JP2011152054A - Concentration-measuring method, chip for measuring concentration and method for using the chip for the measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a concentration-measuring method, by which the concentration can quantitatively be measured with a chip, and the like. <P>SOLUTION: A reaction for binding specific probe molecules to target molecules is performed in a condition for binding the probe molecules to the target molecules with a calibration chip having the same performance as that of a measurement chip to determine the relation of the concentration of the target molecules in a calibration solution with a measurement quantity of light. The concentration of the target molecules in a measurement target solution is calculated on the basis of the measurement quantity of light determined at the step for determining the measurement quantity of light with the relation determined about the calibration chip. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a concentration measuring method for measuring the concentration of a target molecule using a measuring chip provided with a probe molecule fixed on a substrate.

  A method of detecting a target DNA using a DNA chip in which sites where probe molecules are immobilized on a substrate is arranged is widely used. By labeling the target DNA that binds to the probe molecule by hybridization with a fluorescent substance, the target DNA can be detected using an optical reader.

  In such a DNA chip, different types of target DNA can be simultaneously detected by one measurement by assigning different probe molecules to each site. The amount of fluorescence at each site varies according to the frequency of each target DNA.

JP 2007-212478 A

  When using a DNA chip, it is possible to detect a large number of genes simultaneously. However, the amount of fluorescent light measured by the reader is merely a relative indication of the frequency of the target DNA, and the absolute concentration of the target DNA cannot be determined. Therefore, the current situation is that the DNA chip is used only for screening of genes and is not used for absolute concentration measurement.

  That is, in the measurement using the DNA chip, the appearance of each site is only relatively confirmed by visually grasping the brightness of each site, and based on the output signal characteristics of the camera, the saturation characteristics of the probe DNA, and the like. Therefore, quantitatively quantifying the concentration of each site is not common. Moreover, the chip manufacturing and measurement methods are not controlled so that the numerical value has absolute significance (for example, the concentration of the target molecule).

  On the other hand, by using real-time PCR (polymerase chain reaction), it is possible to quantitatively measure the expression of a specific gene, its change, and the like. However, due to the problem of interference during amplification, a plurality of genes cannot be measured simultaneously, and it is substantially difficult to apply real-time PCR to a large number of genes. Furthermore, it is necessary to prepare a calibration curve for correcting the difference in enzyme activity for each measurement, and the work load is large.

  An object of the present invention is to provide a concentration measurement method and the like capable of quantitative measurement using a chip.

The concentration measurement method of the present invention is the same as the measurement chip in the concentration measurement method for measuring the concentration of a target molecule using a measurement chip comprising a base material and a probe molecule fixed on the base material. Determining the relationship between the concentration of the target molecule of the calibration solution and the measurement light quantity for the calibration chip by performing hybridization under specific hybridization conditions using the performance calibration chip; and And using the relationship obtained for the calibration chip, the measurement light quantity when the measurement chip is hybridized to the measurement target solution under the specific hybridization conditions, The measurement target solution based on the measured light quantity obtained in the obtaining step Characterized in that it comprises the steps of: calculating a concentration of the definitive target molecule.
According to this concentration measurement method, at the time of the measurement obtained in the step of obtaining the measurement light quantity by the measurement chip, using the relationship between the concentration of the target molecule of the calibration solution obtained by the calibration chip and the measurement light quantity for calibration. Since the concentration of the target molecule in the solution to be measured is calculated based on the measured light quantity, quantitative concentration measurement can be performed.

  The hybridization conditions may include a labeling method of the hybridization temperature or target molecule.

The concentration measuring chip of the present invention comprises a substrate and a probe molecule fixed on the substrate, and is a concentration measuring chip capable of measuring the concentration of a target molecule hybridized to the probe molecule, For the calibration chip having the same performance as the concentration measurement chip, the relationship between the concentration of the target molecule of the calibration solution under a specific hybridization condition and the measured light amount is obtained as calibration information in the concentration measurement chip. And
According to this concentration measurement chip, the relationship between the concentration of the target molecule of the calibration solution under a specific hybridization condition and the measurement light amount can be obtained as calibration information in the concentration measurement chip, so quantitative concentration measurement is performed. be able to.

  Using the concentration measurement chip, the measurement light amount when hybridized to the solution to be measured under the specific hybridization condition, and using the relationship obtained for the calibration chip, You may calculate the density | concentration of the target molecule in the said measuring object solution based on a measurement light quantity.

  The hybridization conditions may include a labeling method of the hybridization temperature or target molecule.

The method of using the measurement chip of the present invention is a method of using a measurement chip that measures the concentration of a target molecule using a measurement chip comprising a base material and a probe molecule fixed on the base material. A step of obtaining a measurement light amount when the measurement chip is hybridized with a measurement target solution under a specific hybridization condition, and a concentration of a calibration solution obtained for a calibration chip having the same performance as the measurement chip And calculating the concentration of the target molecule in the measurement target solution based on the measurement light quantity obtained in the step of obtaining the measurement light quantity by the measurement chip using the relationship with the measurement light quantity, The relationship is defined as hybridization under the specific hybridization conditions using the calibration chip. And wherein the obtained by performing down.
According to the method of using the measurement chip, the measurement light quantity at the time of measurement obtained in the step of obtaining the measurement light quantity by the measurement chip using the relationship between the concentration of the calibration solution obtained by the calibration chip and the measurement light quantity. Since the concentration of the target molecule in the measurement target solution is calculated based on the above, quantitative concentration measurement can be performed.

  The hybridization conditions may include a labeling method of the hybridization temperature or target molecule.

  According to the concentration measuring method of the present invention, measurement is performed based on the measurement light amount obtained in the step of obtaining the measurement light amount, using the relationship between the concentration of the target molecule of the calibration solution obtained for the calibration chip and the measurement light amount. Since the concentration of the target molecule in the target solution is calculated, quantitative concentration measurement can be performed.

  According to the concentration measuring chip of the present invention, since the relationship between the concentration of the target molecule of the calibration solution under a specific hybridization condition and the measurement light amount can be obtained as calibration information in the concentration measuring chip, quantitative concentration measurement It can be performed.

  According to the method of using the measuring chip of the present invention, measurement is performed based on the measured light quantity obtained in the step of obtaining the measured light quantity using the relationship between the concentration of the calibration solution obtained for the calibration chip and the measured light quantity. Since the concentration of the target molecule in the target solution is calculated, quantitative concentration measurement can be performed.

  In the step of obtaining the measurement light quantity using the measurement chip, the measurement light quantity may be measured by an absolute light quantity measurement system of the fluorescence light quantity.

  The binding between the probe molecule and the target molecule may include not only hybridization between nucleic acids such as DNA and RNA but also an antigen-antibody reaction between a protein and an antibody.

It is a figure which shows a DNA chip, (a) is a top view which shows the structure of a DNA chip, (b) is a figure which shows the manufactured DNA chip group of the same performance. The flowchart which shows the work procedure at the time of using a DNA chip. It is a figure for demonstrating the principle of the calibration method for obtaining an absolute value light quantity system, (a) is a figure which shows the state at the time of calibration, (b) is a figure which shows the state at the time of measurement, (c) is light reception The figure which illustrates the relationship between the absolute light quantity per unit area of an element, and the gradation of the luminance signal output of a camera. The top view which shows the structure of the cartridge for chemical reaction which accommodated the chip | tip. Sectional drawing in the VV line | wire of FIG. It is a figure which shows the structure of the chip | tip for target molecule detection, (a) is a perspective view, (b) is sectional drawing in the BB line of (a). The figure which shows operation at the time of solution transfer.

  Hereinafter, an embodiment of a concentration measuring method according to the present invention will be described.

  FIG. 1A is a plan view showing the structure of a DNA chip, and FIG. 1B is a view showing a manufactured DNA chip group having the same performance.

  As shown in FIG. 1A, the surface of the DNA chip 10 is provided with sites 10a, 10b,... Where probe DNA having a sequence complementary to the target DNA is arranged. The number of sites can be selected according to the application of the chip, and can be, for example, about 2 to 30,000 sites. By assigning different probes to each site, simultaneous measurement can be performed for a plurality of types of target DNA. In this embodiment, a plurality of DNA chips 10, 10,... Having the same performance are manufactured (FIG. 2B), a part of which is used as a calibration chip, and the rest is used as a measurement chip. .

  Next, a method for using the DNA chip 10 will be described.

  FIG. 2 is a flowchart showing an operation procedure when the DNA chip 10 is used. Steps S0 to S3 show a procedure for calibrating the chip.

  First, DNA chips 10, 10,... (FIG. 2B) having substantially the same performance are manufactured (step S0). In this specification, the fact that the performance is the same means that the performance is substantially the same so that the measurement result at the time of calibration, which will be described later, can be used effectively during the measurement.

