxide layer that protects the surface of the image sensor substrate 34. When the CMOS element or the like of the sensor substrate 34 is formed, the insulating layer (protective layer) is formed by means such as CVD.
The upper filter 33 has such selectivity that the excitation wavelength of the enzyme activity detector 32a is allowed to pass but the fluorescence wavelength is blocked, and the lower filter 35 is excited to pass the fluorescence wavelength of the enzyme activity detector 32a. The wavelength has such selectivity that it blocks.
The chip substrate 31 is formed by CVD or the like, similar to a silicon oxide layer formed by means such as CVD as an insulating layer (protective layer) when forming a CMOS element or the like of the image sensor substrate 34.
In the biochip 30 according to the present embodiment configured as described above, the enzyme activity detection unit 32a of the chip substrate 31 is provided on the projection surface of the lower filter 35 disposed directly on the silicon oxide layer 36 of the image sensor substrate 34. Since the chip substrate 31 is manufactured by using the same semiconductor manufacturing technology as that for forming the CMOS element or the like of the image sensor substrate 34, the manufacturing process can be shortened. .

Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples.
For the amino acids, peptides, protecting groups, solvents, and the like described in the present Examples, abbreviations commonly used in the technical field or abbreviations adopted by the IUPAC-IUB naming committee are used.
For example, the following abbreviations are used. Ala: alanine, Pro: proline, Lys: lysine, Phe: phenylalanine, Aca: aminocaproic acid, Ac: acetyl, ACC: 7-amino-4-carboxymethylcoumarin, Boc: t-butyloxycarbonyl, Lys (Boc): Side chain t-butyloxycarbonyl protected lysine, DCC: dicyclohexylcarbodiimide, DCM: dichloromethane, DIEA: N, N-diisopropylethylamine, DMF: N, N-dimethylformamide, EtOH: ethanol, Fmoc: 9-fluorenylmethyloxy Carbonyl, HATU: o- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, HBTU: o- (benzotriazol-1-yl) -1,1 , 3,3-tetramethyluronium hexafluorophosphate, HOAt: 1-hydroxy-7-azoben Triazole, HOBt: 1-hydroxybenzotriazole, TFA: trifluoroacetic acid.

In Example 1, as a preliminary experiment for forming an enzyme activity detection unit, a peptidyl fluorescent group-bound spherical substrate was synthesized as an enzyme activity detection substrate, and the activity on enzymes (trypsin and chymotrypsin) was measured. The method will be described below.
In addition, a biochip can be configured by filling and arranging such a spherical substrate in each region to be the enzyme activity detection units S1 to Sn of the sample solution flow path formed in a spiral shape or the like.
<Synthesis of peptidyl fluorescent group-bonded spherical substrate>
As a substrate, spherical NH ₂ -PEGA-resin (manufactured by Watanabe Chemical Industry) was used. The vessel for solid phase synthesis was fixed vertically, NH ₂ -PEGA-resin (0.05 mmol / g, 0.5 g) was added, DMF (10 ml) was allowed to flow with the vessel cock open, and the solvent was replaced. Then close the vessel cock and add Fmoc-Aca-OH (0.13 mmol, 44 mg), HBTU (0.13 mmol, 48 mg), DIEA (0.13 mmol, 0.022 ml) dissolved in DMF (2 ml), Reacted overnight. After the reaction, the cock was opened and washed with DMF (10 ml) and methanol (10 ml) to obtain Fmoc-Aca-PEGA resin.

Next, DMF (10 ml) was allowed to flow through the Fmoc-Aca-PEGA resin (0.05 mmol / g, 0.5 g) in the vessel with the cock open, and solvent substitution and washing were performed. The cock was closed and 20% piperidine / DMF was added and reacted for 30 minutes to remove Fmoc. The cock was then opened to remove 20% piperidine / DMF and then washed with DMF (10 ml). Next, with the cock closed, Fmoc-Aca-OH (3 eq), HATU (3 eq), HOAt (3 eq), and DIEA (5 eq) were dissolved in DMF (2 ml) and allowed to react for 3 hours. It was. The cock was then opened and washed with DMF (10 ml) and methanol (10 ml) to obtain Fmoc-Aca-Aca-PEGA resin in which the binding part (Aca-Aca-) was bound to the substrate (PEGA resin). .
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 mL). After washing three times with DCM (1 ml) and once with DMF (1 ml), Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol), DCC (11 mg , 54 mmol), HOBt · H 2 O (8.3 mg, 54 mmol) was added and allowed to react for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give a fluorescent group. Fmoc-Lys (Boc) -ACC-Aca-Aca-PEGA resin in which (ACC) was bound to the binding site (Aca-Aca-) was obtained.
Then, it was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml) and stirred for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to acetylate the unreacted product.

