JP4203826B2 - Automatic analysis method and apparatus - Google Patents

Description

  The present invention relates to a method and apparatus for analyzing a sample by a sensor unit including a piezoelectric element such as a crystal resonator, and more particularly to a method and apparatus capable of analyzing dioxins.

Modern comfortable social life and industrial activities depend on mass production and consumption of many artificial and natural chemicals. These are a wide variety of chemical substances produced and produced as by-products from industrial chemicals, raw materials for polymer compounds, dyes / pigments, pharmaceuticals, agricultural chemicals, food / feed additives, biotechnology products, etc. , Natural products, and compounds made from natural products. On the other hand, there are environmental and safety issues and health promotion related to research and development, manufacturing, sales, use and disposal of chemical substances, environmental pollution caused by chemical substances, side effects due to toxic impurities in pharmaceuticals, food Various problems such as residual pesticides, contamination with toxic substances derived from additives in food and feed, food contamination by mycotoxins, narcotics, abuse of chemical and bioweapons, etc. have occurred. To maintain human health and environmental safety, quality and process management for all processes related to R & D, manufacturing, sales, use and disposal of many chemical substances, and toxicity of chemical substances It is necessary to clarify the permissible amount to humans and foods, the amount of exposure to the environment and biological systems, and to determine the permissible amount, regulatory values, waste disposal and storage methods, etc. This purpose requires a large amount of money and a large number of personnel, and various laws and regulations to be observed have been established.
In general, gas chromatography (GC) analyzer, high-performance liquid chromatography (HPLC) analyzer, gas chromatography mass spectrometry is used to measure chemical substances in samples derived from industrial products, humans, environment, food, livestock and marine products. An analytical instrument such as a device (GC / MS) is used. These analytical instruments are capable of precise and high-precision measurements, but they require an analysis room with an air-conditioning system for installing the analytical instruments, an expensive instrument body, and a skilled analyst for instrument analysis. Analyzing institutions that can be installed are limited, and many days are required for the analysis including the pretreatment of the sample, and there is a big problem that the analysis cost of the sample becomes high. Taking pesticides as an example, the percentage of analysis costs in the price including product research, development, manufacturing, quality control, safety assessment, etc. reaches 15%, and these costs have a significant impact on the international competitiveness of products. is doing.
In order to solve various problems associated with instrumental analysis, it is necessary to develop an economical measurement method that is quick, simple, and highly sensitive, and immunochemical measurement methods are attracting attention. This method uses the specificity of the antigen / antibody reaction and is a new application of the principle that has been used for protein testing in clinical tests to low molecular weight compounds. In Western countries, reagents are being developed for measuring environmental pollutants and residual pesticides in soil and groundwater, measuring food mycotoxins and food additives, and measuring antibiotics and narcotics. In Japan, imports and sales of reagents developed in Europe and the US are also in progress.
For many important chemical substances that support human life and industrial activities, it is desirable to promote the research, development and dissemination of immunochemical measurement reagents that meet domestic and foreign regulations. This method is applicable to environmental products, agricultural chemicals, food and feed additives, mycotoxins, hormones, vitamins, antibiotics for industrial products and environmental, food, livestock and fishery products, human samples, regardless of whether they are water-soluble or water-insoluble. It can be applied to the measurement of a wide range of chemical substances such as substances, sulfa drugs, active ingredients of Chinese medicine, biotechnology products, narcotics and doping agents, general chemical substances, chemical weapons, bioterrorism-related substances.
By the way, a liquid containing a substance to be measured is dropped on a detector using a piezoelectric element such as a quartz vibrator to change the apparent thickness or mass of the piezoelectric element, and thereby the resonance frequency (or oscillation frequency) of the piezoelectric element. ) Is known (see, for example, Japanese Patent Application Laid-Open No. 2000-283905 or International Publication WO00 / 26636 pamphlet). The present inventors have developed an apparatus for inspecting the presence or amount of environmental pollutants such as dioxins using this measurement method (for example, Japanese Patent Application Laid-Open Nos. 2001-242057 and 2002-310873). Gazette and JP-A-2002-310874). However, although it has the advantage of being able to analyze environmental samples at a low cost, it requires skilled workers to perform an accurate analysis and cannot measure multiple samples at the same time. There is a growing need for automatic analyzers from an analysis perspective. As an automatic analyzer, it is conceivable to apply, for example, an apparatus described in Jomotehei 11-502937.