Next, with respect to one calibration chip selected from the DNA chips 10, 10,..., A calibration solution in which dummy target molecules T _{1 to} T _N are prepared at specified concentrations A _{1 to} A _N , respectively. Hybridization is performed using (Step S1). Subscripts 1 to n are numbers corresponding to the type of target DNA. Hybridization is performed under standard conditions in which conditions such as temperature and labeling method (pre-dye / post-dye) are defined. The pre-dying is a method for labeling the target DNA before hybridization, and the post-dying is a method for labeling after hybridization.

Next, the light amount of the DNA chip 10 (calibration chip) is measured, and the light amounts B _{1 to} B _N [W / m ² ] of the sites corresponding to each target DNA are measured. Further, to measure the background values due to the excitation light _{(C 1 ~C N) [W} / m 2] ( step S2).

Next, probe coefficients kp _{1 to} kp _N are calculated (step S3). kp _i (i = 1 to N) is
kp _i = A _i / (B _i −C _i ) [MOL / (W / m ² )] or [molecule / (W / m ² )] (where i = 1 to N)
Defined by The probe coefficients kp _{1 to} kp _N are defined as values corresponding to the ratio between the concentration for each target DNA and the amount of light at the corresponding site, and take different values depending on the type of target DNA.

  Steps S11 to S13 in FIG. 2 show a procedure for performing measurement using the measurement DNA chip 10 (measurement chip).

  First, using a measurement chip, hybridization is performed on the solution to be measured under the above hybridization conditions (the above standard conditions) (step S11).

Next, the light quantity of the DNA chip 10 (measurement chip) is measured, and the light quantities y _{1 to} y _N [W / m ² ] of the sites corresponding to each target DNA are measured (step S12). Here, measurement in the absolute light quantity system is required, but a method for measuring the absolute light quantity system will be described later.

Next, the concentrations A _{1 to} A _N of the respective target DNAs are calculated from the site light amounts y _{1 to} y _N (step S13). Here, the concentrations A _{1 to} A _N of each target DNA in the solution to be measured are calculated backward using the probe coefficients kp _{1 to} kp _N. The concentration A _i (i = 1 to N) is
A _i = kp _i × (y _i −Z _i )
(However, i = 1 to N)
Is calculated by Z _i is the background value.

Thus, in this embodiment, by converting the light amount into a concentration using the probe coefficients kp _{1 to} kp _N calculated using one DNA chip 10 as a calibration chip, a large number of target DNAs are obtained. The concentration in the solution to be measured can be quantitatively determined by one measurement. There is no need to create a calibration curve for each measurement.

Next, a method for obtaining the absolute value light quantity system will be described. The absolute value light quantity system stabilizes the light source and the optical system, thereby guaranteeing the accuracy that the read value of the light quantity of the image itself becomes the same value every time, and making the calibration method of the reading device national trebleable , A physical absolute value notation [W / m ² ] etc. in conformity with the SI unit system).

  FIG. 3 is a diagram for explaining the principle of the calibration method for obtaining the absolute value light quantity system. In FIG. 3A, 100 is a power meter, 200 is an optical microscope, and 300 is an illumination reference light source such as an LED.

  The power meter 100 is traceable to the national standard of optical power, and is compatible with the national standard. The power meter 100 measures the light quantity of the illumination reference light source 300 via the microscope 200 and calibrates the light quantity of the illumination reference light source 300 with reference to the power meter 100.

Next, as shown in FIG. 3B, the light quantity of the calibrated reference light source 300 for illumination is detected by a light receiving element (not shown) of a light quantity detector (for example, camera) 400 through the microscope 200. . Here, by comparing the output value [W] of the power meter 100 with the total value of all the pixels of the output value of the light amount detector 400, the relationship between the received light amount of the light receiving element of the camera 400 and the output signal of the camera is calibrated. be able to. That is, the value of [(W / m ² ) / gradation] can be calibrated.

  FIG. 3C shows an example of a calibration result obtained by such a calibration method. FIG. 3C is a diagram illustrating the relationship between the absolute light amount per unit area of the light receiving element and the gradation of the luminance signal output of the camera. For example, when the light source 300 is green light, Y = 2.61E-06X + 2.57E-05, and when the light source 300 is red light, Y = 1.81E-06X-5.26E-05 Become. In the expression, X is a gradation and Y is a light quantity.

  When the camera calibrated in this way is used in a light receiver of a reading device (for example, a reading device described in Japanese Patent Application Laid-Open No. 2001-31690 proposed by the applicant of the present application), direct fluorescence is obtained from the measured gradation value of the image. The amount of light can be measured.

On the other hand, the amount of light generated from the fluorescent dye can be obtained by the following arithmetic processing.
The power ΔI of light absorbed by the fluorescent dye is given by the following equation.
ΔI = 2.3 × 10 ³ × α × Io × n / (Na × S) [W] (1)
However,
α: molar extinction coefficient 8 × 10 ⁴ [M ⁻¹ cm ⁻¹ ]
Io: Incident light intensity [W]
n: number of molecules [pieces]
Na: Avogadro number 6 × 10 ²³
S: Area [cm ² ]
It is.

  By considering the quantum efficiency and the like, the amount of fluorescent light generated from the fluorescent dye can be estimated.

  Therefore, the number n of fluorescent molecules on the chip can be directly estimated by associating the fluorescence light quantity measured using the camera calibrated by the calibration method with the estimated fluorescence light quantity value.

Thus, in the concentration measuring method of the present invention, the probe coefficient kp _{1 is} obtained by performing measurement in the absolute light quantity system both at the time of calibration (step S2 in FIG. 2) and at the time of measurement (step S12 in FIG. 2). calculation of ~Kp _N, and enables the concentration of back-calculated using the probe coefficient _kp 1 ~kp _N.

  In the above embodiment, the probe coefficients kp1 to kpN are calculated for all target DNAs using a single chip. However, a calibration solution may be prepared for each target DNA and hybridization may be performed. . In this case, it is necessary to use one calibration chip for each target DNA.

In hybridization, there is mutual interference (cross contamination) between a plurality of target DNAs. Therefore, by setting concentration close to the concentration A ₁ to A _N of the dummy target molecule T ₁ through T _N to the state at the time of measurement at the time of calibration, the effect of mutual interference is compensated, thereby improving the measurement accuracy .

  In the present invention, the calibration of the chip can be performed on the chip manufacturer side, for example, and the gene measurement using the chip can be performed on the chip purchaser side. In this case, the result of calibration (calibration information), that is, the relationship between the concentration of the target molecule of the calibration liquid and the measured light quantity (probe coefficient in the above embodiment) is given to the chip purchaser, so that This enables quantitative measurement.

  Also, when manufacturing a custom chip with a target molecule specified, the custom chip can be calibrated and the calibration information can be added to the custom chip for sale.

  In the present invention, it is possible to measure the concentration with high accuracy by matching the hybridization conditions at the time of calibration with the hybridization conditions at the time of measurement as much as possible. Therefore, it is desirable to clearly indicate the hybridization conditions at the time of calibration so that the hybridization temperature, the labeling method of the target molecule, the salt concentration, the hybridization time, and other conditions can be matched.

  Further, by using the same reading device at the time of calibration and measurement, it is possible to suppress errors caused by differences in the reading devices. In particular, by using an absolute value light quantity system, it becomes possible to use versatile light quantity values that conform to international standards that do not depend on manufacturers, models, instrumental differences, and aging.

  In the present invention, as a calibration chip and a measurement chip, a through-type chip in which probe molecules are fixed inside a through-hole formed inside the chip can be used. Various types of penetrating chips can be used. For example, a porous filter in which a probe is solid-phased, a fiber chip (for example, a product “Genopal” (registered trademark) manufactured by Mitsubishi Rayon Co., Ltd.), or the like can be used.

  Furthermore, the measuring chip can be accommodated in a chemical reaction cartridge (Japanese Patent Laid-Open No. 2006-337238) proposed by the present inventor. This cartridge for chemical reaction is provided with a structure in which wells and flow paths are provided so that a desired chemical treatment can be performed by moving the contents by deformation of the cartridge based on external force. A measuring chip is accommodated in the cartridge, and hybridization can be performed in the cartridge.

  Hereinafter, with reference to FIGS. 4 to 7, an example in which the measurement chip is accommodated in the chemical reaction cartridge will be described. A target molecule detection chip 10A described later corresponds to the measurement chip and the calibration chip in the present invention.

  FIG. 4 is a plan view showing the structure of such a chemical reaction cartridge, and FIG. 5 is a cross-sectional view taken along the line VV of FIG.

  As shown in FIGS. 4 and 5, the chemical reaction cartridge includes a substrate 1 and an elastic member 2 that is superimposed on the substrate 1.

