JP2006010391A - Manufacturing method of specimen for organism-related substance, and testing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of a specimen for an organism-related substance capable of detecting and quantitating accurately a sample material to be reacted specifically with a probe, even when using a plurality of specimens having different probe solidification rates relative to each specimen. <P>SOLUTION: Information on the solidification rate of a probe for solidification rate inspection solidified together with a probe for the reaction is preserved in an information preservation part of the specimen. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a test piece manufacturing method and a test apparatus used for analysis of biologically relevant substances such as DNA.

  The human genome project to determine the entire base sequence of a huge human genome of about 3 billion bp and analyze them has shifted the focus from systematic base sequence to systematic functional analysis.

  By the way, the specific contents of the genetic information relate to (1) what kind of protein is synthesized and (2) under what conditions the protein is synthesized. Regarding (1), analysis methods such as Western blotting, Northern blotting, Southern blotting, etc. are widely used. However, these methods analyze only specific proteins extracted from cells, DNA, and RNA, and are not always suitable for simultaneous analysis of other proteins, DNA, and RNA at the same time. Not.

  On the other hand, regarding the condition under which (2) the protein is synthesized, since the control of the transcription level is deeply involved, it is difficult to perform a sufficient analysis with the conventional analysis method. there were. The biggest cause is the lack of data on both the control sequence in DNA and the control content corresponding to it.

However, recently, with the advent of technologies called DNA chips and DNA microarrays, the analysis of gene expression information is expected to make dramatic progress, making it easier to analyze the conditions under which the above proteins are synthesized. It is expected to become. These chips and microarrays are obtained by immobilizing arbitrary oligonucleotides on a carrier surface of about 1 cm ² at high density. A DNA chip is obtained by dividing a silicon chip into a number of sections by photolithography, and directly synthesizing single-stranded DNA having a specific base sequence on each section. In the DNA microarray, a carrier that has been a conventional membrane is used as a slide glass, and the spot size of each spot on the membrane carrier is about 300 μm or more, and is reduced to about 200 μm or less. Therefore, using a DNA microarray makes it easy to analyze many samples at once.

A DNA chip or DNA microarray is connected to a signal reader and a computer system for controlling the signal reader. When a probe arranged on the chip or on the microarray and the sample nucleic acid are hybridized, the sample nucleic acid and the probe are separated from each other. A signal (fluorescence signal or the like) emitted from the complex is detected and analyzed. It can be used for various applications such as gene DNA mutation analysis, polymorphism analysis, nucleotide sequence analysis, and expression analysis, depending on the type of DNA and its arrangement on the DNA chip or DNA microarray.
JP-A-11-187900 Japanese Patent No. 3442327 Japanese Patent No. 3350421

  However, analysis using DNA microarrays has just begun to discuss the fabrication of microarrays and their detection devices, and there are still many problems.

  As a method for producing a DNA microarray, for example, an apparatus called a spotter is used to spot oligo DNA or cDNA on a substrate. Spotting methods include “contact printing method” in which pins are placed in direct contact with a slide glass to place oligo DNA or cDNA, or “contactless printing” in which oligo DNA or cDNA is blotted on a slide glass using inkjet technology. Law ".

  In the above spot method or the like, it is difficult to chemically immobilize oligo DNA or cDNA on a glass slide on a substrate in the first place. In some cases, the oligo DNA or cDNA is simply supplied onto the substrate and may not be solidified at all. Accordingly, the fact is that there is a variation in the amount of immobilized probe between microarrays of different lots, which are produced at different times.

  As described above, if the amount of the solid phase of the probe varies among DNA microarrays (substrates), it may adversely affect the results of analysis using a plurality of DNA microarrays, particularly analysis requiring quantitativeness. In addition to the problem of quality control.

  As a method for confirming the amount of oligo-DNA or cDNA immobilized on a carrier, there is a method of actually judging by hybridization as described in Patent Document 1, for example. Further, in Patent Document 2 and Patent Document 3, more accurate hybridization is performed by obtaining the amount of immobilized probe by fluorescently labeling all probes immobilized on a carrier and using this for correction. The quantity can be quantified. However, in these methods, (1) the cost is increased because all the probes are labeled, and (2) the steric hindrance is caused in the hybridization reaction due to the fluorescent dye for quantifying the amount of immobilized probe. (3) Since detection is performed by two-color fluorescence of a fluorescent dye for hybrid detection and a fluorescent dye for determination of the amount of solid phase, the detection apparatus becomes complicated, and ( 4) There are problems such as the need to correct the luminous efficiency of the fluorescent dye.

The present invention has been made in view of such problems, and is characterized by the following configuration. That is, the method for producing a test piece for a biological substance according to the present invention includes:
Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step (C) of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier;
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating a solid phase immobilization rate (probe solid phase immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
The probe immobilization rate, and the probe supply amount and the probe immobilization amount, as appropriate, are stored in an information storage unit provided in the carrier, or in an information storage unit provided separately from the carrier. Storing together with the identification number of the carrier (F);
Is included.

In addition, the method for producing a test piece for a biological substance according to the present invention includes:
Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier (C);
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating an immobilization rate (probe immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
A step (E ′) of comparing the predetermined minimum immobilization rate with the probe immobilization rate; and
When the probe immobilization rate is equal to or higher than the minimum immobilization rate,
The probe immobilization ratio, as appropriate, the probe supply amount and the probe solid phase immobilization amount are stored in an information storage unit provided in the carrier, or an information storage unit provided separately from the carrier Or the step (F) of storing together with the identification number of the carrier, or if the probe immobilization rate is less than the minimum immobilization rate,
An information storage unit that is excluded from the manufacturing process, or that stores information indicating that the carrier is defective is stored in an information storage unit provided in the carrier, or is provided separately from the carrier And storing together with the identification number of the carrier (F ′);
Is included.

Furthermore, the method for producing a test piece for a biological substance according to the present invention includes:
Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier (C);
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating a solid phase immobilization rate (probe solid phase immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
The probe immobilization ratio, as appropriate, the probe supply amount and the probe solid phase immobilization amount are stored in an information storage unit provided in the carrier, or an information storage unit provided separately from the carrier And storing together with the identification number of the carrier (F);
Compare the probe solid-phase rate stored in the information storage unit with a predetermined minimum solid-phase rate,
When the probe immobilization rate is not less than the minimum immobilization rate, the test piece having the probe immobilization rate is determined as “pass”, or the probe immobilization rate is the minimum immobilization rate. When it is less than the phase rate, the test piece having the probe solid phase rate is determined as “fail”,
A step (G) of separating a pass product and a reject product;
Is included.

