JP2016169957A - Method and device for inspection using ink-jet method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a device for inspection using an ink-jet method that can inspect more than one sample rapidly, sensitively, and accurately even when the samples are present in small amounts.SOLUTION: The method for inspection using an ink-jet method according to the present application includes a first step of applying a first solution containing a subject of the inspection on a substrate by discharging the solution by an ink-jet method and a second step of applying a second solution containing an antibody to the substrate by discharging the solution by the ink-jet method, the first and second solutions being applied to the same spot of the substrate and the antibody and the subject being reacted on the substrate to determine the quantity of the subject.SELECTED DRAWING: None

Description

  The present invention relates to an inspection method using an inkjet method, and an inspection apparatus that performs the inspection method.

  An immunological measurement method (hereinafter referred to as immunoassay) is one of physical and chemical measurement methods, and is a method for quantitatively measuring substances contained in a sample liquid by utilizing the binding ability of an antigen to an antibody. . For example, in the case of performing quantitative analysis using instrumental analysis, a complicated purification operation is required after grinding and extracting the target substance, but in immunoassay, measurement can be performed only with simple pretreatment. As described above, the immunoassay is widely used because it is easy to operate and not only provides an analysis result in a short time but also does not require expensive analytical instruments and reagents.

  Among them, the ELISA method is widely used as a method in which an antigen or antibody is adsorbed and fixed to the bottom surface of a support, for example, a microplate made of polystyrene, and an antigen-antibody reaction is carried out in the well. However, this manual method requires pre-processing that is not as good as instrumental analysis, requires skill in the reaction operation, and further requires specialized equipment for inspection. It can only be used, or the number of specimens that can be examined at one time cannot be increased.

  Therefore, as a simpler method, a so-called chromatography method in which an antigen-antibody reaction is performed on a flat membrane surface is used. In the chromatographic method, a sample to be measured is first reacted with an antigen or antibody that specifically reacts with a specific substance contained in the sample, and the membrane is moved in advance while moving on the membrane surface of the flat plate. The presence or absence of the measurement object is determined by reacting an antigen or an antibody against the measurement object that has been solid-phased in the middle part on the surface. However, in order to obtain a highly accurate test result, it is difficult to immobilize a certain amount of antigen or antibody accurately on a predetermined membrane surface with good reproducibility, and there is a disadvantage that the reaction takes time.

  In this chromatographic method, the method of pre-immobilizing an antigen or antibody on a flat membrane surface is extremely important. In order to obtain an accurate test result, a certain amount of antigen or antibody is reproducibly reproduced. It is necessary to immobilize on a predetermined membrane surface accurately. Therefore, a method using an ink jet has been studied.

  For example, Patent Document 1 discloses a method in which a protein solution is attached as a droplet to a solid phase by an inkjet method, and the protein is stably immobilized on a substrate by this method. In addition, it is described that the function / activity of the protein is stably maintained without being deactivated. Patent Document 2 also discloses a technique for immobilizing a reactive material using an ink jet.

  Further, in Patent Document 3, a film or carrier having a protein adsorption ability such as a nitrocellulose film is used when an antibody or antigen is solid-phased using an ink jet printer having a piezo element as a printing mechanism. It is disclosed to use.

  Patent Document 4 discloses a chip for detecting a target substance by an antigen-antibody reaction, and the chip has one or more determination areas in which detection of the target substance is recognized, and each determination area includes In addition, a target substance detection chip is disclosed that includes two or more immobilization regions that are independently immobilized with a detection protein that causes an antigen-antibody reaction with a target substance.

Japanese Patent Laying-Open No. 2005-069988 JP 2001-116750 A JP 09-54093 A JP 2010-008109 A

  However, in these techniques, when the number of samples and reagents used for the measurement is reduced, the detection sensitivity is lowered. Therefore, in order to perform quantitative analysis, further improvement in sensitivity and accuracy is desired.

  In view of this, some aspects of the present invention provide an inkjet method capable of inspecting a plurality of samples in a short time even with a small amount of sample by solving at least a part of the above-described problems. It is an object of the present invention to provide an inspection method used and an inspection apparatus for performing the inspection method.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following aspects or application examples.

[Application Example 1]
One aspect of the inspection method using the inkjet method according to the present invention is:
A first step of applying a first solution containing a subject on a substrate by discharging the solution using an inkjet method;
A second step of applying the second solution containing the antibody onto the substrate by ejecting the solution by an inkjet method,
The first solution and the second solution are applied to the same position on the substrate;
The analyte is quantified by reacting the antibody and the analyte on the substrate.

  According to the aspect of application example 1, since the first solution and the second solution are applied to the same position on the substrate by the ink jet method, even a small amount of sample is highly sensitive and accurate in a short time. It is possible to inspect the sample.

[Application Example 2]
In the inspection method using the inkjet method of Application Example 1,
The first solution and the second solution may be mixed on the substrate by a plurality of ejections.