However, in the above-described conventional technology, the liquid sample is sucked into the probe, and the sucked sample is merely discharged to the sensor unit having a piezoelectric element (quartz crystal unit, etc.). There is a problem that the distribution of environmental pollutants in the country differs every time the inspection is performed, and as a result, an accurate inspection cannot be performed.
The above and other features and advantages of the present invention will become more apparent from the following description when considered in conjunction with the accompanying drawings.

FIG. 1 is a perspective view showing an embodiment of an analyzer according to the present invention.
FIG. 2 is a plan view showing an embodiment of a quartz crystal substrate used in the analyzer shown in FIG.
FIGS. 3A and 3B are enlarged cross-sectional views showing an embodiment of the sensor unit, in which FIG. 3A shows a state in which the sample has been (re) discharged, and FIG. 3B shows a state in which the sample has been (re) sucked.
FIG. 4 is an enlarged plan view showing an embodiment of the sensor unit.
FIG. 5 is a sectional view showing another embodiment of the quartz substrate.
FIG. 6 is a plan view showing another embodiment of the sensor unit.
FIG. 7 is a side view showing another embodiment of the sensor unit.
FIG. 8 is a perspective view showing another embodiment of the sensor unit.
FIG. 9 is a chart showing specific steps of an analysis method using the apparatus of the present invention.
(A) shows a method of subtracting a crystal resonator in each step, and (b) is a chart diagram showing a method of measurement using all crystal resonators.
FIG. 10 is a chart showing specific steps of an analysis method using the apparatus of the present invention.
FIG. 11 is a chart showing specific steps of an analysis method using the apparatus of the present invention.
FIG. 12 is a chart showing specific steps of an analysis method using the apparatus of the present invention.
FIG. 13 is a chart showing specific steps of an analysis method by a direct reaction method using the apparatus of the present invention.
FIG. 14 is a chart showing specific steps of an analysis method based on a chemical amplification reaction of a sensor response by a sandwich reaction after a direct reaction method using the apparatus of the present invention.
FIG. 15 is a chart showing specific steps of an analysis method based on a competitive reaction method using the apparatus of the present invention.
FIG. 16 is a chart showing specific steps of an analysis method based on a chemical amplification reaction of a sensor response by a sandwich reaction after a competitive reaction method using the apparatus of the present invention.
FIG. 17 is a chart showing specific steps of an analysis method by an amplification reaction (ATRP method) of a sensor response by an antibody immobilized on a polymer with a clear molecular structure using the apparatus of the present invention.
FIG. 18 is a chart showing specific steps of an analysis method based on a DNA hybridization measurement method using the apparatus of the present invention.
FIG. 19 is a graph showing the results of dioxin analysis of the example.
FIG. 20 is a graph showing the results of fenitrothion analysis of the examples.
FIG. 21 is a graph showing the results of carbofuran analysis of the example.
FIG. 22 is a graph showing the results of simazine analysis in Examples.
FIG. 23 is a graph showing the results of nonylphenol analysis in Examples.
FIG. 24 is a graph showing the results of 2,4-dichlorophenoxyacetic acid analysis of the examples.
FIG. 25 is a graph showing the results of the molinate analysis of the example.
FIG. 26 is a graph showing the results of atrazine analysis in Examples.
FIG. 27 is a graph showing the results of the metolachlor analysis of the example.
FIG. 28 is a graph showing the results of the alachlor analysis of the example.
FIG. 29 is a graph showing the results of diazinon analysis of the example.