  In the above-described method for producing a test piece for a biological substance according to the present invention, the quantification in the steps (B) and (D) may be performed by measuring a spectrum specific to the probe for immobilization rate test. In addition, the measurement of the spectrum unique to the probe for immobilization rate test can be a signal measurement derived from a fluorescent dye introduced in advance to the probe for immobilization rate test.

The method for measuring a biological substance of the present invention includes:
It is a carrier having an information storage unit in which the probe immobilization rate on the carrier, and the probe supply amount and the probe immobilization amount are appropriately recorded, or the probe immobilization rate, and the probe supply amount and A probe in which the amount of immobilized probe is recorded in an information storage unit provided separately from the carrier together with the identification number of the carrier, and a probe that specifically reacts to the sample substance Supplying the sample substance to the carrier immobilized on the spot (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
The measured intrinsic spectral intensity is corrected by the probe immobilization rate or probe immobilization amount recorded in the information storage unit, and a predetermined standard immobilization rate or value per standard immobilization amount. Converting to (IV);
Is included.

The method for measuring a biological substance of the present invention includes:
It is a carrier having an information storage unit in which the probe immobilization rate on the carrier and, if appropriate, the probe supply amount and the probe immobilization amount are recorded, or the probe immobilization rate, and the probe supply amount as appropriate In addition, the amount of immobilized probe and the identification number of the carrier are recorded in the information storage unit provided separately from the carrier, and a probe that specifically reacts to the sample substance is predetermined. Supplying the sample substance to the carrier immobilized on the spot of (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
In a method for measuring a biological substance,
Ratio of probe immobilization rate or probe immobilization amount of test piece to standard value (standard immobilization rate or standard immobilization amount) of test sample probe immobilization rate or probe immobilization amount (standard immobilization rate or standard immobilization amount) Calculated by dividing the predetermined standard reaction time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization rate by the calculated ratio). The steps (I) and (II) are performed by using the time as the reaction time from the start of the step (I) to the start of the step (II).

Furthermore, the method for measuring a biological substance of the present invention includes:
It is a carrier having an information storage unit in which the probe immobilization rate on the carrier and, if appropriate, the probe supply amount and the probe immobilization amount are recorded, or the probe immobilization rate, and the probe supply amount as appropriate And a probe that is recorded in an information storage unit provided separately from the carrier, in which the amount of immobilized probe is combined with the identification number of the carrier, and a probe that specifically reacts to a sample substance. Supplying a sample substance to a carrier immobilized on a predetermined spot (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
In a method for measuring a biological substance,
Ratio of probe immobilization rate or probe immobilization amount of test piece to standard value (standard immobilization rate or standard immobilization amount) of test sample probe immobilization rate or probe immobilization amount (standard immobilization rate or standard immobilization amount) Calculated by dividing the predetermined standard measurement time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization rate or solid phase immobilization amount by the calculation ratio). The step (III) is performed using the time as the measurement time of the step (III).

  In the above-described method for measuring a biological substance according to the present invention, the measurement of the intrinsic spectrum can be a measurement of a signal derived from a fluorescent dye introduced in advance into a sample substance.

Moreover, the detection apparatus for biologically related substances of the present invention is
A fluid transport unit for supplying a fluid to a test piece for detecting a biological substance and for removing the fluid from the test piece;
A temperature control unit for controlling the reaction temperature in the test piece;
Light source to the specimen;
A spectrum detector for detecting a spectrum from the test piece;
An information storage unit provided in the test piece, or a test piece information reading unit for reading information stored in an information storage unit provided separately from the test piece; and a signal detected by the spectrum detection unit A storage unit storing a signal correction application for correcting the intensity based on the information stored in the test piece information reading unit;
And a biologically relevant substance detection device controlled by a computer.

Furthermore, the measurement signal correction program of the present invention includes:
On the computer,
A procedure for inputting a standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. A procedure for reading and obtaining a ratio (calculation ratio) of the probe immobilization rate or the probe immobilization amount to the standard immobilization rate or the standard immobilization amount;
A procedure for obtaining a standard solid phase immobilization rate or a value per standard solid phase immobilization amount (conversion value) by reading a measured signal value from the test piece and dividing this by the calculated ratio; and
Procedure for outputting the converted value;
Is a program for executing

The correction program of the present invention is
On the computer,
The standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece and the probe immobilization rate or probe immobilization amount of the test piece are standard. A procedure for inputting a reaction time of the test piece or a measurement time of a signal from the test piece in the case where the solidification rate or the standard solidification amount is the same;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. A procedure for reading and determining the ratio (calculation ratio) of the probe immobilization rate or the amount of probe immobilization to the standard immobilization rate or the standard immobilization amount;
Procedure for determining the actual reaction time or measurement time in the test piece by dividing the reaction time or measurement time by the calculated ratio, and instructing this to the reaction time control unit or signal measurement unit of the test piece ;
Is a program for executing

  The present invention further provides a computer-readable recording medium in which the above program is recorded.

  According to the present invention, it is possible to produce a test piece in which the solid-phase rate of the probe on the carrier is known, the quality control becomes easy, and further, when a fluorescent label or the like is added to the probe. Since it is essentially possible to label only the solid-phase-immobilization rate test probe and not label the reaction probe, it is possible to reduce the manufacturing cost as compared with the conventional manufacturing method. In addition, if the test piece manufactured in this way is used together with the apparatus and program of the present invention, even when a measurement is performed using a plurality of carriers having different solid phase immobilization rates, the measurement result is more accurate. Quantitative analysis can be performed, and analysis with higher reliability can be provided.

(Definition)
In the present application, the “sample substance” means a biological substance to be analyzed and prepared for analysis. This includes nucleic acids such as DNA, cDNA, and RNA; various hormones, tumor markers, proteins such as enzymes, antibodies, and antigens; and complexes such as PNA, which are naturally derived substances. May also be prepared artificially.