[Application Example 3]
In the inspection method using the inkjet method of Application Example 1 or Application Example 2,
The adherend surface of the substrate may have a water repellent region.

[Application Example 4]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 3,
The substrate may be transparent.

[Application Example 5]
In the inspection method using the inkjet method of any one of Application Examples 1 to 4,
The antibody can be attached to a particle.

[Application Example 6]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 5,
The particle diameter of the particles may be 10 nm or more and 1 μm or less.

[Application Example 7]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 6,
The reaction can be performed while being heated by a heating means.

[Application Example 8]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 7,
The method of discharging the first and second solutions can be a method of using a head using an electrostrictive element.

[Application Example 9]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 8,
The first solution and the second solution can be discharged from different nozzles.

[Application Example 10]
In the inspection method using the inkjet method according to any one of Application Examples 1 to 9,
Further, a third solution that is at least one selected from the group consisting of a cleaning solution, a reagent solution, an amplification solution, and a detection solution is applied to the same position as the position where the first and second solutions are applied. Can have steps.

[Application Example 11]
One aspect of the inspection apparatus using the ink jet method according to the present invention is:
A first nozzle that discharges a first liquid containing a subject onto a substrate by an inkjet method;
A second nozzle that discharges a second liquid containing an antibody onto the substrate by an inkjet method;
The first nozzle and the second nozzle are configured such that the first solution and the second solution are discharged to the same position on the substrate.

The figure which shows the structure of the test | inspection apparatus used for this Embodiment. The figure which shows the structure of a droplet discharge head. The top view which shows the solution arrange | positioned at the array form on the board | substrate. The figure which shows a mode that a solution is discharged on a board | substrate.

  Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, this invention is not limited to the following embodiment, Various modifications implemented in the range which does not change the summary of this invention are also included.

1. INSPECTION METHOD USING INKJET METHOD AND INSPECTION DEVICE An inspection method using an ink jet method according to an embodiment of the present invention is a method in which a first solution containing a subject is applied on a substrate by being ejected by an ink jet method. And a second step of applying a second solution containing an antibody onto the substrate by ejecting the antibody using an inkjet method. The first solution and the second solution are on the substrate. The sample is applied at the same position, and the sample is quantified by reacting the antibody and the sample on the substrate.

  Hereinafter, the inspection method using the ink jet method according to the present embodiment will be described in detail in the order of the configuration of the apparatus capable of implementing this, the substrate, and the inspection method.

1.1. Inspection Device An inspection device using an ink jet method in which the inspection method according to the present embodiment is implemented includes a first nozzle that discharges a first liquid containing a subject onto a substrate by an ink jet method, and a first nozzle that contains an antibody. A second nozzle that discharges the second liquid onto the substrate by an ink jet method, and the first nozzle and the second nozzle eject the first solution and the second solution at the same position on the substrate. Configured to be. Here, “the same position on the substrate” refers to an area on the substrate where the droplet of the first solution and the droplet of the second solution can come into contact. As the inspection apparatus according to the present embodiment, for example, a droplet discharge apparatus shown in FIG.

  FIG. 1 is a diagram showing a configuration of a droplet discharge device used as an inspection device according to the present embodiment.

  As shown in FIG. 1, the droplet discharge apparatus includes a droplet discharge unit 100, a stage (work table) 101, a Y direction drive shaft 102, an X direction guide shaft 104, a stage drive motor 105, a base 106, and a control unit 107. The substrate 108 is placed on the stage 101. A first solution containing a subject and a second solution containing an antibody are applied onto the substrate 108 by ejecting them using an inkjet method, and the first solution and the second solution are mixed. The antibody and the analyte are reacted on the substrate.

  A Y-direction drive motor 103 is connected to the Y-direction drive shaft 102. The Y-direction drive motor 103 is a stepping motor, for example, and rotates the Y-direction drive shaft 102 when an operation signal facing in the Y-axis direction is supplied from the control unit 107. When the Y-direction drive shaft 102 is rotated, the droplet discharge unit 100 moves in the Y direction.

  The X-axis direction guide shaft 104 is fixed to the base 106. A stage drive motor 105 is connected to the stage 101. The stage drive motor 105 is a stepping motor, for example, and moves the stage 101 in the X direction when a drive signal corresponding to the X axis direction is supplied from the control unit 107. That is, by driving the stage 101 in the X direction and driving the droplet discharge unit 100 in the Y direction, the droplet discharge unit 100 can be freely moved to a desired location on the substrate 108. The means for relatively moving the droplet discharge section 100 and the substrate 108 is not limited to the above-described configuration, and can be changed as appropriate.

  The control unit 107 supplies a drive signal for controlling the discharge timing, the number of discharges, and the like of the solution to be discharged to the droplet discharge unit 100. The control unit 107 supplies drive signals for controlling these operations to the Y-direction drive motor 103 and the stage drive motor 105, respectively.