FIG. 30 is a graph showing the results of anti-syphilis (TP) antibody analysis in Examples.
FIG. 31 is a graph showing the results of FDP analysis of the example.
FIG. 32 is a graph showing the results of CRP analysis of the example.
FIG. 33 is a graph showing the results of the AFP analysis of the example.
FIG. 34 is a graph showing the results of ASO analysis of the example.
FIG. 35 is a graph showing the results of RA analysis of the example.
FIG. 36 is a graph showing the results of ferritin analysis in Examples.
FIG. 37 is a graph showing the results of Legionella analysis of Examples.
FIG. 38 is a graph showing the results of the Escherichia coli analysis of the example.
FIG. 39 is a graph showing the results of 2,3,7,8-TCDD analysis of Examples.
FIG. 40 is a graph showing the results of the Escherichia coli analysis of the example.
FIG. 41 is a graph showing the results of the α-fetoprotein analysis of the example, and shows the results of measuring the α-fetoprotein concentration in human serum. FIG. 42 is a graph showing the results of atrazine analysis of the example, and shows an example of measurement by competitive reaction of atrazine concentration in environmental water. 41 (a) and 42 (a) are examples of measurement by a manual operation by an expert, and FIGS. 41 (b) and 42 (b) show examples of measurement with the automatic analyzer of the present invention. Each measurement point is an average value when six independent crystal units are used, and an error bar on the vertical axis indicates (standard deviation).
FIG. 43 is a front view showing the shape of the distal end portion (nozzle, 100) of the dispensing probe. In FIG. 43, reference numeral 101 denotes a water repellent processed portion such as Teflon (registered trademark). In FIGS. 43 (1) to 43 (7), the right hand side of the paper is the tip (nozzle tip).

According to the present invention, the following means are provided:
(1) The sample in the container is sucked into the probe, and the mass change on the sensor is changed to the fundamental resonance frequency or the oscillation frequency change in the overtone mode, the resonance frequency change in each mode, the impedance change, and the phase. Change, current change, voltage change, capacitance change, reactance change, admittance change, susceptance change, or magnetic moment change, etc. Discharging and re-sucking the discharged sample to the probe and re-discharge the re-suctioned sample to the sensor unit, thereby causing a chemical reaction (for example, covalent bond, Physiological adsorption, immune reaction, hybridization, sugar chain recognition reaction, etc.) Alternatively, the progress of the detachment reaction of the substance to be measured (analytical substance) can be promoted from the sensor part by using an unreacted substance or a detachment reaction reagent (for example, any reagent such as urea or glycine hydrochloride solution). Including analytical methods.
(2) The suction of the sample in the container to the probe, the discharge of the sample from the probe to the sensor unit, the re-suction of the discharged sample to the probe, and the re-sucked sample The analysis method according to (1), wherein the re-discharge is performed by an automatic analyzer including the plurality of containers, the plurality of probes, and the plurality of sensor units.
(3) Further, a cleaning liquid (for example, a liquid such as ultrapure water, pure water, ion exchange water, distilled water, heavy water, methanol, ethanol, propanol, etc., or one or more solutions thereof is disposed inside and outside the probe. (1), including washing with ultrapure water, pure water, ion-exchanged water, and distilled water, and most preferably ultrapure water and pure water. The analysis method described in 1.
(4) The analysis method according to (1), further comprising spraying a cleaning liquid or gas from the probe or another probe to the sensor unit to clean the sensor unit.
(5) A container placement section for placing a container for containing a sample, a probe for sucking and discharging a sample for analysis in the placed container, and a mass change on the sensor, a fundamental resonance frequency or an overtone (overtone) ) Mode oscillation frequency change, resonance frequency change in each mode, impedance change, phase change, current change, voltage change, capacitance change, reactance change, admittance change, susceptance change, or magnetic moment change, etc. And a sensor unit having a piezoelectric element that detects (quantifies)
The probe has a first position for sucking the sample in the container, and a second position for performing re-suction of the discharged sample and re-discharge of the re-sucked sample after discharging the sucked sample to the sensor unit. An analyzer that is selectively movable in time and / or space to a position.