  The “probe” is a substance known to have a property of specifically binding to the above sample substance. For example, when the sample substance is a nucleic acid, the probe is a base of the nucleic acid. A nucleic acid having a base sequence complementary to the sequence. Other examples of combinations of probes and sample substances include antigens for antibodies and vice versa, receptors for ligands and vice versa, and regulatory sequence-containing nucleic acid molecules for gene expression control proteins such as transcription factors The reverse is the case.

  In the probe immobilization method for biologically relevant substances of the present invention, as a pre-stage, pretreatment can be performed on the solid phase immobilization part of the carrier on which the probe is solid phased. Pretreatment includes treating the solid phase part of the carrier with a substance such as poly-L-lysine. Pretreatment is performed in advance to impart a property of reacting with a group in the probe (a group originally provided in the probe, or an artificially introduced group such as an amino group) on the surface of the carrier. Alternatively, instead of such pretreatment, it is also possible to use a carrier having a metal oxide film or the like having reactivity with the probe on the surface.

And in the solid-phase immobilization method of the probe for biological substances of the present invention, first, the above step (A) is carried out. In step (A), two types of probes, “reaction probe” and “immobilization rate test probe”, are immobilized on a carrier. The reaction probe is a probe that specifically binds to a sample substance. On the other hand, the immobilization rate test probe is the same class as the reaction probe and satisfies the following conditions. That is,
(1) The solid phase reaction on the carrier has substantially the same properties as the reaction probe; and (2) has sufficient quantitativeness in the system in which the probe is used, preferably Must emit a unique spectrum that can be distinguished from other substances in the system.

  As a specific example of the probe for immobilization rate satisfying these conditions, when the reaction probe is a nucleic acid molecule, the immobilization rate test probe is also a nucleic acid molecule, and the reaction probe is a protein. The solid phase-immobilization rate test probe is also a protein and can be further labeled with a fluorescent label or the like. More preferably, the immobilization rate test probe is immobilized on a carrier, and is the same type of probe as one of a plurality of types of reaction probes that are also immobilized on the carrier, and further a fluorescent label The thing which attached | subjected etc. can be mentioned. As a label to be added, in addition to a fluorescent dye, a group that emits a characteristic spectroscopic spectrum or a radioactive label can be used. Here, the label or group to be attached to the probe for immobilization rate inspection needs to be attached to the probe in a form that does not substantially affect the reaction with the sample substance and the reaction with the carrier. In the present invention, it is essentially possible to give a label only to the probe for immobilization rate test and not to give any label to the probe for reaction.

  The probe can be immobilized on the support using a spotter or the like in a method known in the art. More specifically, when the probe is a nucleic acid molecule, for example, a necessary amount of a probe having an amino group introduced in advance at the 3 ′ end is supplied to a predetermined spot on the carrier, The probe can be solid-phased by forming a bond with poly-L-lysine previously introduced onto the carrier.

  After supplying the probe to the carrier, the above step (B) is performed. The quantification is performed by measuring a probe-specific spectrum that can be distinguished from other substances in the system in which the probe is used, and more specifically, derived from a probe label, such as a fluorescent label. This is done by measuring the signal to be transmitted. For quantification, the measured value (signal intensity) is used as it is indirectly representing the probe supply amount, or the probe supplied in step (A) is applied to a calibration curve prepared in advance. It is also possible to convert to the absolute amount. When using a calibration curve, it is desirable that the linearity be ensured between the probe supply amount and the measurement signal. Unlike these methods, when the uniformity of the probe solution to be supplied and the accuracy of the probe supply amount by the spotter are guaranteed, the step of checking the concentration of the probe solution and the supply amount of the spotter You can also In this case, in this step, the spectrum specific to the probe for immobilization rate inspection is not measured, but the probe solution concentration and the probe supply amount are appropriately set according to the following information storage unit or a separate test piece manufacturing system. This can be recorded in a recording unit or the like of a computer connected to, and used as a process for calculating the solid phase rate later.

  Next, said process (C) is performed. The washing is desirably performed after a lapse of time considered that the solid phase reaction between the probe and the carrier supplied in the step (A) reaches equilibrium. Usually, it is preferable to perform the above steps (A) and (B) under the condition that the reaction has reached equilibrium after the elapse of time for performing steps (A) and (B). Is considered to be longer than the total time of the above steps (A) and (B), in this step (C), after an appropriate time has elapsed, that is, after the time when it is considered that an equilibrium state has been reached. It is desirable to start cleaning. In particular, when there is a time difference in the cleaning of each spot, if the cleaning is started before the reaction reaches equilibrium, the amount of the solid phase for each spot will vary, and the solidification rate test will be performed. Since each reaction probe may be immobilized on the carrier at a solid phase rate different from the solid phase rate of the probe, it is not desirable.

  Next, said process (D) is performed. In this step (D), the amount of the probe remaining on the carrier after washing, that is, only the probe considered to be immobilized on the carrier among the probes supplied to the carrier before washing is quantified. As in the above step (B), the quantification here can be carried out using the measured value (signal intensity) as it is to represent the supply amount (relative amount) or using a calibration curve prepared in advance. It is also possible to convert to the absolute amount of the probe immobilized on the carrier. The former case is effective when the amount of probe supplied to the carrier is always constant. On the other hand, the latter case is also effective when the amount of probe supplied to the carrier varies depending on the carrier, or the amount supplied varies from carrier to carrier. The amount of immobilized probe calculated in this step is appropriately recorded together with the above-mentioned probe supply amount in the following information storage unit, a recording unit of a computer separately connected to the test piece manufacturing system, etc. It can use for the calculation process of a phase conversion rate.

  Next, said process (E) is performed. The immobilization rate calculated here is the ratio of the measured value in the step (D) to the measured value in the step (B), that is, a signal derived from the probe that is considered to be immobilized on the carrier. It means the ratio of the value (probe amount) to the signal value (probe amount) derived from all probes supplied to the carrier. When the probe supply amount to the carrier is constant regardless of the carrier, the ratio of the signal values may be obtained. On the other hand, when the probe supply amount to the carrier is changed or when the fluctuation amount cannot be ignored. In the steps (B) and (D), signal measurement is performed under the condition that linearity is ensured between the measurement signal and the actual amount of the probe, and the amount of the probe is appropriately quantified with a calibration curve. It is desirable to determine the quantitative ratio.