Subsequently, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Pro-OH (18 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin.
Hereinafter, Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin was used to repeat the same procedure using Fmoc-Ala-OH, and the peptide was extended to attach the binding moiety (Ala-Ala -Pro-Lys) bound to Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin. Then, the cock was closed and DIEA (2.5 mmol, 0.435 ml) and acetic anhydride (1.25 mmol, 0.117 ml) were diluted in DMF (2 ml) and reacted for 1 hour to acetylate the terminal group of the bond. Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin was obtained.
Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-PEGA resin (0.05 mmol, 0.5 mg) was placed in the vessel, and DCM (10 ml) was flowed with the cock open to replace the solvent. Then, close the cock and add 25% TFA / DCM (2 ml), react for 30 minutes, remove Boc, wash with DCM (10 ml), H2O (10 ml) The peptidyl fluorescent group-bonded spherical substrate (Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin) of Example 1 was obtained.

<Measurement of enzyme activity>
The peptidyl fluorescent group-bound spherical substrate Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin as the enzyme activity detection substrate of Example 1 was added to wells A to D of a 96-well fluorescence measurement microplate. The following solutions were added, and the difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes was measured. The fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVO ™ SX 1420 multilabel counter (manufactured by PerkinElmer).
A: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 240 μl, trypsin (1 mg / 1 ml) to 10 μl
B: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 240 μl, chymotrypsin (1 mg / 1 ml) to 10 μl
C: Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca-PEGA resin (wet) 5 mg, 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2) 250 μl
D: 250 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2),
The difference between well A and well B is the type of enzyme, and the difference between well A (or well B) and well C is the presence or absence of an enzyme. Well A (or well B, C) and well The difference from D is the presence or absence of a substrate for enzyme activity detection.
The difference between the fluorescence value at the start of the experiment and the fluorescence value after 30 minutes is shown in Table 1 for each well.

Comparing the fluorescence values of wells A and B and well C in Table 1, the enzyme activity detection substrate of Example 1 was about 315 times in well A where trypsin was present, and about 315 times in well B where chymotrypsin was present. It was confirmed that the difference was 10 times. Thus, it was shown that the enzyme activity corresponding to the amount of enzyme, the type of enzyme, and the like can be detected by applying the enzyme activity detection substrate of Example 1. Further, comparing the fluorescence values of well A and well B, the enzyme activity detection substrate of Example 1 has a fluorescence value in the case of trypsin that is approximately 30 times or more larger than that in the case of chymotrypsin. confirmed. This is because the amino acid of the binding part that binds to the fluorescent group of the enzyme activity detection substrate of Example 1 is lysine, and trypsin has the specificity of selectively cleaving the peptide bond mainly on the C-terminal side of lysine. It is guessed that it was expressed from On the other hand, chymotrypsin mainly has the specificity of selectively cleaving the peptide bond on the C-terminal side of the aromatic amino acid residue, so it is presumed that the change in fluorescence value before and after the reaction was small.
Accordingly, it was shown that the enzyme activity detection substrate of Example 1 has specificity depending on the enzyme, so that qualitative analysis of the enzyme having activity is possible. In addition, if a sample solution containing the same type of enzyme is brought into contact with the same type of enzyme activity detection substrate and the fluorescence value is measured after a predetermined time, the change in the fluorescence value is affected by the action of the enzyme. It was speculated that quantitative analysis of the enzyme was possible.

  In Example 2, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded planar substrate as a substrate for enzyme activity detection. The method will be described below. The enzyme activity detector can be formed by applying the synthesis method described below, and enzyme activity detectors having different enzyme specificities can be arranged in a predetermined pattern in each region of the sample solution flow path.