(6) The container placement section is capable of placing a plurality of containers, the plurality of probes are provided to be selectively movable to the first and second positions, and the plurality of sensor sections are provided. The analyzer according to (5), which is provided.
(7) Furthermore, a second container placement section for placing a second container containing a cleaning liquid is included, and the plurality of probes can be further moved to a third position for cleaning the probes with the cleaning liquid. The analyzer according to item (6).
(8) The analyzer according to (7), further including a cleaning device that sprays cleaning liquid or gas onto the sensor unit to clean the sensor unit.
(9) Furthermore, it includes a plurality of second probes that blow cleaning or drying gas onto the sensor unit, and the second probe has a third position that blows the gas onto the sensor unit; The analyzer according to (5) or (6), wherein the analyzer is selectively movable from a position 3 to a fourth position spaced from the position 3.
(10) By using the molecular recognition reaction of the immune reaction or receptor molecular recognition, hybridization of DNA or DNA aptamer, sugar chain recognition of lectin, peptide array of peptide or combinatorial synthesis, or synthetic receptor molecular recognition reaction Any one of the above (1) to (9) related to a novel rapid, simple, and highly sensitive measurement method for specific chemical substances, viruses, bacteria, etc. that interact with molecules immobilized on the sensor The analyzer described.
(11) The number of suction / discharge is preferably 1 to 10,000 times / minute, more preferably 1 to 1,000 times / minute, and most preferably 1 to 1,000 times / minute. The analyzer according to any one of (10) to (10).
(12) Any one of the above (1) to (11), wherein the preferable suction / discharge repetition time is 0.1 to 100 minutes, more preferably 1 to 30 minutes, and most preferably 1 to 10 minutes. 2. The analyzer according to item 1.
(13) A preferable suction / discharge amount is 0.01 to 100 microliters, more preferably 0.1 to 50 microliters, and most preferably 1 to 30 microliters (1) to (12). The analyzer according to any one of the above.
(14) As shown in FIG. 37, for example, the preferable probe tip (nozzle) has a cylindrical shape (for example, FIG. 37 (1)), a bevel tip type (for example, FIG. 37 (2)), and a spherical shape. (E.g., Fig. 37 (3)), conical type (e.g., Figs. 37 (4) to 37 (5)), needle type (e.g., Fig. 37 (6)), cylindrical type coated with fluororesin (e.g., figure 37 (7)), a polyhedral type such as a quadrilateral cross section, etc., more preferably a cylindrical type, a bevel tip type or a conical type, and most preferably a cylindrical type or a conical type. The analyzer according to any one of the above.
(15) The positional relationship between the preferred nozzle and the piezoelectric element sensor during suction / discharge (the distance between the top and bottom, for example, the distance between the tip of 30 and the top surface of 50 in FIG. 3) is preferably 0.01 to 10 mm The analysis apparatus according to any one of (1) to (14), which is more preferably 0.1 to 10 mm, and most preferably a distance of 0.5 to 3 mm.
(16) The preferred shape of the piezoelectric element is platinum, gold, silver, copper in a groove formed by processing a quartz base plate into a concave shape on both sides or one side by chemical treatment or machining, or a flat shape such as # 4000 finish, mirror finish, etc. Integrate multiple oscillators created by vapor deposition of electrodes, etc., and crystal base plate by chemical treatment or machining to form concave shape on both sides or only one side or flat shape such as # 4000 finish, mirror finish etc. The analyzer according to any one of (1) to (15), wherein the device is a single element formed by vapor deposition of electrodes such as platinum, gold, silver, and copper in the groove.
(17) Preferred piezoelectric elements include a quartz resonator, a surface acoustic wave element, a flexural plate wave (FPW) element, an acoustic plate wave (APM) element, a shear horizonal acoustic plate (SH-APM) element, a flexural low element, A resonator or a surface transverse wave (STW) surface shear wave device, more preferably a crystal resonator, a surface acoustic wave device, a ceramic resonator, or an STW device, and most preferably a crystal resonator ( The analyzer according to any one of 1) to (16).
(18) A preferable method for detecting the mass load on the piezoelectric element is preferably a fundamental resonance frequency or an oscillation frequency change in an overtone mode, a resonance frequency change in each mode, an impedance change, a phase change, and a current. Change, voltage change, capacitance change, reactance change, admittance change, susceptance change, or electrical change such as magnetic moment change, and more preferably basic resonance frequency or overtone mode. Oscillation frequency change, resonance frequency change in each mode, impedance change, phase change, current change, or voltage change, most preferably oscillation frequency change, resonance frequency change, impedance change, phase change at the basic resonance frequency Said change, current change, or voltage change 1) Analysis device according to any one of - (17).
(19) The sensor body for detecting the characteristics of the piezoelectric element preferably has an oscillation comprising one or more piezoelectric elements such as 20 in FIG. 1 or 20 in FIG. 6, FIG. 7, and FIG. It is a frequency measurement device or a detection device as an electrical change such as resonance frequency change, impedance change, phase change, current change, voltage change, capacitance change, reactance change, admittance change, susceptance change, or magnetic moment change, and more Preferably, it is a detecting device for oscillation frequency change in fundamental resonance frequency or overtone (overtone) mode, resonance frequency change in each mode, impedance change, phase change, current change, or voltage change, and most preferably basic Oscillation frequency change at resonance frequency, resonance frequency change, impedance change, phase change, current change Or wherein the detection device of the voltage change (1) Analysis apparatus according to any one of - (17).
(20) Drying of the piezoelectric element is preferably performed by dry clean air, dry air, high purity inert gas (for example, helium, argon, xenon, neon, krypton, etc.), high purity nitrogen gas, nitrogen gas, hydrogen It is carried out using a gas selected from gas, halogen gas, oxygen gas or using vacuum suction, more preferably dry clean air, dry air, nitrogen gas, high purity inert gas (for example, helium Or argon), using a gas selected from high purity nitrogen gas, or using vacuum suction, most preferably dry clean air, high purity nitrogen gas, high purity inert gas (eg, argon) The analyzer according to any one of (1) to (19), wherein the analyzer is performed using a gas selected from
(21) The oscillation or resonance frequency of the piezoelectric element is preferably 0.01 MHz to 100,000 MHz, more preferably 1 MHz to 1,000 MHz, and most preferably 1 MHz to 160 MHz. The analyzer according to any one of the above.
(22) The molecular recognition element immobilized on the piezoelectric element is preferably a monoclonal or polyclonal antibody, probe DNA, peptide DNA, peptide, lectin, sugar that can specifically react with and capture the molecule to be detected. Chain, protein, antigen molecule for detection antibody, synthetic or natural receptor molecule, more preferably monoclonal molecule or polyclonal antibody, probe DNA, antigen molecule for detection antibody that can specifically react with and capture the detection target molecule Most preferably, it is a monoclonal or polyclonal antibody capable of specifically reacting and capturing with a molecule to be detected, and an antigen molecule for the detection antibody according to any one of the above (1) to (21) Analysis equipment.
(23) The number of piezoelectric elements and their detection circuits used for simultaneous detection is preferably 1 to 1,000,000, more preferably 1 to 1,000, and most preferably 1 to 100. The analysis device according to any one of (1) to (22), wherein the analysis device is a single piece.
(24) The number of probes for suction and discharge used for simultaneous detection is preferably 1 to 1,000,000, more preferably 1 to 1,000, and most preferably 1 to 100. The analyzer according to any one of (1) to (23), which is a book.
(25) The number used for detecting the piezoelectric element is preferably 1 to 1,000,000, more preferably 1 to 1,000, and most preferably 1 to 100 ( The analyzer according to any one of 1) to (24).
(26) C-reactive protein (CRP), anti-streptidine O (ASO), anti-syphilis antibody (TP), fibrin degradation product (FDP), α-fetoprotein (AFP), rheumatoid factor (RA), which are disease marker substances ), The analyzer according to any one of (1) to (25), wherein ferritin is detected.
(27) The above-mentioned (1) to detect dioxins, PCBs, fenitrothion, carbofuran, simazine, nonylphenol, 2,4-dichlorophenoxyacetic acid, molinate, atrazine, metolachlor, alachlor, diazinon which are environmental pollutants. (25) The analyzer according to any one of (25).
(28) The analyzer according to any one of (1) to (25), which detects the number of Legionella spp. Or E. coli, which are microorganisms detected in food inspection.
(29) a non-labeled antibody (first antibody) in which a polymer or metal fine particles introduced onto a piezoelectric element (for example, a quartz crystal resonator) by a chemical bond such as a covalent bond or physical adsorption is not immobilized; Amplification of sensor response (ie, amplification by chemical reaction of electrical signals) by sandwich reaction using polymer or metal fine particles (for example, nanoparticles, micron particles, etc.) immobilized on two or second antibodies The analyzer according to any one of (1) to (25) to be detected.
(30) An unlabeled probe DNA (first probe DNA) on which a polymer or metal fine particle introduced onto a piezoelectric element (for example, a quartz crystal resonator) by chemical bonding such as covalent bonding or physical adsorption is not immobilized; Amplification of sensor response (chemical amplification of electrical signal) by sandwich reaction using second probe DNA or polymer immobilized on second probe DNA or metal fine particles (for example, nanoparticles, micron particles, etc.) The analyzer according to any one of (1) to (25), wherein the reaction is detected.
(31) Via an active esterification reaction on a polyacrylic acid polymer chain with a well-defined molecular structure synthesized by an atom transfer radical polymerization method in an academic literature (printed by the present inventors, Biosensors & Bioelectronics) on a piezoelectric element. An antibody immobilized by a covalent bond method was used (for example, 10 times the antibody immobilization density by the three-dimensional immobilization reaction of the antibody, see the ATRP method described later), and the amplification of the sensor response (electric signal (Analytical amplification) The analyzer according to any one of (1) to (25), which detects a reaction.
(32) The temperature range used for detection with a piezoelectric element (for example, detection of immune reaction) is preferably −20 degrees to 100 degrees Celsius, more preferably 0 degrees to 50 degrees Celsius, and most preferably The analyzer according to any one of (1) to (25), which is 5 degrees to 40 degrees Celsius.
(33) The measurement time used for detection with a piezoelectric element (for example, detection of immune reaction) is preferably 1 minute to 1,000 minutes, more preferably 5 minutes to 100 minutes, and most preferably 10 minutes. The analysis device according to any one of (1) to (25), wherein the analysis device has a minute to 30 minute period.
(34) As a distance between a dispensing pipette (for example, 30 in FIG. 3) used for detection with a piezoelectric element (for example, detection of immune reaction) and a piezoelectric element (for example, 50 in FIG. 3), The analyzer according to any one of (1) to (25), which is 0.01 mm to 100 mm, more preferably 0.1 mm to 10 mm, and most preferably 0.5 mm to 2 mm.
(35) The angle between the dispensing pipette used for detection with the piezoelectric element (for example, detection of immune reaction) and the surface of the piezoelectric element is preferably in the range of 10 degrees to 270 degrees, more preferably 10 degrees to The analysis apparatus according to any one of (1) to (25), which is in a range of 180 degrees, and most preferably in a range of 90 degrees to 30 degrees. The angle here refers to an angle formed by a dispensing pipette with the horizontal plane when the surface of the piezoelectric element is a horizontal plane (0 degree).