  Next, a configuration example of the droplet discharge unit 100 will be described. FIG. 2 is a diagram illustrating a configuration of the droplet discharge head. Such a droplet discharge head 30 is built in the droplet discharge unit 100 so that the nozzle 321 faces the substrate 108. This droplet discharge head is a so-called electrostatic drive system.

  As shown in FIG. 2A, the droplet discharge head 30 includes a cover glass 31, a pressurizing chamber substrate 32, and an electrode substrate 33. Further, as shown in FIG. 2B, the pressurizing chamber substrate 32 is formed so that a receiving port 323 for liquid supplied from a tank (not shown), and the pressurizing chamber 322 and the nozzle 321 communicate with each other. Has been. A diaphragm is provided on a part of the wall of the pressurizing chamber 322, and an electrode 334 (counter electrode) is provided on the side opposite to the diaphragm and opposite to the flow path via a gap. It has been. When a potential difference is applied to the diaphragm and the counter electrode 334, the diaphragm is attracted to the counter electrode 334 by electrostatic pressure, and generates a negative pressure in the pressurizing chamber. When the potential difference is canceled, the liquid in the pressurizing chamber is made a droplet having a small particle size and discharged from the nozzle 321 by the restoring force of the diaphragm.

  By discharging each solution onto the substrate 108 from the nozzle 321, a plurality of solution droplets can be applied onto the substrate 108 (see FIG. 3). FIG. 3 is a top view showing solutions arranged in an array on the substrate 108.

  A configuration in which a plurality of droplet discharge heads 30 are built in the droplet discharge unit 100 and a tank is individually connected to each head is preferable. By disposing the tanks individually, it is possible to apply various discharge solutions, and it is possible to apply various solutions used in the inspection method described later for each head. One tank may be arranged in common for a plurality of heads. If the tank is shared, the same analyte solution can be discharged onto the substrate 108.

  In FIG. 2, an electrostatic drive type head is exemplified as the droplet discharge head 30, but the present invention is not limited to this, and a piezo type or bubble type head using an electrostrictive element (piezo element) is also possible. Although it can be employed, it is preferable to employ an electrostatically driven or piezo-type head that does not generate instantaneous heat generation in order to prevent degradation or alteration of the antibody reacted with the analyte. The discharge using the electrostatic drive method or the piezo method does not generate heat and does not adhere to a heat source unlike a thermal head, so that it is possible to prevent deterioration of antigens and antibodies that are sensitive to heat. The

  The droplet discharge head 30 is preferably a serial head for full-color printing that ejects a plurality of colors of ink and includes a large number of nozzles for each color. For example, a plurality of nozzles (nozzle openings) for discharging a solution are provided on a nozzle surface (not shown) formed on the surface of the droplet discharge head 30 that faces the substrate 108, and a plurality of nozzles are arranged in a predetermined direction to form a nozzle array. May be configured. In this case, by providing a plurality of nozzle rows, different solutions can be filled one by one.

  Thus, in the case of a configuration including a plurality of nozzles 321, one of them is a first nozzle (not shown) that discharges a first solution containing a subject onto a substrate by an inkjet method, Another one can be assigned to a second nozzle (not shown) that discharges the second solution containing the antibody onto the substrate 108 by an inkjet method. Further, other nozzles are assigned to nozzles that discharge a cleaning solution for washing the reaction product between the antibody and the analyte, and a third solution such as a reagent solution, an amplification solution, and a detection solution onto the substrate 108 by an ink jet method. Can do. Although other solutions can be discharged without using the ink jet method, it is preferable that the third solution is also discharged from the ink jet head from the viewpoint of stably discharging the solution in a constant amount.

  In the droplet discharge apparatus used as the inspection apparatus of the present embodiment, the droplet discharge unit 100 includes a plurality of nozzles 321, so that various types of analytes and various solutions can be discharged onto the substrate 108. It becomes possible to improve work efficiency.

  Note that in the droplet discharge apparatus used in this embodiment, a heating unit 110 is connected to a stage 101 on which a substrate 108 is mounted (see FIG. 1). Such components will be described with reference to the schematic diagram of FIG. FIG. 4 is a cross-sectional view of a main part of the droplet discharge device according to the present embodiment.

  As shown in FIG. 4, the substrate 108 is disposed below the nozzle 321 of the droplet discharge unit 100. The substrate 108 is fitted and fixed on the stage 101. A heating element 110A is connected to the back surface of the stage 101 as a heater 110, and is connected to an external power source (not shown) by a wiring 110B. The heating element 110A can be used without any particular limitation as long as it is used as a heating means. For example, a resistance heating element using a metal can be used as the heating element 110A. However, the heating element 110A is heated to such an extent that it can maintain the optimum temperature of the antigen-antibody reaction, for example, 37 ° C. What is possible is preferred.