<Synthesis of a peptidyl fluorescent group-bonded planar substrate>
As the substrate, a synthetic resin carrier made by separating only one plate from a multi-plate synthetic resin carrier for peptide synthesis (Lantern series (registered trademark) manufactured by Mimotops Co., Ltd.) was used.
Put one synthetic resin carrier lantern (Mimotops D-series, introduction rate 18 mmol / piece) as a substrate in the screw tube and stir for 30 minutes with 20% piperidine / DCM (1 mL) to remove the Fmoc group. Went. After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Aca-OH (19 mg 54 mmol), DCC (17 mg 81 mmol), HOBt · H2O (8 mg 54 mmol) were DMF. It was dissolved in (1 ml) and added to react for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give Fmoc- Obtained Aca-lantern.
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Aca-OH (19 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Aca-Aca-lantern with one end of the binding site (Aca-Aca) immobilized on the substrate (lantern) It was.

Subsequently, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 mL). Fmoc-Lys (Boc) -ACC-OH (36 mg, 54 mmol), washed with DCM (1 ml) three times and DMF (1 ml) once to swell lantern and dissolved in DMF 1 ml, DCC (11 mg, 54 mmol) and HOBt · H2O (8.3 mg, 54 mmol) were added and reacted for 24 hours. After completion of the reaction, it was washed twice with DMF (1 ml), twice with DCM (1 ml), twice with EtOH (1 ml) and twice with DCM (1 ml), and then dried under reduced pressure to give a binding site. Fmoc-Lys (Boc) -ACC-Aca-Aca-lantern in which a fluorescent group (ACC) was bound to (Aca-Aca) was obtained.
Then, it was washed once with DMF (1 ml), DIEA (32 ml, 183 mmol) and acetic anhydride (8.5 ml, 90 mmol) were diluted in DMF (1 ml) and stirred for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to acetylate the unreacted product.
Next, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing 3 times with DCM (1 ml) and DMF (1 ml), Fmoc-Pro-OH (18 mg, 54 mmol), HATU (20 mg, 54 mmol), HOAt (7 mg, 54 mmol) ), DIEA (16 ml, 90 mmol) was dissolved in DMF (1 ml) and reacted for 1 hour. Then, it was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml) to obtain Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern.

Hereinafter, Fmoc-Pro-Lys (Boc) -ACC-Aca-Aca-lantern was repeated twice using Fmoc-Ala-OH to elongate the peptide, and bound to the fluorescent group (ACC) (Ala Fmoc-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-lantern to which -Ala-Pro-Lys) was bound was obtained.
Thereafter, the Fmoc group was removed by stirring for 30 minutes using 20% piperidine / DCM (1 ml). After washing with DMF (1 ml) 3 times with DCM (1 ml), DMF (1 mL), DIEA 32 ml (183 mmol) and acetic anhydride 8.5 ml (90 mmol) were added to DMF (1 mL). Diluted and added for 1 hour. Thereafter, the lantern was washed 3 times with DMF (1 ml) and 3 times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then, wash 3 times with DCM (1 ml), 5 times with H2O (1 ml), 3 times with DCM (1 ml), dry under reduced pressure, and end group of the binding site (Ala-Ala-Pro-Lys) Ac-Ala-Ala-Pro-Lys (Boc) -ACC-Aca-Aca-lantern was obtained in which was acetylated.
The Boc group of Lys side chain was removed using 25% TFA / DCM (1 ml), and the target enzyme activity detection substrate of Example 2 (Ac-Ala-Ala-Pro-Lys-ACC-Aca-Aca- lantern).

<Measurement of enzyme activity>
100 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl2), 100 μl of methanol, and 50 μl of trypsin (1 mg / 1 ml) were added to the enzyme activity detection substrate of Example 2 and reacted. The fluorescence values before and after the reaction were measured. The fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVO ™ SX 1420 multilabel counter (manufactured by PerkinElmer).
As a result, the initial fluorescence value was 39000, the fluorescence value after reaction for 18 hours was 145000, and it was confirmed that the fluorescence value changed about 4 times. Thereby, it was shown that the enzyme having activity can be detected even in the enzyme activity detection substrate of Example 2 using lantern as the substrate.