  On the back surface (lower surface in FIG. 5) of the elastic member 2, a concave portion having a predetermined shape is formed that is recessed toward the front surface (upper surface in FIG. 5). This recess creates a space between the cartridge substrate 1 and the elastic member 2, and as shown in FIGS. 4 and 5, the chambers 21, 22, and 23 for storing the solution and the target molecule detection chip 10 </ b> A have a solution. The supply unit 24 for supplying the fluid and the like, and the flow paths 25, 26, 27, and 28 are configured. As shown in FIG. 4, the flow path 25, the chamber 21, the flow path 26, the chamber 22, the flow path 27, and the supply unit 24 are sequentially communicated.

  In the region other than the concave portion, the cartridge substrate 1 and the elastic member 2 are bonded to each other. For this reason, the solution accommodated in the recess is sealed in the cartridge, and leakage to the outside is prevented.

  As shown in FIGS. 4 and 5, the substrate 1 is provided on the back side (lower side in FIG. 5) of the target molecule detection chip 10 </ b> A and the storage unit 11 in which the target molecule detection chip 10 </ b> A is stored. A discharge part 12 for storing the solution discharged from the molecule detection chip 10A is formed. A support member 4 that supports the target molecule detection chip 10 </ b> A is fixed to the back surface of the elastic member 2. The target molecule detection chip 10A housed in the housing part 11 is bonded to the cartridge substrate 1 and the support member 4 and fixed inside the cartridge. With such a structure, the target molecule detection chip 10A is fixed inside the cartridge while preventing leakage of the solution in the thickness direction of the target molecule detection chip 10A from the periphery of the target molecule detection chip 10A. Further, as shown in FIG. 5, the discharge portion 12 of the cartridge substrate 1 is communicated with the flow path 28.

  The target molecule detection chip 10A is a penetrating chip that detects a target molecule by hybridization.

  6A and 6B are diagrams showing the structure of the target molecule detection chip 10A, where FIG. 6A is a perspective view, and FIG. 6B is a cross-sectional view taken along line BB in FIG. 6A. As shown in FIGS. 6 (a) and 6 (b), in the target molecule detection chip 10A, detection sites 31 to which probes corresponding to individual target molecules are fixed are two-dimensionally arranged, and each detection site is arranged. 31 is partitioned by a partition wall 32. Each detection site 31 detects a target molecule in a solution penetrating in the thickness direction of the target molecule detection chip 10A (vertical direction in FIG. 6B) by hybridization.

  Next, the operation of transferring the solution in the cartridge will be described. FIG. 7 is a diagram illustrating an operation during solution transfer.

  A solution to be inspected is injected into the chamber 21 formed in the cartridge in advance. Injection of the solution is performed by inserting an injection needle through the flow path 25. A stopper (not shown) made of an elastic body is attached to the flow path 25 in a sealed state, and an injection needle is inserted into the stopper when injecting the solution. When the injection needle is pulled out after the solution is injected, the needle hole of the stopper is closed, and a sealed state is secured.

  Next, as shown in FIGS. 7 and 4, the elastic member 2 is pressed against the substrate 1 by the rollers 51 and 52 provided at regular intervals, and the rollers 51 and 52 are moved to the right. By such an operation, the solution injected into the chamber 21 is sent into the supply unit 24 via the flow channel 26, the chamber 26, and the flow channel 27. Further, as shown in FIG. 7, the solution penetrates downward from each supply site 31 of the target molecule detection chip 10 </ b> A from the supply unit 24 and reaches the chamber 23 via the discharge unit 12 and the flow path 28.

  Next, the roller 51 and the roller 52 are reciprocated a predetermined number of times in the left-right direction in FIG. 7 while being pressed against the elastic member 2. By such an operation, the solution is reciprocated between the chamber 22 and the chamber 26 and passed through the detection site 31 of the target molecule detection chip 10A a plurality of times. This gives ample opportunity for the solution to contact the detection site 31 required for hybridization. It also reduces false hybridization by molecules other than the target molecule. If the hybridization conditions are appropriate, the reciprocation of the roller 51 and the roller 52 can be omitted.

  As shown in FIG. 7, during the hybridization, the temperature of the solution may be maintained at a value suitable for hybridization using a heater HT.

  After performing such an operation for hybridization, the target molecule hybridized in the target molecule detection chip 10A is detected using a predetermined reader or the like. The target molecule may be detected inside the cartridge without removing the target molecule detection chip 10A from the cartridge, or the target molecule may be detected after the target molecule detection chip 10A is removed from the cartridge. Also good.

  In this case, the calibration conditions (the target molecule detection chip 10A) are accommodated in the same chemical processing cartridge, and the hybridization conditions can be matched by causing hybridization in the cartridge during calibration. When the chip (calibration chip and measurement chip) is accommodated in the chemical processing cartridge, the hybridization conditions can be easily matched with high accuracy, and the concentration measurement accuracy can be improved. Further, even when the chip is accommodated in the chemical processing cartridge, the target molecule may be detected using the same reading device at the time of calibration and measurement.

  As described above, according to the concentration measurement method of the present invention, the measurement light quantity is obtained in the step of obtaining the measurement light quantity using the relationship between the concentration of the target molecule of the calibration liquid obtained for the calibration chip and the measurement light quantity. Since the concentration of the target molecule in the measurement target solution is calculated based on the measurement light quantity, quantitative concentration measurement can be performed.

  The scope of application of the present invention is not limited to the above embodiment. The present invention can be widely applied to a concentration measuring method for measuring the concentration of a target molecule using a measuring chip provided with a probe molecule fixed on a substrate. The present invention is not limited to application to a DNA chip. The probe molecules and target molecules of the chip in the present invention include not only nucleic acids but also proteins and antibodies.

10 DNA chip (calibration chip, measurement chip)
10A Target molecule detection chip (calibration chip, measurement chip)

Claims (9)

In a concentration measurement method for measuring the concentration of a target molecule using a measurement chip comprising a substrate and a probe molecule fixed on the substrate,
Performing a hybridization under specific hybridization conditions using a calibration chip having the same performance as the measurement chip, thereby obtaining a relationship between the concentration of the target molecule of the calibration solution and the measurement light amount for the calibration chip; ,
Using the measurement chip, obtaining a measurement light amount when hybridized to the measurement target solution under the specific hybridization conditions;
Calculating the concentration of the target molecule in the measurement target solution based on the measurement light quantity obtained in the step of obtaining the measurement light quantity by the measurement chip, using the relationship obtained for the calibration chip;
A concentration measurement method comprising: The concentration measurement method according to claim 1, wherein the hybridization condition includes a labeling method of the hybridization temperature or a target molecule. A concentration measuring chip comprising a substrate and a probe molecule fixed on the substrate, and capable of measuring the concentration of a target molecule hybridized to the probe molecule,
For the calibration chip having the same performance as the concentration measurement chip, the relationship between the concentration of the target molecule of the calibration solution under a specific hybridization condition and the measured light amount is obtained as calibration information in the concentration measurement chip. Concentration measuring chip. Using the concentration measurement chip, the measurement light amount when hybridized to the solution to be measured under the specific hybridization condition, and using the relationship obtained for the calibration chip, The concentration measuring chip according to claim 3, wherein the concentration of the target molecule in the measurement target solution is calculated based on a measurement light amount. 5. The concentration measuring chip according to claim 3, wherein the hybridization condition includes a labeling method of the hybridization temperature or a target molecule. In a method of using a measurement chip that measures a concentration of a target molecule with a fluorescent label bonded to a target molecule using a measurement chip including a base material and a probe molecule fixed on the base material,
Using the measurement chip, obtaining a measurement light amount when the probe molecule and the target molecule undergo a binding reaction with respect to the solution to be measured under a condition in which the specific probe molecule and the target molecule undergo a binding reaction;
Based on the measurement light quantity obtained in the step of obtaining the measurement light quantity by the measurement chip using the relationship between the concentration of the calibration solution obtained for the calibration chip having the same performance as the measurement chip and the measurement light quantity. Calculating the concentration of the target molecule in the measurement target solution,
With
The method for using a measuring chip, wherein the relationship is obtained by performing a binding reaction between the probe molecule and the target molecule under a condition in which the specific probe molecule and the target molecule perform a binding reaction using the calibration chip. . The measurement chip according to claim 6, wherein the conditions for the binding reaction between the probe molecule and the target molecule include a temperature at which the probe molecule and the target molecule bind to each other or a labeling method of the target molecule. how to use. The method for using the measuring chip according to claim 6 or 7, wherein in the step of obtaining the measuring light amount using the measuring chip, the measuring light amount is measured by an absolute light amount measuring system of the fluorescent light amount. 9. The measurement chip according to claim 6, wherein the binding reaction between the probe molecule and the target molecule is a hybridization between nucleic acids or an antigen-antibody reaction between a protein and an antibody. how to use.

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