  Next, said process (F) is performed. The solid-phase ratio calculated in the above step (E) is useful information in terms of quality control of the biological related substance test piece. Therefore, by storing this in the information storage unit provided in the carrier for which the solidification rate has been calculated, and using this information appropriately in subsequent experiments and quality control, correction of analysis results and testing It becomes easy to perform quality control of the pieces. Together with the probe immobilization rate, the probe supply amount and the probe immobilization amount can also be stored in the information storage unit. Here, the “information storage unit provided on the carrier” includes an IC chip, an IC tag, a barcode, and the like. On the other hand, the “information storage unit provided separately from the carrier” may be a storage medium that can be read by a computer system used in an analysis system using a test piece for a biological substance. In this case, it is necessary to record the solid phase rate in the reading unit in combination with an identification number assigned in advance to the carrier for which the solid phase rate has been calculated.

  Through the above steps (A) to (F), it is possible to produce a test piece for a biological substance with a solid phase ratio of the probe clarified.

The present invention also provides the above steps (A) to (E), and
A step (E ′) for comparing the predetermined minimum immobilization rate with the probe immobilization rate calculated in the step (E); and
When the probe immobilization rate is equal to or higher than the minimum immobilization rate,
The probe immobilization ratio, as appropriate, the probe supply amount and the probe solid phase immobilization amount are stored in an information storage unit provided in the carrier, or an information storage unit provided separately from the carrier (F) storing together with the identification number of the carrier, or when the probe immobilization rate is less than the minimum immobilization rate,
An information storage unit that is excluded from the manufacturing process, or that stores information indicating that the carrier is defective is stored in an information storage unit provided in the carrier, or is provided separately from the carrier And storing together with the identification number of the carrier (F ′);
There is also provided a method for producing a test piece for a biological substance, comprising:

  In this manufacturing method, after the steps (A) to (E), the predetermined solid phase immobilization rate is compared with the probe solid phase immobilization rate. If it is equal to or higher than the minimum immobilization rate, it is determined that a necessary and sufficient amount of the probe has been immobilized, and the immobilization rate, as appropriate, the probe supply amount and the probe immobilization amount, The step (F) of storing in the information storage unit provided in the carrier or storing the information in the information storage unit provided separately from the carrier together with the identification number of the carrier is performed. On the other hand, if the probe immobilization rate is less than the minimum solid phase immobilization rate, it is a quality control problem, so the processing for the carrier is stopped, and the carrier is discarded and reprocessed. Or the information indicating that the carrier is defective is stored in the information storage unit provided in the carrier, or the carrier identification number is provided in the information storage unit provided separately from the carrier. And the step (F ′) of storing together. When information indicating a defect is stored in the information storage unit, it can be distinguished from other normal products as a defective product in a quality control process after the manufacturing process.

The present invention also compares the above-described steps (A) to (E) and (F), and the probe immobilization rate stored in the information storage unit with a predetermined minimum immobilization rate,
When the probe immobilization rate is not less than the minimum immobilization rate, the test piece having the probe immobilization rate is determined as “pass”, or the probe immobilization rate is the minimum immobilization rate. When it is less than the phase rate, the test piece having the probe solid phase rate is determined as “fail”,
A step (G) of separating a pass product and a reject product;
There is also provided a method for producing a test piece for a biological substance, comprising:

  In the manufacturing method described above, even when the steps (A) to (F) are performed, even if the probe immobilization rate is less than the minimum immobilization rate, the probe immobilization rate is stored in the information storage unit. . Therefore, in order to exclude test pieces of a certain quality or lower from the production line, it is necessary to perform a quality control process after the production by comparing the probe solidification rate with the minimum solidification rate. Regardless of whether it is a product or a rejected product, a test piece is once completed, and then a quality control process is performed. Thus, the production line can be simplified because the pure production process is not affected by the quality control process. Furthermore, in the case of this production method, it is possible to flexibly set the minimum solid phase immobilization rate.

The present invention also provides
It is a carrier having an information storage unit in which the probe immobilization rate on the carrier, and the probe supply amount and the probe immobilization amount are appropriately recorded, or the probe immobilization rate, and the probe supply amount and A probe in which the amount of immobilized probe is recorded in an information storage unit provided separately from the carrier together with the identification number of the carrier, and a probe that specifically reacts to the sample substance Supplying the sample substance to the carrier immobilized on the spot (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
The measured intrinsic spectral intensity is corrected by the probe immobilization rate or probe immobilization amount recorded in the information storage unit, and a predetermined standard immobilization rate or value per standard immobilization amount. Converting to (IV);
(In this case, when correcting with the probe immobilization rate recorded in the information storage unit, it is converted to a value per standard solid phase immobilization rate, and the information storage unit When correcting with the recorded probe immobilization amount, it is converted into a value per standard immobilization amount).

  In the biological material measuring method of the present invention, first, the step (I) is performed. In this step, if the reaction probe contains a substance that specifically reacts with the sample substance, it is necessary to set conditions under which a complex is formed and exists stably. is there. Here, unlike conventional methods (for example, those described in Patent Document 2 and Patent Document 3), the reaction probe does not need to be labeled. The immobilization rate (or amount of immobilization) of the reaction probe to the carrier is indirectly determined from the immobilization rate (or immobilization amount) of the probe (for immobilization rate test) recorded in the information storage unit. Stipulated in

  However, in the step (I), those that do not react with the reaction probe and those that react non-specifically with the reaction probe or the carrier surface are mixed, so the above washing step (II) is then performed. Do. Washing is performed under a composition and conditions that are considered not to dissociate the formed complex. More specifically, a solution having the same composition as the sample solution except that it does not contain the sample substance can be used as the cleaning solution, and the conditions (temperature, etc.) can be the same as in the above step (I). The volume is preferably at least several times that of the sample solution.

  After this step (II), the above step (III) is performed to quantify the complex, and a spectrum for confirming the formation of the complex is measured. Depending on the combination of the probe and the sample material, it may be possible to measure the spectrum specific to the complex between them, in which case it is not necessary to introduce a label in advance in the sample material, Usually, a signal derived from a fluorescent dye or the like previously introduced into the sample substance is measured.