In Example 3, an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for enzyme activity detection. The method will be described below.
<Synthesis of a peptidyl fluorescent group-bonded planar substrate>
As the substrate, a synthetic resin carrier made flat by separating only one plate from a multi-plate synthetic resin carrier for peptide synthesis (Lantern series (registered trademark) manufactured by Mimotopus Co., Ltd.) was used.
Put 1 lantern of synthetic resin carrier lantern (Mimotops D-series, introduction rate 18 mmol / piece) into the screw tube and stir for 30 minutes with 20% piperidine / DCM (1 mL) to cut out the Fmoc group. It was. Fmoc-Aca-OH 20.0 mg (56.6 mmol), DCC 17.5 mg (84.5 mmol), HOBt in which lantern was swollen with DMF (1 ml × 1) and dissolved in 1 ml of DMF after DCM washing (1 ml × 3) -H2O 8.8 mg (57.5 mmol) was added and it stirred for 23 hours. DMF (1 ml x 2), DCM (1 ml x 2), DCM / EtOH = 1: 1 (1 ml x 2), EtOH (1 ml x 2), DCM (1 ml x 1), diethyl ether (1 The resin was washed with ml × 1) and dried to obtain Fmoc-Aca-lantern.

Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to excise the Fmoc group, washed with DCM (1 ml × 3), and then 23.0 mg of Fmoc-Aca-OH dissolved in 1 ml of DMF ( 65.1 mmol), HATU 21.6 mg (56.8 mmol), HOAt 8.1 mg (59.5 mmol), and DIEA 15.7 ml (90.2 mmol) were added and stirred for 1 hour. The plate was washed with DMF (1 ml × 3) and DCM (1 ml × 3) to obtain Fmoc-Aca-Aca-lantern in which one end of the binding portion (Aca-Aca) was immobilized on the substrate (lantern). Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 ml × 3) to obtain H-Aca-Aca-lantern.
Subsequently, Fmoc-Phe-ACC-OH (33.4 mg 56.7 mmol), DCC 12.5 mg (60.6 mmol), HOBt * H2O 8.9 mg (58.0 mmol) dissolved in 1 ml of DMF were added and stirred for 22 hours. DMF (1 ml x 2), DCM (1 ml x 2), DCM / EtOH = 1: 1 (1 ml x 2), EtOH (1 ml x 2), DCM (1 ml x 1), diethyl ether (1 After washing with ml × 1), drying was performed to obtain Fmoc-Phe-ACC-Aca-Aca-Lantern in which the fluorescent group (ACC) was bound to the binding portion (Aca-Aca).

Next, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, and washed with DCM (1 ml × 3). Next, Fmoc-Pro-OH 24.7 mg (73.2 mmol), HATU 21.4 mg (56.2 mmol), HOAt 8.2 mg (60.2 mmol), DIEA 15.7 ml (90.2 mmol) dissolved in DMF 1 ml were added and stirred for 1 hour. After washing with DMF (1 ml × 3) and DCM (1 ml × 3), Fmoc-Pro-Phe-ACC-Aca-Aca-lantern was obtained.
Thereafter, Fmoc-Ala-OH was introduced twice by introducing Fmoc-Ala-Pro-Phe with the binding moiety (Ala-Ala-Pro-Phe) bound to the fluorescent group (ACC). -Obtained ACC-Aca-Aca-lantern.
Then, the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group, washed with DCM (1 ml × 3), DMF (1 mL), DIEA 32 ml (183 mmol) and anhydrous Acetic acid 8.5 ml (90 mmol) was added and allowed to stir for 1 hour. Thereafter, washing with DMF (1 ml × 3) and DCM (1 ml × 3) is performed, and the terminal group of the binding site (Ala-Ala-Pro-Phe) is acetylated to achieve the target enzyme activity of Example 3 A peptidyl fluorescent group-bonded planar substrate Ac-Ala-Ala-Pro-Phe-ACC-Aca-Aca-lantern was obtained as a detection substrate.