  As described above, in the droplet discharge device used in this embodiment, the substrate 108 is heated via the stage 101 by the heater 110, and the solution 350a discharged from the nozzle 321 onto the substrate 108 is heated. it can. For this reason, it becomes possible to promote an antigen-antibody reaction, which will be described later, and other color development and color development stop reactions on the substrate 108. A piezoelectric element may be used instead of the heating element 110A. When a piezoelectric element is used, mixing and reaction of each solution can be promoted by piezoelectric vibration.

1.2. Substrate Next, a substrate used in the inspection method using the inkjet method according to the present embodiment will be described.

As the substrate 108 used in this embodiment, a glass substrate or the like can be used, for example. The substrate 108 is preferably provided with a water-repellent region (not shown) on its surface (attached surface). As will be described later, since the first and second solutions used in the present embodiment are aqueous solutions, when the substrate 108 has a water-repellent region on the surface, as shown in FIG. By discharging, the contact angle of the applied solution 350a with the surface of the substrate 108 is increased, and each reaction such as an antigen-antibody reaction is promoted.
As a substrate

  If the contact angle becomes too large, the solution becomes spherical and difficult to mix, so the contact angle preferably does not exceed 150 °. In addition, the water repellent region may be formed on the entire surface of the substrate 108, or may be configured to have a water repellent region and a water-immersed region on the deposition surface of the substrate 108 by being partially formed. For example, the first solution containing the subject and the second solution containing the antibody may be applied to the water immersion area on the substrate 108 and reacted there. In the water immersion area, the contact angle of the applied solution is 90 ° or less, and there is wettability. Therefore, even when each solution protrudes into the water repellent area, it easily returns to the water immersion area. For this reason, each reaction including the antigen-antibody reaction between the first solution containing the analyte and the second solution containing the antibody is reliably performed.

  The water-repellent region of the substrate 108 can be formed by, for example, forming a metal alkoxide molecular film having water repellency and then performing a drying process, an annealing process, or the like. Further, for example, the surface of the glass substrate may be subjected to water repellent treatment with silicone. When forming a substrate having a water-repellent region and a submerged region on the surface to be adhered, for example, a mask having a circular hole of about 5 mm on the substrate after water-repellent treatment with plasma using fluorine resin or the like The circular portion may be immersed in oxygen plasma, and the mask may be peeled off to form a substrate having a circular immersed region and other water-repellent regions. Alternatively, the water-repellent-treated substrate surface may be coated with cellulose nanoparticles using an ink jet method, and then dried to form a water immersion region.

  Note that the substrate 108 may be transparent. When the substrate 108 is transparent, the degree of color development of each solution can be visually recognized from the back without measuring the absorbance with a plate reader, which will be described later, so that the outline of the reaction result can be known in a short time. Become.

1.3. First Solution Next, the first solution used in the inspection method using the ink jet method according to the present embodiment will be described. The first solution includes an analyte that specifically reacts with the antibody contained in the second solution.

  For example, in the direct competitive inhibition ELISA method, an analyte such as an agricultural chemical and a conjugate of the hapten and the enzyme (enzyme-labeled hapten) react with the antibody against them competitively, and then the enzyme-labeled hapten bound to the antibody. It is an inspection method that measures the concentration of agricultural chemicals using enzyme activity, has few reaction steps at the time of measurement, and is highly sensitive and highly accurate. Usually, the antibody is immobilized on a carrier such as each well of a 96-well plate, and the analyte is dispensed there and reacted in the well. In the inspection method using the inkjet method according to the present embodiment, The antibody is not immobilized on the carrier, and is applied onto the substrate by being ejected by the inkjet method at the same position as the position where the first solution containing the analyte is applied. The analyte is quantified by reacting the second solution.

  As the first solution containing the specimen, for example, when quantifying the agricultural chemicals contained in the vegetables, the liquid obtained by pulverizing and homogenizing the vegetables with a juicer for 1 minute is used as is or filtered if necessary. Or by diluting with water. Conventionally used methods require pretreatment such as adding an organic solvent such as methanol to a pulverized and homogenized sample, shaking and extracting, and then filtering. On the other hand, when the ink jet method is used, pulverization and homogenization can be used as they are without pretreatment, so that it is possible to perform inspection in a short time without the labor of inspection.

  The viscosity of the first solution is preferably adjusted to 1 mPa · s or more and 100 mPa · s or less, for example, by adding glycerin or the like. When the viscosity of the solution is smaller than 1 mPa · s, the ejection becomes unstable, and the quantitative ejection becomes difficult. Further, when the viscosity of the solution exceeds 100 mPa · s, the discharge amount tends to vary depending on the environmental temperature. Preferably, the viscosity of the solution is 2 mPa · s or more and 20 mPa · s or less, and more preferably 3 mPa · s or more and 10 mPa · s or less.