  Next, the above step (IV) is performed, and the measured spectral intensity is corrected in consideration of the probe immobilization rate or the amount of probe immobilization in the carrier subjected to the measurement. Since the carrier used in this method stores the probe immobilization rate and the probe supply amount and the probe immobilization amount as appropriate in the information storage unit, the probe immobilization rate etc. Read. By dividing the read probe immobilization rate or probe immobilization amount by the predetermined standard immobilization rate or standard immobilization amount, respectively, the standard immobilization rate or standard immobilization amount Convert to value. When the amount of probe supplied to the carrier is always constant, the probe immobilization rate and the standard immobilization rate can be used. On the other hand, the probe supply amount to the carrier is different for each carrier. If it fluctuates, conversion is performed using the probe supply amount and the standard solid phase amount.

  In the above measurement method, the measured signal is corrected after measurement, but unlike this, the reaction time between the sample substance and the probe on the test piece in consideration of the solidification rate or solidification amount. It is also possible to adjust the measurement time of the signal from the test piece.

That is, in the measurement method including the steps (I) to (IV),
The probe immobilization rate or probe immobilization amount of the test piece relative to the standard values (standard immobilization rate and standard immobilization amount, respectively) for the probe immobilization rate or probe immobilization amount of the test piece, respectively. The predetermined standard reaction time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization amount as the standard solid immobilization rate or the standard solid immobilization amount is divided by the ratio (calculation ratio) of By performing the steps (I) and (II) as the reaction time from the start of the step (I) to the start of the step (II) It is possible to determine and measure the reaction time in consideration of the difference in the amount of acidification.

In the measurement method including the steps (I) to (IV),
The probe immobilization rate or probe immobilization amount of the test piece relative to the standard values (standard immobilization rate and standard immobilization amount, respectively) for the probe immobilization rate or probe immobilization amount of the test piece, respectively. The standard measurement time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization amount as the standard solid immobilization rate or the standard solid immobilization amount is divided by the ratio (calculation ratio) of By performing the step (III) with the time obtained by the above as the measurement time of the step (III), the measurement time considering the difference in the probe immobilization rate or the amount of probe immobilization is determined and measured. It is possible.

The present invention further relates to an apparatus for detecting a biological substance as shown in FIG.
A fluid transport unit for supplying a fluid to a test piece for detecting a biological substance and for removing the fluid from the test piece;
A temperature control unit for controlling the reaction temperature in the test piece;
Light source to the specimen;
A spectrum detector for detecting a spectrum from the test piece;
An information storage unit provided in the test piece, or a test piece information reading unit for reading information stored in an information storage unit provided separately from the test piece; and a signal detected by the spectrum detection unit A storage unit storing a signal correction application for correcting the intensity based on the information stored in the test piece information reading unit;
And those controlled by a computer are also provided.

  The fluid transport unit provided in the biological material detecting device of the present invention supplies the sample material solution stored in the sample material solution storage unit (not shown) to the test piece without leakage, and the test piece. This is to eliminate this more. This is preferably such that the supply amount, supply speed (flow velocity), etc. are adjusted by a flow rate control system using a pump suitable for transporting a solution of μl. Moreover, it is preferable that the sample solution storage part and the fluid transport part have a temperature control function.

  The temperature control unit provided in the biological substance detection device of the present invention controls the reaction temperature performed on the test piece, and preferably can set the temperature in units of about 0.1 ° C. is there. In addition, it is desirable to link with the temperature control function of the sample solution storage unit and the fluid transport unit.

  The light source provided in the biological material detection apparatus of the present invention emits a light beam having a wavelength suitable for generating a spectrum to be detected, and depends on spectroscopic properties such as a label to be detected. Is selected.

  The spectrum detection unit provided in the biologically relevant substance detection device of the present invention is for detecting a signal emitted from a probe-sample substance complex, for example, from a fluorescent label introduced into the sample substance. In the case of detecting a signal, a CCD camera or the like can be used. The signal detected by the spectrum detection unit is converted into an appropriate electrical signal and transmitted to a control device such as a computer.

  The test piece information reading unit provided in the biologically relevant substance detection device of the present invention is a dedicated reader when the information storage unit is an IC chip or an IC tag, and the information storage unit is a barcode. Is a bar code reader.

  The storage unit in which the signal correction application stored in the biological material-detecting device of the present invention is stored is a computer-readable storage medium that controls the detection device.

The present invention also provides a measurement signal correction program,
On the computer,
A procedure for inputting a standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. Reading, obtaining the ratio (calculation ratio) of the probe immobilization rate or the probe immobilization amount with respect to the standard immobilization rate or the standard immobilization amount, and appropriately calculating the calculated ratio as the information storage unit Procedure for storing and reading this;
A procedure for obtaining a standard solid phase immobilization rate or a value per standard solid phase immobilization amount (conversion value) by reading a measured signal value from the test piece and dividing this by the calculated ratio; and
Procedure for outputting the converted value;
A measurement signal correction program is also provided.

  The CPU of the computer in which the measurement signal correction program of the present invention is installed receives a command from the program in the main memory, and first asks the user from the input device such as a keyboard, the probe solid phase ratio or the probe. The user is prompted to input a standard value (standard solidification rate or standard solidification amount) regarding the amount of solid phase, and the input value is displayed on the output device.

  The CPU determines whether or not the above value has been input. If the value is input, the CPU receives an instruction from the program, and reads the information storage unit provided in the test piece or the test piece via the reading unit. Read out the probe immobilization rate or the amount of probe immobilization unique to the test piece, which is stored in an information storage unit provided separately. When the probe immobilization rate or the probe immobilization amount is read, the CPU calculates a ratio (calculation ratio) of any of these to the standard immobilization rate or the standard immobilization amount. The calculation ratio is recorded in the information storage unit as appropriate.

  Next, the CPU determines whether the signal measurement value is transmitted from the signal measuring means for measuring the signal from the test piece, and when it is transmitted, receives the signal and holds it in the memory or stored in the information storage unit. Read the calculated ratio. And CPU calculates the value (conversion value) per standard solidification rate or standard solidification amount by performing the calculation which divides the said signal measurement value by the said calculation ratio, This is a user and an output device. Output to.

  By executing the above procedure, it becomes possible to obtain a correction value of the signal measurement value in consideration of the probe immobilization rate that varies from specimen to specimen. Even when a plurality of test pieces having different conversion rates are used, the sample substance can be detected quantitatively.

The present invention further comprises a program comprising:
On the computer,
The standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece and the probe immobilization rate or probe immobilization amount of the test piece are standard. A procedure for inputting a reaction time in the test piece or a measurement time of a signal from the test piece in the case where the solidification rate or the standard solidification amount is the same;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. Reading, obtaining the ratio (calculation ratio) of the probe immobilization rate or the amount of probe immobilization to the standard immobilization ratio or the standard immobilization amount, and appropriately storing the calculated ratio in the information storage unit And further reading this procedure;
Procedure for determining the actual reaction time or measurement time in the test piece by dividing the reaction time or measurement time by the calculated ratio, and instructing this to the reaction time control unit or signal measurement unit of the test piece ;
Provide a program to execute.

  The CPU of the computer in which the program of the present invention is installed receives a command from the program in the main memory, and first asks the user from the input device such as a keyboard, the probe immobilization rate of the test piece or the probe immobilization. The standard value (standard immobilization rate or standard immobilization amount) for the amount and the probe immobilization rate or probe immobilization amount of the test piece are the same as the standard immobilization rate or standard immobilization amount. In this case, the user is prompted to input the reaction time of the test piece or the measurement time of the signal from the test piece, and the input value is displayed on the output device.

  The CPU determines whether or not the above value has been input. If the value is input, the CPU receives an instruction from the program, and reads the information storage unit provided in the test piece or the test piece via the reading unit. Reads out the probe immobilization rate or the amount of probe immobilization specific to the test piece, which is stored in an information storage unit provided separately. When the probe immobilization rate or the amount of probe immobilization is read, the CPU calculates the ratio (calculation ratio) of any of these to the standard immobilization rate or the standard immobilization amount, The calculation ratio is recorded in the information storage unit as appropriate.

  Next, the CPU calculates the actual reaction time or measurement time in the test piece by dividing the reaction time or the measurement time by the calculation ratio, and calculates the reaction time control unit of the test piece. Alternatively, the signal measurement unit is instructed.

  By executing the above procedure, the present program can correct the sample reaction time or the signal measurement time in consideration of the probe immobilization rate that varies from specimen to specimen. Therefore, the signal value measured while executing this program is a value converted in consideration of the probe immobilization rate, so when using multiple test pieces with different probe immobilization rates Even so, it is possible to quantitatively detect the sample substance.

  The computer that executes the program and the computer that is included in the detection apparatus read and execute the program and other necessary programs recorded on a computer-readable recording medium. Each of them is subjected to appropriate processing and an appropriate function is exhibited. Here, the computer-readable recording medium means a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like. Further, the program may be transmitted to a computer via a communication line, and the computer that receives the distribution may execute the program.

  A slide glass carrier is coated with poly-L-lysine, 100 kinds of known specific binding substances, unlabeled oligo DNA (probe for reaction), and one kind of known 5 ′ FITC-labeled oligo DNA The (immobilization rate inspection probe) was supplied to a predetermined position on the carrier using an inkjet spotter. In both the reaction probe and the probe for inspecting the solid phase, an amino group was added to 3 '. The reaction probe was a 60 mer, 50 μM aqueous solution, and the solid phase immobilization rate test probe was a 30 mer, 5 μM aqueous solution.

  The FITC label of the probe for solid phase immobilization rate was excited with a fluorescence microscope (BX51WI, cube WIBA manufactured by OLYMPUS), photographed with a CCD camera (photographing time 500 ms), and the fluorescence intensity was measured. This value is hereinafter referred to as “probe supply amount”.

  The slide glass is sequentially immersed in PBS buffer solution and sterilized water, and the reaction probe and the solid phase immobilization rate test probe that have not been immobilized on the slide glass are removed from the slide glass, and further the slide The glass was dried with a dryer to produce a DNA chip.

  After drying, the FITC label of the probe for immobilization test was excited with a fluorescence microscope (BX51WI, cube WIBA manufactured by OLYMPUS), photographed with a CCD camera, and the fluorescence intensity was measured. This value is hereinafter referred to as “probe immobilized amount”.

Next, the probe immobilization rate was determined using the following equation.
Probe immobilization rate (%) = (probe immobilization amount / probe supply amount) × 100

  The above-described series of operations was performed on 10 slide glasses per day, and this was continuously performed for 5 days, thereby producing a total of 50 DNA chips. The results of the probe immobilization rate for each of these DNA chips are shown in FIG.

  In FIG. 1, the chips No. 1 to 10 are on the first day, the chips No. 11 to 20 are the second day, the chips No. 21 to 30 are the third day, and the chips No. 31 to 40 are On the fourth day, and the chips No. 41 to 50 indicate those produced on the fifth day.

As can be seen from FIG. 1, it can be seen that there is a variation in the probe immobilization rate depending on the date of preparation. This is presumed to be because the conditions such as temperature and humidity at the time of spotting differ depending on the day when the chip was manufactured, and there were non-negligible differences in the solid phase reaction rate and the like. Another possible cause is that the amount of poly-L-lysine coated on the slide glass varies depending on the environment and conditions of the day of manufacture.
In addition, the variation in the amount of poly-L-lysine coated on the slide glass on the same production date is not as large as the variation on the different production dates, but it is It is thought that it has become.

  Next, all the prepared DNA chips were immersed in FITC-labeled random oligo DNA (sample material; 30 mer, 100 nmol) in 1 M NaCl / 50 mM phosphate buffer at 25 ° C. for 3 hours to be immobilized on a carrier. Hybridization reaction with the reaction probe was performed. Thereafter, the slide glass was washed with 1 M NaCl / 50 mM phosphate buffer, photographed with a fluorescence microscope (BX51WI, cube WIBA manufactured by OLYMPUS) and a CCD camera, and the fluorescence intensity of the sample substance hybridized with the probe was measured.

  FIG. 2 shows the use of a plurality of carriers having different probe immobilization rates (showing the immobilization rate in FIG. 1) for the hybridization reaction with reaction probes on these carriers. The relationship between the fluorescence intensity about 4 types of sample materials selected from the 100 types of sample materials and the probe solid-phase rate of each support | carrier is shown. From this figure, it can be seen that the fluorescence intensity, that is, the amount of the sample substance hybridized to the carrier, is proportional to the solidification rate of the probe for immobilization rate test regardless of the type of the probe for reaction. This indicates that the reaction probe and the immobilization rate test probe in each DNA chip have the same immobilization rate on the carrier, and the immobilization rate test probe is immobilized. This means that the solidification rate of the reaction probe is defined by the phase rate and can be effectively used as quality information of the DNA chip (test piece).

  Here, it is desirable that the test results when hybridizing under the same conditions are the same for all DNA chips. However, in reality, since there are conditions that cannot always be controlled, it is difficult to make the solid phase immobilization ratios of the probes uniform in all DNA chips. Therefore, as a countermeasure against this, it is possible to reduce the variation in the hybridization ability of the test specimens to be manufactured by providing acceptance criteria within a certain range for the solid phase ratio of the probe for solid phase ratio test. For example, if the acceptable range of the immobilization rate of the probe for immobilization rate is the reference value +/− 5%, the amount of immobilization of the reaction probe varies, that is, the sample substance hybridizes to the reaction probe. Similarly, the amount is considered to be suppressed to about +/− 5%, and it is possible to give a highly accurate result in an experiment that requires quantitativeness.

For the 50 DNA chips prepared in Example 1, the standard value of the solid phase immobilization rate was set to 45%, and the ratio of the solid phase immobilization rate of each DNA chip to this standard value (called correction value) was calculated. Correction data was calculated using the following formula for the fluorescence intensity (referred to as raw data) derived from the sample substance hybridized to each reaction probe.
Correction data: Raw data / correction value

  FIG. 3 shows the fluorescence intensity from the sample substance hybridized with a certain reaction probe on each DNA chip as raw data. FIG. 4 shows the result of FIG. 3 using correction data.

  As can be seen from the comparison between FIG. 3 and FIG. 4, the quantification of the results was further improved by using the correction value obtained in consideration of the solid phase ratio of the probe. In the graph of FIG. 3, the variation of the raw data was about 20% of the CV value. However, when the correction data of FIG. 4 was used, the variation was reduced to about 1% of the CV value.

Correction values were obtained for 50 DNA chips prepared in Example 1 in the same manner as in Example 2. However, in this example, correction data is not used for correction of measured raw data, but is fixed. Considering the ratio of the phase ratio, the optimum shooting time at each spot was determined. That is, the optimum photographing time for each DNA chip was calculated using the following formula.
Optimal shooting time: 500 ms / correction value

  Using the optimal imaging time for each spot calculated in this way, all DNA chips were imaged again, and the results of outputting the amount of hybridization for each probe (hereinafter referred to as appropriate imaging time data) are shown in FIG. This is shown in FIG. Also in this figure, as in the case of FIG. 4, the results were obtained in which the amount of hybridization was small. In FIG. 5, the variation between the data was about 1% of the CV value.

  As described above, it is possible to reduce the variation in data caused by the probe immobilization rate by taking an image with an appropriate imaging time calculated by considering the probe immobilization rate.

50 DNA chips were produced in the same manner as in Example 1. All DNA chips are attached with barcodes in which the values of the respective probe immobilization rates are input.
All DNA chips were hybridized under the same conditions as in Example 1 using the measurement system schematically shown in FIG. The control device in the figure is provided with a recording unit, in which raw data of all 50 DNA chips were recorded. Each raw data was corrected by the same method as in Example 2 with the probe solidification rate specific to each DNA chip read by the barcode reader, and the correction value was output to the output device and recorded in the recording unit.

  FIG. 7 shows correction values for all DNA chips. The variation between the DNA chips was about 1% of the CV value.

  In the same manner as in Example 4, measurement was performed using 50 DNA chips. The measurement time for each DNA chip was set to the optimum imaging time calculated by the control device based on the probe solid-phase rate of each DNA chip read from the barcode provided on the DNA chip. The results are shown in FIG. As can be seen from this figure, the variation in the correction data for each DNA chip was small and the CV value was about 1%.

This figure shows the solidification rate of the probe for immobilization rate test in each of 50 DNA chips produced in Example 1. This figure shows the relationship between the immobilization rate and the quantification result of the sample substance for four types of probes out of the 100 types of probes immobilized on the DNA chip prepared in Example 1. It is shown. This figure shows the measurement results (before correction) in Example 2. This figure shows the measurement results (after correction) in Example 2. This figure shows the measurement results in Example 3. This figure is a diagram showing one embodiment of the detection apparatus for biologically related substances of the present invention. This figure shows the measurement results in Example 4. This figure shows the measurement results in Example 5.

Explanation of symbols

1 ... Detection device for biological materials,
10: Output device,
11 ... Control device (computer),
12 ... Spectrum detection unit,
13 ... light source,
14: DNA chip (test piece),
15 ... Barcode (information storage unit),
16 ... CCD,
17 ... Mirror unit for fluorescence observation,
18 ... Objective lens 19 ... Barcode reader

Claims (13)

Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step (C) of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier;
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating a solid phase immobilization rate (probe solid phase immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
The probe immobilization rate, and the probe supply amount and the probe immobilization amount, as appropriate, are stored in an information storage unit provided in the carrier, or in an information storage unit provided separately from the carrier. Storing together with the identification number of the carrier (F);
The manufacturing method of the test piece for biologically relevant substances containing this. Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier (C);
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating an immobilization rate (probe immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
A step (E ′) of comparing the predetermined minimum immobilization rate with the probe immobilization rate; and
When the probe immobilization rate is equal to or higher than the minimum immobilization rate,
The probe immobilization ratio, as appropriate, the probe supply amount and the probe solid phase immobilization amount are stored in an information storage unit provided in the carrier, or an information storage unit provided separately from the carrier (F) storing together with the identification number of the carrier, or when the probe immobilization rate is less than the minimum immobilization rate,
An information storage unit that is excluded from the manufacturing process, or that stores information indicating that the carrier is defective is stored in an information storage unit provided in the carrier, or is provided separately from the carrier And storing together with the identification number of the carrier (F ′);
The manufacturing method of the test piece for biologically relevant substances containing this. Supplying a reaction probe to be bound to the sample substance and a probe for immobilization rate to a predetermined spot on the carrier (A);
A step (B) of quantifying the amount of the probe for immobilization rate supplied to the carrier (probe supply amount);
A step of washing each spot of the carrier on which the reaction probe and the immobilization rate test probe are immobilized, and removing the probe not immobilized on the carrier from the carrier (C);
A step (D) of quantifying the amount of the probe for immobilization rate test present on the carrier after spot washing (probe immobilization amount);
A step (E) of calculating a solid phase immobilization rate (probe solid phase immobilization rate) of the probe for immobilization rate on the carrier based on the quantitative results of the steps (B) and (D);
The probe immobilization ratio, as appropriate, the probe supply amount and the probe solid phase immobilization amount are stored in an information storage unit provided in the carrier, or an information storage unit provided separately from the carrier And storing together with the identification number of the carrier (F);
Compare the probe solid-phase rate stored in the information storage unit with a predetermined minimum solid-phase rate,
When the probe immobilization rate is not less than the minimum immobilization rate, the test piece having the probe immobilization rate is determined as “pass”, or the probe immobilization rate is the minimum immobilization rate. When it is less than the phase rate, the test piece having the probe solid phase rate is determined as “fail”,
A step (G) of separating a pass product and a reject product;
The manufacturing method of the test piece for biologically relevant substances containing this.   The method according to any one of claims 1 to 3, wherein the quantification in the steps (B) and (D) is performed by measuring a spectrum specific to the probe for immobilization test.   5. The method according to claim 4, wherein the measurement of the spectrum unique to the probe for immobilization rate test is a signal measurement derived from a fluorescent dye previously introduced into the probe for immobilization rate test. It is a carrier having an information storage unit in which the probe immobilization rate on the carrier, and the probe supply amount and the probe immobilization amount are appropriately recorded, or the probe immobilization rate, and the probe supply amount and A probe in which the amount of immobilized probe is recorded in an information storage unit provided separately from the carrier together with the identification number of the carrier, and a probe that specifically reacts to the sample substance Supplying the sample substance to the carrier immobilized on the spot (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
The measured intrinsic spectral intensity is corrected by the probe immobilization rate or probe immobilization amount recorded in the information storage unit, and a predetermined standard immobilization rate or value per standard immobilization amount. Converting to (IV);
A method for measuring a biological substance, comprising: It is a carrier having an information storage unit in which the probe immobilization rate on the carrier and, if appropriate, the probe supply amount and the probe immobilization amount are recorded, or the probe immobilization rate, and the probe supply amount as appropriate In addition, the amount of immobilized probe and the identification number of the carrier are recorded in the information storage unit provided separately from the carrier, and a probe that specifically reacts to the sample substance is predetermined. Supplying the sample substance to the carrier immobilized on the spot of (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex that may be formed in the spot of the carrier after a certain period of time has not dissociated;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
In a method for measuring a biological substance,
Ratio of probe immobilization rate or probe immobilization amount of test piece to standard value (standard immobilization rate or standard immobilization amount) of test sample probe immobilization rate or probe immobilization amount (standard immobilization rate or standard immobilization amount) Calculated by dividing the predetermined standard reaction time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization rate by the calculated ratio). The method for measuring a biological substance, characterized in that the steps (I) and (II) are performed with time as a reaction time from the start of the step (I) to the start of the step (II). It is a carrier having an information storage unit in which the probe immobilization rate to the carrier and, if appropriate, the probe supply amount and the probe immobilization amount are recorded, or the probe immobilization rate and the probe supply amount as appropriate And a probe that is recorded in an information storage unit provided separately from the carrier, together with the identification number of the carrier, and a probe that specifically reacts to a sample substance. Supplying a sample substance to a carrier immobilized on a predetermined spot (I);
A step (II) of washing each spot of the carrier under a condition in which the probe-sample substance complex which may be formed in the spot of the carrier after a certain period of time does not dissociate;
Measuring the spectrum specific to the probe-sample substance complex or the spectrum specific to the sample substance remaining on the carrier after washing (III);
In a method for measuring a biological substance,
Ratio of the ratio of probe immobilization of the test piece or the amount of immobilization of the probe to the standard value (standard immobilization ratio or amount of standard immobilization of the test piece) Calculated by dividing the predetermined standard measurement time in the case of a test piece having the same solid phase immobilization rate or solid phase immobilization rate or solid phase immobilization amount. The method for measuring a biological substance, wherein the step (III) is performed with the time as the measurement time of the step (III).   The method according to any one of claims 6 to 8, wherein the measurement of the intrinsic spectrum is a measurement of a signal derived from a fluorescent dye previously introduced into a sample substance. A fluid transport unit for supplying a fluid to a test piece for detecting a biological substance and for removing the fluid from the test piece;
A temperature control unit for controlling the reaction temperature in the test piece;
Light source to the specimen;
A spectrum detector for detecting a spectrum from the test piece;
An information storage unit provided in the test piece, or a test piece information reading unit for reading information stored in an information storage unit provided separately from the test piece; and a signal detected by the spectrum detection unit A storage unit storing a signal correction application for correcting the intensity based on the information stored in the test piece information reading unit;
And a biologically relevant substance detection device controlled by a computer. On the computer,
A procedure for inputting a standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. A procedure for reading and obtaining a ratio (calculation ratio) of the probe immobilization rate or the probe immobilization amount to the standard immobilization rate or the standard immobilization amount;
A procedure for obtaining a standard solid phase immobilization rate or a value per standard solid phase immobilization amount (conversion value) by reading a measured signal value from the test piece and dividing this by the calculated ratio; and
Procedure for outputting the converted value;
Measurement signal correction program to execute On the computer,
The standard value (standard immobilization rate or standard immobilization amount) for the probe immobilization rate or probe immobilization amount of the test piece and the probe immobilization rate or probe immobilization amount of the test piece are standard. A procedure for inputting a reaction time in the test piece or a measurement time of a signal from the test piece in the case where the solidification rate or the standard solidification amount is the same;
The probe immobilization ratio or the amount of probe immobilization specific to the test piece stored in the information storage unit provided in the test piece or in the information storage unit provided separately from the test piece. Reading, obtaining the ratio (calculation ratio) of the probe immobilization rate or the amount of probe immobilization to the standard immobilization ratio or the standard immobilization amount, and appropriately storing the calculated ratio in the information storage unit And further reading this procedure;
Procedure for determining the actual reaction time or measurement time in the test piece by dividing the reaction time or measurement time by the calculated ratio, and instructing this to the reaction time control unit or signal measurement unit of the test piece ;
A program for running A computer-readable recording medium on which the program according to claim 11 or 12 is recorded.

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