<Measurement of enzyme activity>
The substrate for enzyme activity detection of Example 2 (introduction rate 2 μmol / substrate) in wells A and C of the microplate for fluorescence measurement, and the substrate for enzyme activity detection in Example 3 (introduction rate 2 μmol / substrate) in wells B and D Each was added with 100 μl of 20 mM Tris HCl buffer (pH 7.2, 100 mM NaCl, 50 mM CaCl 2) and 100 μl of methanol, and further 50 μg of the following enzyme was added and reacted.
A: Chymotrypsin B: Chymotrypsin C: Trypsin D: Trypsin The measurement was performed using a WALLAC ARVOTM SX 1420 multilabel counter (manufactured by PerkinElmer) at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm. The fluorescence value after 30 minutes was measured and the difference was obtained.
The difference in the fluorescence value calculated in each well is shown in (Table 2).

  According to Table 2, for chymotrypsin, a large change in the fluorescence value was confirmed on the enzyme activity detection substrate of Example 3 in which the amino acid at the binding portion that binds to the fluorescent group was one kind of phenylalanine of the aromatic amino acid. It was. In addition, for trypsin, a large change in fluorescence value was confirmed on the enzyme activity detection substrate of Example 2 in which the amino acid at the binding site that binds to the fluorescent group was lysine. The difference between these changes is that chymotrypsin has the specificity of selectively cleaving peptide bonds mainly on the C-terminal side of aromatic amino acid residues, and trypsin mainly reduces peptide bonds on the C-terminal side of lysine. It is presumed that it was expressed because of its specificity for selective cleavage. As a result, it became clear that the activity detection ability for the type of enzyme can be changed by changing the type of amino acid at the binding site that binds to the fluorescent group.

0.3 g of Aca was introduced into 1 g of ordinary resin for peptide synthesis as in Example 1 and divided into 361 groups. ACC was introduced into each of these resins using a peptide synthesizer, then 19 kinds of amino acid residues except cysteine were introduced twice, and finally capping with an acetyl group was performed after deprotection. Thereby, 361 kinds of peptides were synthesized on the resin.
Peptides were cut out from each resin to obtain 361 types of C-terminal unprotected peptides.
On a glass substrate or a silicon oxide layer (chip substrate) constructed as a protective layer (insulating layer) on an image sensor, a spiral sample solution flow path having a flow path width of 500 μm and a flow path depth of 100 μm is formed by etching. Formed. In addition, as a sample supply portion, a concave portion having a depth of 100 μm and a vertical and horizontal length of 2.5 mm was continuously formed at one end portion of the sample solution flow channel by etching.
The entire surface of the sample solution flow channel was coated with amino groups, and 361 types of peptides as the enzyme activity detection unit were introduced into the sample solution flow channel using a condensing agent (HATU). For the introduction, an automatic spotter (model: 222XL) manufactured by Gilson was used, and one kind of peptide was introduced into a circle of about 200 μm at the center of a flow path width of 500 μm, and another kind of peptide was spaced at an interval of about 100 μm. Peptides were introduced one after the other.
A filter (which cuts off wavelengths longer than 350 ± 10 nm) that presses the cover (protective layer) and transmits fluorescence excitation light was pressure-bonded to the upper surface of the sample solution flow channel into which the peptide was introduced. A fluorescent chip (which cuts off wavelengths shorter than 450 ± 10 nm) was adhered to the image sensor substrate, and a chip substrate was further disposed thereon to manufacture a biochip similar to that described in the second embodiment.

As a test sample, a solution of enzyme group 1 centered on trypsin and enzyme group 2 centered on chymotrypsin (20 nM, 5 mM HEPES pH 7.4) was prepared. This was introduced into each of the two biochips using a microsyringe, and the output of the image sensor from the start of introduction was recorded.
The fluorescence intensity of each spot (enzyme activity detection unit) was calculated every 10 msec from the output of the image sensor, and the enzyme activity (fluorescence intensity) for each spot was calculated based on that.