(36) As an analysis sensor, a quartz oscillator, a surface acoustic wave element (SAW), a flexural plate wave (FPW) element, an acoustic plate wave (APM) element, a shear horizonal acoustic plate (SH-APM) element, a flexure element Using the piezoelectric effect of elements, ceramic vibrators, surface transverse wave (STW) surface shear wave elements, etc., the molecular recognition reaction on the sensor can be performed by changing current, voltage, capacitance, reactance, impedance, admittance, and susceptance. The analyzer according to any one of (1) to (35), wherein the analyzer is detected as an electrical change such as a magnetic moment change.
(37) Optical changes such as surface plasmon resonance method, optical waveguide spectroscopy, slab optical waveguide spectroscopy, etc., which measure molecular recognition reactions on biologically derived molecules or artificial receptors immobilized on sensors as optical minute changes. The analyzer according to any one of (1) to (35), which is detected as a quantity.
(38) In the sensor unit, a 96-well crystal resonator is formed on a single crystal plate on the housing, or individual crystal resonators are horizontally installed on the side surface of the housing. The analyzer according to any one of (1) to (35), wherein:
(39) The size of the droplet obtained on the sensor element by dispensing with a dispensing pipette is preferably 0.01 μm to 30 mm in diameter, more preferably 1 μm to 10 mm in diameter, and most preferably The analyzer according to any one of (1) to (35), which has a diameter of 10 μm to 5 mm.
(40) The ambient pressure range of the analyzer is preferably 0.001 Pa to 1,000,000 Pa, more preferably 1 Pa to 10,000 Pa, and most preferably 10 Pa to 1,000 Pa. The analyzer according to any one of 1) to (35).
(41) The cleaning liquid used in the cleaning step is preferably a mixture of ultrapure water, pure water, ion-exchanged water, distilled water, heavy water, methanol, ethanol, propanol, etc., or one or more solutions thereof. It is liquid, More preferably, it is ultrapure water, pure water, ion-exchange water, distilled water, Most preferably, it is ultrapure water, pure water, or any one of said (1)-(35) Analysis equipment.
(42) In order to retain the activity of the antibody immobilized on the piezoelectric element for a long period of time, preferably, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, StabilGuard (registered trademark), BlockAce (registered trademark), glycerin, bovine The analysis apparatus according to any one of (1) to (35), wherein serum albumin is more preferably MPC polymer, StabilGuard (registered trademark), glycerin, and most preferably MPC polymer.
(43) As conditions used for the washing reaction, preferably, 1 to 100 μL of fresh ultrapure water or pure water is repeatedly dispensed and sucked once, and this is repeated 1000 times with individual solutions. More preferably, 5 to 70 μL of fresh ultrapure water or pure water is repeatedly dispensed and sucked once, and this is repeated 100 times with an individual solution, and most preferably fresh ultrapure water or pure water The analytical apparatus according to any one of (1) to (35), wherein dispensing and aspiration are repeated once with about 50 μL of water, and this is repeated five times with an individual solution.
(44) The gas used for the drying reaction is preferably pure nitrogen, dry air, argon, helium, xenon, neon, oxygen, nitrogen, hydrogen, chlorofluorocarbon, hydrofluorocarbon, perfluorocarbon, fluorine-containing ether, blower agent, More preferably, pure nitrogen, dry air, argon, and nitrogen, which are relatively inexpensive, are used. Most preferably, at least one of dry air and nitrogen is used, or one or more of these gases are mixed. The analyzer according to any one of (1) to (35), wherein the analyzer is used.
(45) As conditions used for the drying reaction using dry air and nitrogen as the drying gas, the gas flow rate is preferably 0.1 to 10 L / min, the drying time is 0.1 to 100 minutes, and more Preferably, the gas flow rate is 1 to 5 L / min and the drying time is 1 to 30 minutes, and most preferably the gas flow rate is 2.5 L / min and the drying time is 5 minutes. 2. The analyzer according to claim 1.
(46) The installation environment suitable for the analyzer is preferably an environment free from external electrical / mechanical vibrations or noise, more preferably free from external electrical / mechanical vibrations, and having humidity and temperature. It is an environment kept constant, most preferably an environment where there is no external electrical, mechanical and physical vibration, humidity and temperature are kept constant, and there is no dust or fine particles drifting in the atmosphere (1 The analyzer according to any one of (35) to (35).