1.4. Second Solution Next, a second solution containing an antibody used in the inspection method using the ink jet method according to the present embodiment will be described. The second solution contains an antibody that specifically reacts with the analyte contained in the first solution.

  The second solution is prepared by adding the antibody to an aqueous solvent such as distilled water so as to have a predetermined concentration. Although any antibody can be used as long as it reacts specifically with the analyte contained in the first solution, a monoclonal antibody is preferably used. For example, when agricultural chemicals in agricultural products are used as the subject, measurement of residual agricultural chemicals in agricultural products is likely to cause errors due to the influence of contaminants derived from agricultural products. Therefore, when a monoclonal antibody having high reactivity with the pesticide as the specimen is used, it becomes difficult to be affected by impurities, and sensitivity and accuracy can be further improved.

In addition, the antibody is preferably attached to fine particles such as polypropylene, polyethylene, and polystyrene so that the antibody can easily adhere to the substrate. In this case, the diameter of the fine particles is preferably a size that can be ejected by an ink jet method, and for example, the diameter of the fine particles is preferably 10 nm or more and 1 μm or less. When the diameter of the fine particles is less than 10 nm, the antibody immobilized on the fine particles tends to vary, and when the diameter exceeds 1 μm, ejection by the ink jet tends to be unstable. The diameter of the fine particles is preferably 15 nm or more and 500 nm or less, and more preferably 20 nm or more and 300 nm or less. The antibody is immobilized on the microparticles by, for example, covalent bonding between the antibody and a functional group of the microparticles that are adsorbed or solid phase.

  As in the first solution, the viscosity of the second solution is preferably adjusted to 1 mPa · s or more and 100 mPa · s or less by adding, for example, glycerin or the like. When the viscosity of the solution is smaller than 1 mPa · s, the ejection becomes unstable, and the quantitative ejection becomes difficult. Further, when the viscosity of the solution exceeds 100 mPa · s, the discharge amount tends to vary depending on the environmental temperature. Preferably, the viscosity of the liquid is 2 mPa · s or more and 20 mPa · s or less, and more preferably 3 mPa · s or more and 10 mPa · s or less.

1.5. Third Solution Next, in the inspection method using the inkjet method according to the present embodiment, a third solution other than the first and second solutions may be used.

  Examples of the third solution include ion-exchanged water as a washing liquid for washing away unreacted substances after the antigen-antibody reaction, a color developing solution for coloring the antigen or antibody, and a reaction terminator for stopping the color reaction. Can be given. In the case of a test method using the PCR method (polymerase chain reaction method), an amplification solution for amplifying DNA can be mentioned. In addition, detection solutions containing various detection substances may be used.

  Also in the third solution, as in the first and second solutions, the viscosity is preferably adjusted to 1 mPa · s or more and 100 mPa · s or less, for example, by adding glycerin or the like.

  The various solutions can be prepared by themselves, but commercially available kits may be used. As a commercially available kit, for example, for agricultural chemicals, residual agricultural chemical measurement ELIZA kit Smart Assay series (manufactured by Horiba Biotechnology), EnviroGard residual agricultural chemical kit (manufactured by SDI), RaPI Dassay residual agricultural chemical kit (manufactured by SDI), TubeKit Residual agricultural chemical kit (manufactured by Beacon) and the like. By using these commercially available kits, it is possible to perform inspection more simply.

1.6. Inspection Method Next, an inspection method using the ink jet method according to the present embodiment will be described.

  The inspection method using the ink jet method according to the present embodiment includes a first step of applying a first solution containing a subject on a substrate by discharging the solution using the ink jet method, and a second solution containing an antibody. A second step of applying the first solution and the second solution on the substrate by discharging them by an inkjet method, and applying the antibody and the analyte to the same position on the substrate. This is done by quantifying the analyte by reacting on the substrate.

In the inspection method using the ink jet method according to the present embodiment, since the ink jet method is used as a method of applying the solution on the substrate in the first and second steps, a variety of methods can be formed on the substrate in a short time. The solution can be applied to a small area. For this reason, it is possible to increase the number of specimens that can be examined at one time. By increasing the number of samples, for example, it is possible not only to examine a plurality of types of analytes at a time, but also to use a plurality of types of antibodies or to create dilution columns with different dilution ratios. As described above, by using the inkjet method, it is possible to inspect a plurality of types of analytes at a time with a plurality of types of reagents and various concentrations.

  When using a micropipette to dispense a solution into a sample bottle or microplate, a mixing / shaking process is required, but when using the inkjet method, a separate mixing / shaking process must be performed. Instead, the first solution and the second solution can be mixed uniformly. Furthermore, by using the inkjet method, it is possible to inspect with a small amount of sample, and furthermore, since the size of dots formed by one ejection can be adjusted as appropriate, a plurality of samples with high sensitivity and high accuracy can be obtained in a short time. Quantitative inspection is possible.

  As described above, “the same position on the substrate” refers to a region on the substrate 108 where the droplet of the first solution and the droplet of the second solution can come into contact. That is, the first solution droplet and the second solution droplet applied on the substrate 108 come into contact with each other to be mixed into one droplet, thereby indicating a reactable region.

  In the inspection method using the inkjet method according to the present embodiment, the first step and the second step may be performed simultaneously, or either step may be performed first. In the case where the first step and the second step are performed at the same time, the first solution and the second solution can be uniformly mixed at the same time as the solution is applied onto the substrate. The antigen-antibody reaction is started simultaneously with the application of the solution, and the test time can be further shortened.

  In the first step and the second step, the amount of one dot is preferably 1 pl or more and 10 μl or less, respectively. If the amount of one dot is less than 1 pl, the dot tends to be foggy and stable ejection becomes difficult, and if it exceeds 10 μl, the mixing of the first solution and the second solution tends to be unstable. More preferably, the amount of one dot is 10 pl or more and 1 μl or less. In this way, when using a micropipette to dispense a solution into a sample bottle or microplate, the amount of the solution increases and the manual variation increases. Less and less variation.

  Further, the first solution and the second solution may be mixed on the substrate by a plurality of ejections. By discharging a plurality of times, the first solution and the second solution on the substrate can be mixed more uniformly.

  In the inspection method using the inkjet method according to the present embodiment, the antigen-antibody reaction may be performed while being heated by a heating means. As described above, the antigen-antibody reaction is promoted by heating with the heating means. Further, by heating during other reactions, it is possible to promote other color development and color development termination reactions. Each reaction may be performed not only by the heating means but also by piezoelectric vibration using a piezoelectric element or the like. Moreover, you may irradiate light for reaction promotion.

  Furthermore, a third step of applying a third solution, which is at least one selected from the group consisting of a cleaning solution, a reagent solution, an amplification solution, and a detection solution, to the same position as the position where the first and second solutions are applied. You may have. By having the third step, the same apparatus can be operated, and the inspection becomes easy. In the third step, it is possible to apply another solution without using the ink jet method, but the third solution is also ejected from the ink jet head from the viewpoint of stably dispensing the solution at a constant rate. It is preferable.

  In the third step, for example, when ion-exchanged water is used as the cleaning liquid, a certain amount of ion-exchanged water may be discharged onto the substrate and the substrate may be moved.

When the reaction product obtained through these steps is developed using, for example, a coloring reagent, for example, the absorbance at a specific wavelength can be measured with a plate reader, and the concentration can be measured by comparison with a calibration curve. .

  As described above, in the inspection method using the ink jet method according to the present embodiment, it is possible to detect a plurality of types and analytes having different concentrations at a high sensitivity and high accuracy at a time by using the ink jet method. For this reason, in an immunoassay in which it is difficult to increase the number of specimens, a test can be performed easily and accurately with a small amount of sample, and further, can be performed in a short time and at a low cost. Thereby, for example, it is possible to simultaneously detect a trace amount and a large number of analytes that could not be analyzed conventionally, and it is possible to easily analyze the interaction between proteins and the interaction between proteins and other molecules. In addition, it can be used for prevention and diagnosis of diseases in a short time in a medical field. Furthermore, since a large number of samples can be measured at one time, it becomes possible to analyze a residual pesticide with many measuring objects in a short time.

2. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to experimental examples and comparative examples, but the present invention is not limited to these examples. In this example, tomato was used as a subject for examining residual pesticides using a competitive enzyme immunoassay. The tomato used was cut into edible portions, ground and homogenized with a juicer for 1 minute, and then filtered through a membrane filter having a pore size of 5 μm as needed to use as tomato juice. Tomato juice was used in advance after confirming that no residual pesticide was detected.

2.1. Preparation of standard solution for preparing calibration curve Using the agrochemical reagents contained in the following commercially available kits 1 to 4, standard solutions for preparing calibration curves of four types of pesticides (acetamipride, isoxanthion, thiamethoxam, malathion) were prepared. First, put the tomato juice into a 5 g test tube, add the agrochemical reagent to a concentration of 20 ppb, then add the enzyme complex solution contained in the following commercial kits 1 to 4 to this mixture, etc. The mixture was diluted and used as a standard solution for preparing a calibration curve. As an inspection device, an inkjet printer PX-G930 (manufactured by Seiko Epson Corporation) using a piezo element head was used, and the obtained standard curve preparation standard solutions were put in four ink containers, and glycerin was put in each ink container. The viscosity of the solution was adjusted to 4 mPa · s, and the discharge amount was quantified. In addition, as the commercially available kits 1 to 4, 96-well plate specifications of the residual pesticide measurement ELIZA kit Smart Assay series (manufactured by Horiba Biotechnology) were used.
<Immunoassay commercial kit HORIBA>
-Commercial kit 1: Acetamiprid measurement kit-Commercial kit 2: Isoxanthion measurement kit-Commercial kit 3: thiamethoxam measurement kit-Commercial kit 4: Malathion measurement kit

2.2. Preparation of analyte solution 2.2.1. Example 1
Similar to the standard solution for preparing the calibration curve, 5 g of tomato juice is put in a test tube, and acetamiprid is added as an agrochemical component to a concentration of 1 ppb. In the same manner as above, an enzyme complex solution was added and mixed with an equal amount to prepare a sample solution. Then, it was put in another ink container of an ink jet printer, glycerin was added, the viscosity of the sample solution was adjusted to 4 mPa · s, and the discharge amount was quantified.

2.2.2. Example 2
Example 2 was prepared in the same manner as in Example 1 except that isoxanthion was added to a concentration of 5 ppb instead of acetamiprid added as an agrochemical component in Example 1.

2.2.3. Example 3
Example 3 was prepared in the same manner as in Example 1 except that thiamethoxam was added instead of acetamiprid added as an agrochemical component in Example 1.

2.2.4. Example 4
Example 4 was prepared in the same manner as in Example 1 except that malathion was added to a concentration of 100 ppb instead of acetamiprid added as an agrochemical component in Example 1.

2.2.5. Example 5
Example 5 was prepared in the same manner as in Example 1 except that a mixture of acetamiprid and thiamethoxam was added instead of acetamiprid added as an agrochemical component in Example 1.

2.3. Preparation of antibody solution Put the antibody solution contained in the above-mentioned commercial kit 1 in an ink container different from the container containing the standard solution for preparing the calibration curve and the sample solution, add glycerin, and the viscosity of the sample solution Was adjusted to 4 mPa · s to quantify the discharge rate. Similarly, in the other three ink containers, put the antibody solutions contained in the above-mentioned commercially available kits 2 to 4, respectively, add glycerin, adjust the viscosity of each antibody solution to 4 mPa · s, and discharge the amount. Quantified.

2.4. Preparation of other solutions The ion exchange water for washing, the color developing solution, and the reaction terminator were separately put in an ink container different from the standard solution for preparing the calibration curve, the sample solution, and the antibody solution. To the solutions in these ink containers, glycerin was added to adjust the viscosity of each solution to 4 mPa · s and the discharge amount was quantified in the same manner as the standard curve preparation standard solution, the sample solution, and the antibody solution. .

2.5. Quantitative determination of pesticides 2.5.1. Preparation of calibration curve At room temperature, each antibody solution was applied to a glass substrate whose surface was water-repellent treated with silicone by the inkjet method so that the number of dots was 50 μl. On the applied antibody solution, apply 50 μl of the standard solution for preparing the calibration curve to the number of dots, and leave it in that state for 30 minutes. The antibody in the antibody solution and the agrochemical component in the standard solution for preparing the calibration curve are used as antigens. Antibody reaction was performed. While left at room temperature, most of the water in the reaction solution reacted on the substrate was evaporated. Next, ion exchange water was applied onto the reaction product by an ink jet method to wash the mixed portion on the substrate, and unreacted agricultural chemical components were removed. Thereafter, a color developing solution was applied onto the washed reaction product by an ink jet method and allowed to stand for 10 minutes for color development reaction. And the reaction stop liquid was apply | coated to the location which carried out color reaction by the inkjet method, and reaction was stopped. Finally, the absorbance at a wavelength of 450 nm was measured with a Multiskan GO microplate spectrophotometer manufactured by Thermo Scientific to create a calibration curve.

2.5.2. Measurement of Agricultural Chemical Components in Sample Solution Similar to the creation of a calibration curve, each antibody solution was applied to a glass substrate whose surface was water-repellent treated with silicone at room temperature by the ink jet method at a dot number of 50 μl. On the applied antibody solution, the sample solution was applied in a dot number of 50 μl, and left in that state for 30 minutes to cause an antibody-antibody reaction between the antibody in the antibody solution and the agrochemical component in the sample solution. While left at room temperature, most of the water in the reaction solution reacted on the substrate was evaporated. Next, ion exchange water was applied onto the reaction product by an ink jet method to wash the mixed portion on the substrate, and unreacted agricultural chemical components were removed. Thereafter, a color developing solution was applied onto the washed reaction product by an ink jet method and allowed to stand for 10 minutes for color development reaction. And the reaction stop liquid was apply | coated to the location which carried out color reaction by the inkjet method, and reaction was stopped. Finally, the absorbance at a wavelength of 450 nm was measured with a plate reader, the concentration was calculated from the prepared calibration curve, and the dilution was converted to the concentration of the pesticide contained in the sample. The same process was performed three times.

2.6.1. Comparative Example 1
In Example 1 above, each solution was ejected using the inkjet method, but in Comparative Example 1, each solution was dispensed using a micropipette. Specifically, a sample solution was prepared in the same manner as in Example 1 above, and 10 μl of the obtained sample solution was placed in the well of the antibody plate of the commercial kit 1 using a micropipette and incubated at room temperature for 60 minutes. Next, the well plate was washed with ion exchange water. Thereafter, 100 μl of the color developing solution was dropped into each well and incubated for 10 minutes, and further 100 μl of a reaction stop solution was dropped to stop the reaction. Next, the absorbance of each well at a wavelength of 450 nm was measured with a plate reader, the concentration was calculated from a calibration curve prepared in advance, and the dilution was converted to the concentration of the pesticide contained in the sample. The same process was performed three times. In preparing the calibration curve, 150 μl of the enzyme complex solution was used.

2.6.2. Example 6
Example 1 was performed in the same manner as Example 1 except that a thermal head was used instead of a printer using a piezo element head.

2.7. Evaluation result In Examples 1-5, each agrochemical component was detected within 10% of reproducibility. On the other hand, in Comparative Example 1, the reproducibility exceeded 20%, and the variation was large. This is presumably because Comparative Example 1 was performed manually and it was necessary to perform dilution and extraction many times. Moreover, the sample amount required in Comparative Example 1 was 100 times or more that in Examples 1 to 5, and it took 6 hours until the measurement result was obtained. It was not possible to measure in Example 6. This is probably because the antibody was inactivated by heat because a thermal head was used.

  As described above, the present invention can easily and accurately perform a test with a small amount of sample in an immunoassay in which it is difficult to increase the number of specimens. It becomes possible to inspect. In this example, the preparation of the calibration curve and the measurement of the agrochemical component in the sample solution were performed separately, but they can also be performed by a single operation. In this case, the inspection time can be further shortened.

  The present invention is not limited to the embodiments described above, and various modifications are possible. For example, the present invention includes substantially the same configuration (for example, a configuration having the same function, method, and result, or a configuration having the same purpose and effect) as the configuration described in the embodiment. In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that achieves the same effect as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

DESCRIPTION OF SYMBOLS 30 ... Droplet discharge head, 31 ... Cover glass, 32 ... Pressurization chamber board | substrate, 33 ... Electrode substrate, 100 ... Droplet discharge part, 101 ... Stage, 102 ... Y direction drive shaft, 103 ... Y direction drive motor, 104 DESCRIPTION OF SYMBOLS X direction guide shaft, 105 ... Stage drive motor, 106 ... Base, 107 ... Control part, 108 ... Substrate, 110 ... Heater, 110A ... Heating element, 110B ... Wiring, 321 ... Nozzle, 322 ... Pressure chamber, 323 ... Reception port, 334 ... Electrode (counter electrode), 350 ... Solution (droplet), 350a ... Solution

Claims (11)

A first step of applying a first solution containing a subject on a substrate by discharging it by an ink jet method and a second step of applying a second solution containing an antibody on the substrate by discharging an ink jet method 2 steps,
The first solution and the second solution are applied to the same position on the substrate;
An inspection method using an inkjet method, wherein the analyte is quantified by reacting the antibody and the analyte on the substrate. In claim 1,
An inspection method using an inkjet method, wherein the first solution and the second solution are mixed on the substrate by a plurality of ejections. In claim 1 or claim 2,
An inspection method using an ink jet method, wherein a surface to be attached of the substrate has a water-repellent region. In any one of Claims 1 thru | or 3,
An inspection method using an inkjet method, wherein the substrate is transparent. In any one of Claims 1 thru | or 4,
An inspection method using an inkjet method in which the antibody is attached to particles. In any one of Claims 1 thru | or 5,
An inspection method using an ink jet method, wherein the particle diameter of the particles is 10 nm or more and 1 μm or less. In any one of Claims 1 thru | or 6,
The said reaction is the inspection method using the inkjet method performed while heating with a heating means. In any one of Claims 1 thru | or 7,
An inspection method using an ink jet method, wherein the first and second solutions are ejected using a head using an electrostrictive element. In any one of Claims 1 thru | or 8,
The inspection method using an inkjet method in which the first solution and the second solution are discharged from different nozzles. In any one of Claims 1 thru | or 9,
Further, a third solution that is at least one selected from the group consisting of a cleaning solution, a reagent solution, an amplification solution, and a detection solution is applied to the same position as the position where the first and second solutions are applied. An inspection method using an inkjet method, which includes a step. A first nozzle that discharges a first liquid containing a subject onto a substrate by an inkjet method;
A second nozzle that discharges a second liquid containing an antibody onto the substrate by an inkjet method;
The first nozzle and the second nozzle are configured such that the first solution and the second solution are ejected to the same position on the substrate, and an inspection apparatus using an inkjet method .

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