FIG. 8 is a graph in which the fluorescence intensity is plotted for each amino acid constituting each spot. In FIG. 8, the x-axis and the y-axis indicate the combination sequences of the respective amino acids, and the z-axis indicates the fluorescence intensity corresponding to the combination of the amino acids.
From FIG. 8, it was confirmed that the fluorescence intensity patterns output from the biochips having the same enzyme activity detection unit array differ between enzyme group 1 and enzyme group 2. Thus, for example, by using human body fluid as a sample solution and detecting the enzyme activity as image data periodically using the biochip of the present invention, it is determined whether or not the enzyme activity in the body fluid has changed. It was confirmed that comprehensive monitoring was possible and pathological diagnosis and the like could be performed accurately and promptly.

  The biochip of the present invention is applied to the analysis of sample solutions containing enzymes, proteins, and genes related in the fields of pathology, medical care, drug discovery, food, and environment, and comprehensively includes characteristic data such as enzyme activity. Can be evaluated and specified, and can contribute to medical fields such as pathological diagnosis and research such as proteome analysis.

Schematic diagram showing the enzyme activity detection principle of the enzyme activity detector Schematic diagram of enzyme activity detector with different arrangement Schematic perspective view of the biochip in the first embodiment Schematic diagram showing the state of the enzyme activity detectors arranged in the sample solution flow path Configuration diagram for measuring enzyme activity of sample solution introduced into biochip in embodiment 1 using image sensor Schematic diagram of biochip in embodiment 2 Schematic diagram showing a modification of the biochip in the second embodiment Graph plotting fluorescence intensity for each amino acid combination

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chip substrate 2 Fluorescent group 3 Oligopeptide 4 Enzyme 5 Fluorescent group in which the fluorescence wavelength was changed 6 Oligopeptide 7 First fluorescent group 7a First fluorescent group in which fluorescence is observed 8 Second oligopeptide 9 Second fluorescent group 10 Biochip 11 Chip substrate 12 Sample solution flow path 13 Enzyme activity detection unit 14 Liquid supply unit 15 Liquid storage unit 20 Image sensor 21 Lens 22, 23 Filter 24 Image sensor 30 Biochip 31 Chip substrate 32 Sample solution Flow path 32a Enzyme activity detection unit 33 Upper filter 34 Image sensor substrate 35 Lower filter 36 Silicon oxide layer

Claims (6)

  A biochip for detecting an enzyme in a sample solution introduced into a chip substrate, the sample solution flow path formed on the chip substrate in a flow path pattern such as a spiral shape, a tree shape, or a radial shape, and A biochip comprising a plurality of enzyme activity detectors arranged in a sample solution flow path in a predetermined order and having different activities with respect to a predetermined enzyme.   The enzyme activity detection unit is (a) a fluorescence whose fluorescence characteristics such as a fluorescence frequency and a fluorescence intensity are changed depending on a chemical bond state with a peptide chain that is bonded to the chip substrate on the base end side and to the other end side. And (b) an oligopeptide that is bound to the fluorescent substrate via an enzyme-specific binding portion that is imparted by a peptide chain having a specific amino acid sequence composed of a combination of amino acids. Item 10. The biochip according to item 1.   The biochip according to claim 1 or 2, wherein the fluorescent substrate is an aminomethylcoumarin-based fluorescent substrate and is bonded onto the chip substrate chemically modified with a silane coupling agent or the like.   4. The image sensor substrate according to claim 1, wherein the chip substrate is translucent, and an image sensor substrate on which a CCD, a CMOS element, and the like are arranged is laminated on the back side thereof. 5. Biochip.   5. The light-transmitting or non-light-transmitting protective layer is provided on a surface side of the chip substrate on which the sample solution flow path is formed. 5. Biochip. A sample solution supplying step of supplying a sample solution to the sample solution channel of the biochip according to any one of claims 1 to 5 under a predetermined condition;
A two-dimensional image on the chip substrate formed by a state change for each of the enzyme activity detectors arranged on the sample solution flow path is processed with a filter that removes the specific wavelength region, and the time series data or after a predetermined time A data acquisition process for acquiring the result data of
A data determination step of comparing the time series data or the result data with the library data stored in advance under the same measurement conditions to determine the degree of pattern matching with each library data by the pattern recognition means or the statistical data processing means,
A method for testing the functionality of a sample solution, comprising: