JP2003344401A - Inspection method of organism-related material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inspection method capable of inspecting an organism- related material, simply, simultaneously, accurately in a short time. <P>SOLUTION: Genetic screening which is an example of organism-related material inspection is performed by following procedures: first of all, a nucleic acid sample 114 which is a specimen liquid is dropped onto a DNA micro-array 101; then, a hybridization reaction of the DNA micro-array 101 is generated; thereafter, an excessive nucleic acid sample 114 is removed from the DNA micro-array 101; finally, fluorescence observation of the DNA micro-array 101 is performed by a microscope 102. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inspecting a biological substance, that is, a method for detecting information by a reaction of a biological substance.

[0002]

2. Description of the Related Art In recent years, gene analysis technology has advanced, and gene sequences in many organisms including humans have been revealed. In addition, the causal relationship between the analyzed gene product and disease is gradually being elucidated.

The genetic testing methods currently used are the steps of extracting a nucleic acid from a biological sample, amplifying a target gene to be inspected using a nucleic acid amplification method called PCR, NASBA method, etc. It comprises a step of labeling with an element or a fluorescent molecule, and a step of measuring the base sequence of the labeled target gene or its concentration.

Recently, many capillary electrophoresis apparatuses have been used which can process a large number of samples at high speed by using a plurality of capillaries for fluorescently labeled nucleic acids.
As a result, it can be performed in about one-third to one-quarter of the time required by the method using the conventional electrophoresis apparatus.

Furthermore, in recent years, an inspection method using a DNA chip for simultaneously inspecting a plurality of genes has been developed. The DNA chip has a large number of cDNAs on the surface of the glass substrate.
The A probe is immobilized, and a large number of oligo probes synthesized in a minute region on silicon are manufactured by applying a semiconductor manufacturing process. Both are D in the sample
A plurality of NA base sequences and expression amounts can be determined simultaneously.

The application of the DNA chip has made it possible to analyze a large amount of gene expression and a plurality of mutations. In addition, from the data obtained using the DNA chip, many genes are classified into a plurality of groups (that is, clustering), and information on the variation of genes due to development and differentiation is obtained. The gene information thus obtained is used as a database that can be easily accessed through the Internet.

[0007] The gene expression level test and mutation analysis are carried out by using the electrophoresis method and the microarray method.
However, the electrophoresis method has a drawback that the time required for the inspection is long and the number of the inspections that can be performed at one time is limited.

A gene analysis method using a DNA chip is
Although many inspections can be performed at one time, a long inspection time is required, many inspection steps are included, and complicated operations are required. Therefore, it has a drawback that an analysis result with good reproducibility cannot be obtained.

In order to overcome such drawbacks, DNA
A method has been developed in which a porous glass wafer manufactured by using a microfabrication process is used as a carrier for a chip. A method of using such a porous glass wafer as a carrier for a DNA chip is disclosed in, for example, Japanese Patent Publication No. 9-504864, and an inspection similar to a DNA chip can be performed with good reproducibility and in a short time.

[0010]

For example, in the method of using a conventional porous glass wafer as a carrier of a DNA chip for inspecting a gene which is a bio-related substance, a reaction part for carrying out liquid movement and temperature control, and fluorescence detection are used. The measurement part for performing is separate, and various device configurations are required. For this reason,
Not only does it take time and labor to transfer between various devices,
There are problems such as a temperature change occurring during transportation and dust attached during transportation. Care should be taken to ensure reproducibility of measurement results.

In addition, the sample liquid to be inspected may contain dust and the like, which is an obstacle to an accurate inspection.

The biological substance is a protein,
There are cells, tissues, etc., and the above-mentioned problems and obstacles are problems that methods for inspecting these have in common.

In particular, in clinical tests, there is a strong demand for testing genes in a short time with high accuracy and easily, and it is presumed that it is difficult to meet the needs of these users.

The present invention has been made in consideration of such a current situation, and an object thereof is to provide an inspection method capable of easily and accurately inspecting a biological substance in a short time in real time.

[0015]

Means for Solving the Problems The present invention is a method for inspecting a bio-related substance for in real-time inspecting a reaction of a bio-related substance using a solid-phase carrier that can pass a fluid. A supply step of supplying a bio-related substance, a reaction step of causing a reaction of the bio-related substance on the solid-phase carrier, an elimination step of eliminating an excess bio-related substance after the reaction of the bio-related substance, and a solid-phase carrier And an observing step of optically observing the biological substance that has reacted with The reaction step, the exclusion step and the observation step are preferably carried out in a humidified atmosphere.

The excluding step includes, in one example, a step of immersing the solid phase carrier in washing water. The removing step may further include a step of applying ultrasonic vibration to the wash water. The removing step may further include a step of removing the wash water from the upper side of the solid phase carrier.

The excluding step includes, in another example, the step of passing a gas through the solid phase carrier. The gas passed through the solid phase carrier may contain temperature-controlled microdroplets.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

First Embodiment The present embodiment will be described as an example of a method for inspecting a biological substance.
It is directed to genetic testing using DNA microarrays. However, the present invention is not limited to the genetic test using the DNA microarray, and can be similarly applied to the test of other bio-related substances other than the gene, for example, the test of the protein using the proteome array.

The basic principle of the genetic test carried out in this embodiment is to detect a nucleic acid chain having a specific sequence contained in a sample by using an immobilized nucleic acid probe whose sequence is known. It is a thing. For example, a sample of single-stranded nucleic acid is immobilized on a substrate and then brought into contact with the nucleic acid contained in the sample labeled with a fluorescent substance or the like. When the nucleic acid in the sample has a sequence complementary to the nucleic acid probe, the nucleic acid in the sample hybridizes with the nucleic acid probe to form a double strand and is immobilized on the substrate. Therefore, after removing the unreacted nucleic acid chain by washing, by fluorescently detecting the label (rhodamine, FITC, Cy3, Cy5, etc.) attached to the probe, a target having a sequence complementary to the probe is detected. Nucleic acid is detected.

In the present embodiment, a gene testing device 10 used for gene testing using a DNA microarray.
A schematic configuration of 0 is shown in FIG.

As shown in FIG. 1, the genetic testing apparatus 1
00 includes a reaction stage 103 for supporting a reaction vessel 101 containing a DNA microarray and promoting reaction of the DNA microarray, and a microscope 102 for optically observing the DNA microarray.
The microscope 102 has an XY stage, and the reaction stage 103 is placed on the XY stage. The reaction stage 103 is moved by an XY stage, for example, to change the field of view observed by the microscope 102.

The genetic test apparatus 100 further includes a microscope 10.
2 is provided with a CCD (charge coupled device) camera 104 for taking a fluorescence image of the DNA microarray obtained by 2. The CCD camera 104 is preferably a cooling type in order to improve inspection accuracy.

The genetic test apparatus 100 further has a hood 106 for covering the reaction stage 103. The hood 106 is provided to block ambient light that is unnecessary for microscopic observation, prevent changes in the temperature environment, and prevent dust from entering the observation region. Hood 10
Reference numeral 6 has an entrance / exit for inserting and removing the reaction stage 103, and an observation window for enabling microscopic observation.

The reaction stage 103 is pushed out of the hood 106 by the XY stage when the reaction container 101 is installed, and is housed in the hood 106 during observation. The doorway of the hood 106 has an opening / closing door,
After housing the reaction stage 103, the opening / closing door is closed. The environment inside the hood 106 may be kept constant by a temperature controller and a humidity controller provided inside the hood 106.

As shown in FIG. 2, the reaction vessel 101
Includes a thin plate-shaped slide chip 107 having a DNA microarray 110a, and an upper housing 108 and a lower housing 109 which sandwich and hold the slide chip 107 from above and below. The upper housing 108 and the lower housing 109 are preferably made of a light-shielding member, more preferably a dark black member. The upper housing 108 and the lower housing 109 are fixed to each other by fastening means such as screws or adhesives, and hold the slide chip 107.

On the upper and lower surfaces of the slide chip 107, DNA is
An O-ring (not shown) is provided so as to surround the microarray 110a, and the fluid (nucleic acid sample or washing solution) supplied later is supplied to the upper housing 108 and the lower housing 10.
Leakage from the gap 9 may be prevented.

As shown in FIG. 3, the slide chip 1
07 includes, for example, a porous portion 110 made of ceramic, and a pair of support plates 111 for sandwiching and supporting the porous portion 110. The pair of support plates 111 have circular openings 112 at positions corresponding to each other for defining the DNA microarray 110a. That is,
The DNA microarray 110a has a porous portion 1 exposed through a pair of openings 112 formed in a pair of support plates 111 arranged above and below the porous portion 110, respectively.
It refers to part 10.

The porous portion 110 preferably has a large number of fine pores extending in the thickness direction. Here, the porous portion 110 does not necessarily have such a structure, and may have any structure as long as the fluid can pass therethrough. The material of the porous portion 110 may be any material as long as the porous portion 110 is not broken at the temperature and pressure exposed during the gene hybridization reaction.

Further, if the thickness of the porous portion 110 is made sufficiently thin and the pore diameter of the porous portion is made sufficiently small, a required amount of sample or reagent can be distributed and accommodated in a large number of pores. Therefore, if the number of pores required to accommodate a desired amount of liquid is formed on a specific area of the surface of the porous portion 110 and the sample and the reagent are supplied to the predetermined area, the porous portion 110 A stable analysis can be performed within.

As shown in FIG. 4, the upper housing 10
8 is a DNA microarray 1 of the slide chip 107
Taper-shaped opening 113 for exposing 10a
have. In the space inside the opening 113, the DNA
Nucleic acid sample 11 supplied to the microarray 110a
4 can be saved.

The lower housing 109 is a slide chip 1.
07, a concave portion 115 formed in a position corresponding to the DNA microarray 110a, the bottom surface of which is inclined, a waste liquid hole 116 extending from the lowermost portion of the concave portion 115 to the lower surface of the lower housing 109, and a waste liquid. D extends from the middle of the hole 116 to the side surface of the lower housing 109, D
It has a liquid storage hole 117 capable of storing the nucleic acid sample 114 supplied to the NA microarray 110a.

A plurality of droplets of the nucleic acid probe solution are ejected onto the DNA microarray 110a by, for example, a liquid dispensing apparatus using the ink jet ejection principle. One droplet of the nucleic acid probe solution ejected onto the porous portion 110 is instantly sucked into the inside of the fine pores to form a nucleic acid probe spot having a minute diameter.

As shown in FIG. 5, the DNA microarray 110a has a large number of nucleic acid probe spots 118 thus formed. In general, one nucleic acid probe spot 118 has one porous portion 11 included therein.
One kind of nucleic acid probe on the inner wall of the fine porous part of 0
18 is fixed. In other words, one nucleic acid probe spot 118 is a region including fine pores in which one type of nucleic acid probe 118 is fixed on its inner wall.

Usually, different kinds of nucleic acid probes 118 are fixed to different nucleic acid probe spots 118 in the DNA microarray 110a.
The same nucleic acid probe 118 may be fixed to a plurality of nucleic acid probe spots 118 in the DNA microarray 110a. In addition, surface treatment is applied to the inner wall of the fine porous portion of the porous portion 110 to reduce frictional resistance to fluid and nucleic acid probe 1.
The adsorptivity of reagents such as 18 may be adjusted.

The size of the slide chip 107 is, for example, half the size of a normal slide glass, and is about 3
It is 7 mm × about 12 mm × about 1 mm. For example, DNA
The microarray 110a has a diameter of about 4 mm, one nucleic acid probe spot 118 has a diameter of about 100 μm, and 40 in one DNA microarray 110a.
Zero nucleic acid probe spots 118 are arranged at 200 μm intervals.

One nucleic acid probe spot 118 has one
It has 00 to 1000 fine pores, preferably 1000 fine pores, and one pore has a diameter of 0.05 μm, preferably about 0.2 μm.

The DNA microarray 110a corresponds to one reaction unit in which one type of reaction is carried out. A conventional DNA chip is formed by immobilizing a probe on the surface of a slide glass and has a reaction region having a two-dimensional spread. Unlike such a DNA chip, D
The NA microarray 110a has a three-dimensional reaction region having a two-dimensional spread on the surface of the substrate and also a spread in the thickness direction of the substrate.

Nucleic acid probe 118 is typically about 10
It is a polynucleotide or an oligonucleotide consisting of a nucleic acid having a nucleotide length of not less than about 100 nucleotides and is generally used when detecting a nucleic acid by hybridization.

One nucleic acid probe spot 118 included in the DNA microarray 110a has a plurality of minute pores, that is, a three-dimensional space capable of accommodating a minute amount of liquid, as described above. Although one nucleic acid probe spot 118 occupies a very narrow area in the plane when the DNA microarray 110a is viewed from above, the porous portion 11
Since it has a three-dimensional space extending in the thickness direction of 0, the area actually occupied by one nucleic acid probe spot 118 is much larger than it looks.

For example, by changing the conditions for each nucleic acid probe spot 118 (by changing the type of the nucleic acid probe 118 to be fixed, changing the surface treatment, etc.), the size of the small DNA microarray 110a can be greatly reduced. A lot of information can be obtained with sufficient sensitivity.

The analysis using the DNA microarray 110a includes, for example, detection of the presence or absence of a mutation such as an oncogene in a subject, gene expression analysis in the subject,
Expression analysis of drug resistance genes, genotyping of disease-related or susceptibility genes in subjects, determination of intracellular signal gene expression patterns in subjects, clinical therapeutic and diagnostic analysis, and non-clinical basis Also in research, various genetic analyzes and the like are performed.

The slide chip 107 described here has one DNA microarray 110a, but the slide chip 107 has a plurality of DNA microarrays 11a.
It may have 0a. Such a slide chip 1
In 07, a plurality of DNA microarrays 110a
May be used for testing different samples, or may be used for different types of analysis of the same sample.

As shown in FIGS. 6 and 7, the reaction stage 103 includes a base portion 12 on which the reaction vessel 101 is placed.
0 and the cover portion 12 that can be opened and closed with respect to the base portion 120
3 and 3. Base part 120 and cover part 123
Constitute a support mechanism for supporting the reaction vessel 101. The reaction container 101 is placed on the base portion 120, and then the cover portion 123 is closed,
Supported.

The cover portion 123 is a D by the microscope 102.
It has a microscope observation hole 122 that enables observation of the NA microarray 110a, and two optically transparent cover glasses 121 that close the microscope observation hole 122.
The two cover glasses 121 are spaced apart from each other.

The base portion 120 is terminated at a side surface of the base portion 120, which is in fluid communication with the drain passage 119, which is in fluid communication with the waste liquid hole 116 of the reaction vessel 101. The waste liquid pipe 134 and the discharge channel 119
And a solenoid valve 128 for opening and closing the discharge flow path 119, which is provided in the middle.

The discharge flow path 119 terminates at the surface facing the reaction vessel 101, and a circular groove surrounding it is formed at the end, that is, around the liquid supply port, and in the groove. Has an O-ring 130 disposed therein. The discharge flow path 119 is kept fluid-tight by the O-ring 130 and fluidly communicates with the waste liquid hole 116 of the reaction vessel 101 supported by the reaction stage 103.

The reaction stage 103 further includes the reaction container 10
1 has a syringe piston pump unit 125 for transferring a fluid. Syringe piston pump part 1
For example, 25 moves the nucleic acid sample supplied into the reaction container 101 in the reaction container 101, or supplies washing water into the reaction container 101.

The syringe piston pump unit 125 is in fluid communication with the transfer passage 124, which is in fluid communication with the liquid storage hole 117 of the reaction vessel 101,
A syringe piston pump 131 for transferring the fluid in the reaction container 101, and a solenoid valve 127 for opening and closing the transfer channel 124, which is provided in the middle of the transfer channel 124.
And have.

The transfer flow path 124 is terminated at the surface facing the reaction vessel 101, and a circular groove surrounding it is formed at the end, that is, around the liquid supply port, in the groove. Has an O-ring 130 disposed therein. The transfer flow path 124 is fluid-tightly connected to the liquid storage hole 117 of the reaction vessel 101 supported by the reaction stage 103 by the O-ring 130 so as to be fluidly connected.

The syringe piston pump 131 includes a syringe 138 and a piston 1 that can move in the syringe 138.
29, a syringe 138, and a seal 139 for keeping the piston 129 liquid-tight.

The piston 129 of the syringe piston pump portion 125 is driven by, for example, a motor unit (not shown). In other words, the genetic testing device 10
Although not shown, 0 has a motor unit for driving the piston 129 of the syringe piston pump unit 125. The motor unit includes, for example, a stepping motor and a rack and pinion mechanism that converts the movement of the rotation shaft of the stepping motor into a linear movement.

The syringe piston pump section 125 further includes
A wash water flow path extending from the syringe piston pump 131, a wash water pipe 132 that is in fluid communication with the wash water flow path and terminates at a side surface of the syringe piston pump portion 125, and a wash water flow path. And a solenoid valve 126 for opening and closing the wash water flow path.

As schematically shown in FIG. 8, the washing water pipe 132 of the syringe piston pump portion 125 is fluidly connected to the washing water tank 133, and the waste liquid pipe 134 provided in the base portion 120 is a suction pump. It is in fluid communication with the waste liquid tank 136 via 135. In other words,
The gene testing device 100 includes a wash water tank 133 that is in fluid communication with the syringe piston pump unit 125 of the reaction stage 103, and a discharge channel 119 of the reaction stage 103.
A waste liquid tank 136 in fluid communication therewith.

The reaction stage 103 further includes the reaction vessel 10
1 has a temperature adjusting mechanism for adjusting the temperature of the DNA microarray 110a. The temperature adjusting mechanism has a heating means for raising the temperature of the reaction container 101. The heating means is, for example, as shown in FIGS. 6 and 7, a plate-shaped heater 105 such as a silicon rubber heater, a ceramic heater, or a Peltier element.

The plate heater 105 is embedded in the base portion 120 and the cover portion 123, and preferably makes surface contact with the reaction vessel 101 supported by the reaction stage 103. Specifically, as shown in FIGS. 6 and 7, the plate heater 105 embedded in the base portion 120 makes surface contact with the lower housing 109 of the reaction container 101 placed on the plate heater 105. Thereby, the plate heater 105 can raise the temperature of the reaction vessel 101 in a short time.

The temperature adjusting mechanism is preferably the reaction vessel 1.
It further has a cooling means for lowering the temperature of 01.
The cooling means is, for example, the base portion 120 and the cover portion 123.
Is a Peltier device (not shown) provided in the. The Peltier element preferably makes surface contact with the reaction vessel 101 supported by the reaction stage 103.

The temperature adjustment mechanism is drive-controlled by a temperature adjuster (not shown). In other words, the genetic testing device 100 has a temperature adjuster (not shown) for driving and controlling the temperature adjusting mechanism.

The temperature adjusting mechanism may further have a temperature sensor for monitoring the temperature of the reaction vessel 101. In this case, the temperature regulator may drive and control the heating means and the cooling means based on the information from the temperature sensor.

The genetic test apparatus 100 further includes a control computer (not shown) that controls the entire apparatus. The control computer includes an XY stage for adjusting the position of the reaction stage 103, a motor unit for driving the syringe piston pump unit 125, a plurality of solenoid valves for opening and closing the flow path in which these are provided, and the reaction vessel 101. Suction pump 13 for discharging fluid from the
5. Control a temperature controller for driving and controlling the temperature adjusting mechanism.

The genetic test in this embodiment proceeds according to the flow shown in FIG.

That is, in the genetic test, as shown in FIG. 9, first, a nucleic acid sample 114 as a sample liquid is dropped on the DNA microarray 101. Next, the hybridization reaction of the DNA microarray 101 is caused. After that, surplus nucleic acid sample 1
14 is excluded from the DNA microarray 101. Finally, using the microscope 102, the DNA microarray 101
Is observed by fluorescence.

More preferably, all the steps described above are performed in a humidified atmosphere except for the dropping of the nucleic acid sample 114. That is, the genetic test is more preferably performed as shown in FIG.
The process proceeds according to the flow indicated by 0.

That is, in a more preferable genetic test, as shown in FIG. 10, first, a nucleic acid sample 114 as a sample liquid is dropped on the DNA microarray 101. Next, the hybridization reaction of the DNA microarray 101 is caused to occur in a humid atmosphere. After that, the excess nucleic acid sample 114 is removed from the DNA microarray 101 in a humidified atmosphere. Finally, the microscope 1
02, the DNA microarray 101 is subjected to fluorescence observation in a humid atmosphere.

The specific operation of the genetic test apparatus 100 will be described below.

In FIG. 6, the reaction container 101 is placed on the base portion 120 of the reaction stage 103 with the cover portion 123 opened. Then, the nucleic acid sample 114 is supplied into the opening 113 of the reaction container 101. Then, the cover 123 is closed.

As a result, the waste liquid hole 11 of the reaction vessel 101
6 is in fluid communication with the discharge channel 119 provided in the base portion 120, and the liquid storage hole 11 of the reaction vessel 101 is provided.
7 is in fluid communication with the liquid-sending flow path provided in the syringe piston pump unit 125. In addition, all the flow paths inside the syringe piston pump unit 125 are
Is placed in advance with cleaning water 137 before being placed.

When the cover 123 is closed, the lower cover glass 121 provided on the cover 123 is provided.
Makes surface contact with the upper housing 108 of the reaction vessel 101. Thereby, the upper housing 108 of the reaction container 101
The opening 113 formed in is closed.

As a result, foreign matter such as dust in the hood 106 is prevented from entering the opening 1 of the upper housing 108 of the reaction vessel 101.
13 is prevented. As a result, it is possible to prevent erroneous inspection of the DNA microarray 110a by the microscope 102 due to the inclusion of foreign matter.

Then, in order to cause a hybridization reaction, the DNA microarray 110 in the reaction vessel 101 is
The temperature of a is controlled. That is, the four heaters 105 heat the DNA microarray 110a and each liquid to a temperature suitable for the hybridization reaction.

Since the opening 113 of the reaction vessel 101 is sealed by the cover glass 121, the inside thereof becomes a high humidity environment, that is, a humidified atmosphere. As a result, the nucleic acid sample 114 does not evaporate, and the nucleic acid sample 1
The concentration of 14 is kept constant. Therefore, the unwanted change in the concentration of the nucleic acid sample 114 does not occur.

The opening 113 of the reaction vessel 101 is
Since it is shielded from the external environment through the two cover glasses 121 arranged with the sealed air in between, the temperature gradient with the external environment is reduced. This prevents dew condensation on the surface of the cover glass 121 during temperature control.

Subsequently, only the solenoid valve 127 on the DNA microarray 110a side is opened, and the piston 129 of the syringe piston pump 131 is moved to the right side of the drawing, as shown in FIG. Thereby, the nucleic acid sample 11
4 passes through the DNA microarray 110a and moves into the liquid storage hole 117 in the lower housing 109.

Next, as shown in FIG. 6, the piston 1
29 is moved to the left side of the drawing. As a result, the nucleic acid sample 114 passes through the DNA microarray 110a,
It returns to the upper side of the DNA microarray 110a, that is, inside the opening 113 again.

This operation is repeated. As a result, the nucleic acid sample 114 causes a hybridization reaction with the nucleic acid probe spot 118 in the DNA microarray 110a within a short time. As a result, only the predetermined nucleic acid probe spot 118 can emit fluorescence in the subsequent fluorescence observation.

The volume of the liquid storage hole 117 is determined in advance so that the nucleic acid sample 114 does not reach the inside of the syringe piston pump portion 125. Further, the syringe piston pump 131 is driven with the moving distance of the piston 129 limited according to the volume of the liquid storage hole 117. As a result, the nucleic acid sample 114 becomes the syringe piston pump unit 1
It does not invade into 25, and carryover does not occur when another DNA microarray 110a is set.

After the hybridization reaction is completed, the DNA microarray 110a is washed with washing water in order to remove the excess nucleic acid sample 114. The washing of the DNA microarray 110a is performed according to the following procedure.

First, in FIG. 8, the electromagnetic valve 127 on the DNA microarray 110a side and the electromagnetic valve 128 on the suction pump 135 side are closed, the electromagnetic valve 126 on the wash water tank 133 side is opened, and the piston 129 of the syringe piston pump 131 is opened. Is moved to the right side of the page. As a result, the wash water 137 in the wash water tank 133 is sucked into the syringe 138.

Next, the solenoid valve 12 on the wash water tank 133 side
6 is closed, the electromagnetic valve 127 on the DNA microarray 110a side is opened, and the piston 129 is moved to the left side of the drawing. As a result, the cleaning water 137 is injected into the reaction container 101. The injection of the wash water 137 is continued until the wash water 137 passes through the DNA microarray 110a and accumulates thereon. As a result, for example, the washing water is stored in the tapered opening 113 shown in FIG. 6, and the DNA microarray 110a is immersed in the washing water.

Thereafter, the solenoid valve 127 on the DNA microarray 110a side is closed, the solenoid valve 128 on the suction pump 135 side is opened, and the suction pump 135 is operated. As a result, the wash water 137 and the excess nucleic acid sample 114 in the reaction container 101 are discharged to the waste liquid tank 136.

As a result of the series of operations, the DNA microarray 110a is washed. This washing may be repeated. By repeating the washing, the excess nucleic acid sample 114 is more surely removed.

During cleaning, a pressure sensor (not shown) provided in the flow path of the cleaning liquid 137 is used to prevent the pressure of the cleaning liquid 137 from exceeding a predetermined value, and the syringe piston pump 131 and the suction pump 135. May be controlled.

After washing, the DNA microarray 110a
Is observed by fluorescence with the microscope 102.

In this embodiment, since the DNA microarray 110a in the reaction vessel 101 is observed by fluorescence without being transported between various devices, the gene hybridization reaction can be observed in real time.

In addition, since the DNA microarray 110a is washed to remove the excess nucleic acid sample before the fluorescence observation, the background autofluorescence of the DNA microarray 110a is reduced. In other words, it did not cause a hybridization reaction with the nucleic acid probe, but simply DN.
The unwanted fluorescence that has been generated so far due to the nucleic acid sample adhering to the A microarray 110a is completely eliminated. As a result, the S / N ratio of the fluorescence intensity of the nucleic acid probe spot is improved, and an accurate inspection can be performed even with weak fluorescence.

Furthermore, since the hybridization reaction is carried out in a humidified atmosphere, there is no evaporation of the nucleic acid sample, and therefore there is no change in the concentration of the nucleic acid sample, so that a decrease in test accuracy due to the change in the concentration of the nucleic acid sample is prevented. .

Various changes and modifications may be made to the present embodiment without departing from the spirit of the present invention.
For example, in the present embodiment, the DNA microarray 110
Although a is washed with washing water, it may be washed with a liquid other than water. In addition, the DNA microarray 110a
May be washed with a gas such as air. That is, D
The NA microarray 110a may be cleaned by passing a gas through it. For example, cleaning with air can be easily performed by changing the solenoid valve 126 to a three-way valve that is open to the atmosphere.

Further, the DNA microarray 110a is
It may be cleaned with a gas containing temperature-controlled microdroplets, such as steam. For example, cleaning with steam can be easily performed by changing the solenoid valve 126 into a three-way valve and connecting it to a humidified steam tank. The DNA microarray 110a is washed by passing water vapor through it. The temperature of the steam is preferably lower than the temperature suitable for the hybridization reaction.

Second Embodiment This embodiment is directed to cleaning of a DNA microarray using ultrasonic waves.

As shown in FIG. 11, the base portion 120
Is provided with two piezoelectric elements 140 that are in surface contact with the lower housing 109 of the reaction vessel 101 placed on the piezoelectric element 140. The piezoelectric element 140 is, for example, 10 mm × 30 mm ×
It has a size of 1 mm. The piezoelectric element 140 is composed of, for example, a lead silicon titanate (PZT) plate that has been polarized in the thickness direction, and silver electrodes deposited on the two surfaces in the thickness direction.

For the piezoelectric element 140, 100 Vp-
An alternating voltage of a sine wave of about p is applied. Piezoelectric element 14
The frequency of the alternating voltage applied to 0 is preferably a frequency that matches the natural frequency of the stage 103. When an alternating voltage having such a frequency is applied, the piezoelectric element 140
Generates strong ultrasonic vibrations.

As shown in FIG. 11, after completion of the hybridization reaction, the syringe piston pump 131
The washing water 137 is sent to the top of the DNA microarray 110a. Thus, the DNA microarray 110a
While the cleaning water 137 is being supplied to the above, the piezoelectric element 140 incorporated in the base portion 120 is driven by the alternating voltage of the sine wave, and the piezoelectric element 140 generates ultrasonic vibration. As a result, the portion to which the cleaning water is supplied is ultrasonically cleaned.

Thereafter, the waste liquid electromagnetic valve 128 is opened, the other electromagnetic valves 126 and 127 are closed, and the suction pump 135 is operated, whereby the excess nucleic acid sample 114 and the washing water 137 are removed from the reaction vessel 101. .

In the present embodiment, ultrasonic cleaning is applied during cleaning with cleaning water to further improve the cleaning effect. Therefore, the excess nucleic acid sample is more reliably removed before the fluorescence observation. As a result, undesired autofluorescence is further reduced, and S of the fluorescence intensity of the nucleic acid probe spot is
The / N ratio is further improved, and more accurate inspection can be performed.

Third Embodiment This embodiment is directed to supply and suction of washing water from above the DNA microarray.

As shown in FIGS. 12 and 13, the inspection apparatus further includes a cleaning nozzle 141 for supplying the cleaning water to the DNA microarray from above, that is, from the inspection surface side. The cleaning nozzle 141 is in contact with the upper housing 108 of the reaction container 101 in a state where the cover 123 of the reaction stage 103 is opened, and supplies the cleaning liquid to the tapered opening 113 formed in the upper housing 108. Although not shown, the cleaning nozzle 141 is supported by another conveying member, and the cleaning nozzle 141 has a cleaning water tank 13
A pump for sending the cleaning water 137 of No. 3 is connected.

After the completion of the hybridization reaction, as shown in FIG. 12, the cover 123 is opened, the cleaning nozzle 141 is brought into contact with the upper housing 108 of the reaction vessel 101, and the tapered opening formed in the upper housing 108. The cleaning liquid is supplied to the portion 113.

While the cleaning liquid is supplied from the cleaning nozzle 141, only the waste liquid solenoid valve 128 is opened and the suction pump 135 is operated. As a result, the wash water 137 is discharged from the reaction container 101 together with the surplus nucleic acid sample 114.

Similar to the second embodiment, the base portion 120 is provided with the piezoelectric element 140 for enhancing the cleaning effect, and ultrasonic cleaning by the piezoelectric element 140 may be used together during this cleaning operation.

Then, as shown in FIG. 13, while the wash water 137 in the wash water tank 133 is being sent to the DNA microarray 110a from the syringe piston pump 131, the wash water is sucked and removed by the wash nozzle 141. This removes the excess nucleic acid sample.

In this embodiment, since the washing water is flown in one direction during the washing operation, the DNA microarray 110a is
Is always washed with normal wash water. This significantly reduces the cleaning time. In addition, since the cleaning liquid is sucked and removed by the cleaning nozzle located above the DNA microarray 110a, dust existing on the upper side of the DNA microarray 110a, that is, the inspection surface side is also removed.

As a result, the nucleic acid sample 114 exists only in the proper nucleic acid probe spot where the hybridization reaction has occurred, and the surplus nucleic acid sample 114 hardly exists. This further reduces unwanted autofluorescence,
The S / N ratio of fluorescence observation is further improved. Therefore, a more accurate inspection can be performed.

The substance for washing the DNA microarray is not limited to a liquid. That is, the substance supplied from the cleaning nozzle 141 may be temperature-controlled fine droplets. The temperature-controlled microdroplets can also efficiently remove the excess nucleic acid sample 114. Preferably, in order to suppress the pressure load when passing through the DNA microarray, the size of vapor droplets may be about several μm or less. The substance supplied from the cleaning nozzle 141 may be steam or air.

Although the embodiments of the present invention have been described with reference to the drawings, the present invention is not limited to these embodiments, and various modifications and alterations can be made without departing from the scope of the invention. Changes may be made.

[0105]

EFFECTS OF THE INVENTION According to the present invention, since a surplus bio-related substance is eliminated before optical observation, there is provided an inspection method capable of simply and accurately inspecting a bio-related substance in a short time in real time. . According to the inspection method of the present invention, it is possible to measure in real time the degree of reaction of the target bio-related substance while changing the state (concentration, etc.) of the sample liquid in the reaction portion and the temperature conditions. Therefore, it is possible to accurately measure the presence or absence of the expression of the target organism-related substance and the expression amount thereof.

[Brief description of drawings]

FIG. 1 is a schematic perspective view of a genetic testing device according to a first embodiment of the present invention, in which a hood is drawn to show a reaction stage housed in the hood.

FIG. 2 is an exploded perspective view of the reaction container shown in FIG.

3 is an exploded perspective view of the slide chip shown in FIG.

FIG. 4 shows a lateral longitudinal sectional view of the reaction vessel shown in FIG.

FIG. 5 shows a number of nucleic acid probe spots arranged on a DNA microarray.

6 is a lateral vertical cross-sectional view of the reaction stage and the reaction container shown in FIG. 1, showing a state in which the nucleic acid sample is located on the DNA microarray immediately after the reaction container is installed. .

FIG. 7 is a lateral cross-sectional view of the reaction stage and the reaction container shown in FIG. 1, showing a state in which the nucleic acid sample has been moved to the liquid storage hole.

FIG. 8 schematically shows a flow path of the genetic testing device according to the first embodiment of the present invention.

FIG. 9 shows a flow of genetic testing in the present embodiment.

FIG. 10 shows a more preferable genetic test flow in this embodiment.

FIG. 11 is a cross-sectional view of a reaction stage and a reaction container according to the second embodiment of the present invention, showing a state in which a DNA microarray is ultrasonically cleaned by a piezoelectric element provided in a base portion.

FIG. 12 is a cross-sectional view of a reaction stage, a reaction container, and a washing nozzle in the genetic testing device according to the third embodiment of the present invention, showing a state in which washing water is supplied from the washing nozzle.

FIG. 13 is a cross-sectional view of a reaction stage, a reaction container, and a washing nozzle in the genetic testing device according to the third embodiment of the present invention, showing a state in which washing water is sucked by the washing nozzle.

[Explanation of symbols]

100 genetic testing equipment 101 reaction vessel 102 microscope 103 Reaction stage 104 CCD camera 105 plate heater 107 slide tip 108 upper housing 109 Lower housing 110 Porous part 110a DNA microarray 111 support plate 116 waste hole 117 Storage hole 119 discharge channel 120 base 123 Cover 124 Transfer channel 125 syringe piston pump part 131 Syringe Piston Pump 133 wash water tank 135 suction pump 136 waste liquid tank

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hidekazu Ishii             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. (72) Inventor Shiba saki             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. F-term (reference) 4B029 AA07 AA23 BB20 CC03 CC10

Claims (7)

[Claims] 1. A method for inspecting a bio-related substance for real-time inspection of a reaction of a bio-related substance using a solid-phase carrier capable of passing a fluid, the supply step of supplying the bio-related substance to the solid-phase carrier. And a reaction step of causing a reaction of the bio-related substance on the solid phase carrier, an elimination step of removing an excess bio-related substance after the reaction of the bio-related substance, and an optical step of the bio-related substance reacted with the solid-phase carrier. The method for inspecting biologically-relevant substances, which comprises an observing step for observing the object. 2. The inspection method according to claim 1, wherein the reaction step, the exclusion step, and the observation step are performed in a humidified atmosphere. 3. The inspection method according to claim 1, wherein the removing step includes a step of immersing the solid phase carrier in washing water. 4. The inspection method according to claim 3, wherein the removing step further includes a step of applying ultrasonic vibration to the cleaning water. 5. The inspection method according to claim 3, wherein the removing step further includes a step of removing the washing water from the upper side of the solid phase carrier. 6. The inspection method according to claim 1, wherein the excluding step includes a step of passing a gas through the solid phase carrier. 7. The inspection method according to claim 6, wherein the gas passed through the solid phase carrier contains temperature-controlled microdroplets.