(47) Materials suitable for dispensing probes are preferably metals such as glass and stainless steel, artificial plastics, natural resins, resins such as fluororesins, ceramics such as alumina and silicon nitride, clay, cement, natural stone / artificial materials The analyzer according to any one of (1) to (35), which is made of stone, quartz, quartz, or wood, more preferably glass or stainless steel, and most preferably glass.
(48) The material of the analyzer main body is preferably a metal such as aluminum, stainless steel or an alloy (for example, an aluminum alloy), a resin such as an artificial plastic or a natural resin, a ceramic such as alumina, cement, or wood. Preferably, there are metals such as aluminum, stainless steel and alloys, and resins such as artificial plastics and natural resins, most preferably metals such as aluminum, stainless steel and alloys, as described in any one of (1) to (35) above. Analysis equipment.

  The present invention is further described below.
  As a result of intensive studies, the present inventors have made it simple by making the concentration distribution of environmental pollutants and the like in a discharged sample uniform quickly and with high accuracy, and promoting the molecular recognition reaction rate by suction and discharge. It was also found that trace substances can be analyzed quickly. The present invention has been made based on such findings.
  In the inspection method according to the present invention, a sample in a container is sucked by a probe, the sample is discharged from the probe to a sensor unit equipped with a piezoelectric element (quartz crystal vibrator or the like), and the discharged sample is sucked again by the probe. And re-ejecting the re-sucked sample to the sensor unit is performed at least once.
  An analyzer according to the present invention includes a container placement unit for placing a container for storing a sample, a probe for sucking and discharging the sample in the placed container, and a sensor unit including a piezoelectric element (a crystal resonator or the like) including. The probe has a first position for sucking the sample in the container, and a second position for performing re-suction of the discharged sample and re-discharge of the re-sucked sample after discharging the sucked sample to the sensor unit. It is possible to selectively move to a position temporally and / or spatially.
  In the present invention, by using molecular recognition reactions such as immune reaction, receptor, DNA hybridization, lectin sugar chain recognition, etc., it is related to a novel rapid, simple, and sensitive measurement method for chemical substances, viruses, bacteria, etc. Is. Analytical sensor chips to be used include those that measure the molecular recognition reaction on the sensor using the piezoelectric effect such as crystal resonators, surface acoustic wave devices, ceramic resonators, surface shear wave devices, and surface plasmon resonance methods. In the present invention, a crystal resonator is particularly described.
  Next, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. In the description of each drawing, the same elements are denoted by the same reference numerals.
  FIG. 1 is a perspective view showing an embodiment of an analyzer according to the present invention, FIG. 2 is a plan view showing an embodiment of a quartz substrate used in the analyzer shown in FIG. 1, and FIG. FIG. 4 is an enlarged cross-sectional view showing an embodiment (a view showing one sensor portion (42) enlarged in the cross-sectional view taken along line 3-3 in FIG. 2), and FIG. 4 is an enlarged view showing an embodiment of the sensor portion. FIG. 5 is a sectional view showing another embodiment of the quartz substrate.
  Referring to FIGS. 1 to 5, the analyzer 10 is configured as an automatic analyzer that can repeatedly or successively inspect the same sample, the same type of sample, or different samples.
  The analyzer 10 includes a frame-like support 12, two probe assemblies 14 and 16 supported by the support 12 so as to be movable in three directions of X, Y, and Z, and a vial disposed on the support 12. It includes a rack 18, a sensor unit 20 disposed on the support 12, and a cleaning unit 22 disposed on the support 12.
  In the support 12, the vial rack 18, the sensor unit 20, and the cleaning unit 22 are installed on the main body base 24, and the support frame 26 that supports the probe assemblies 14 and 16 is formed from the main body base 24. A support member (connecting frame) 28 is attached to the main body base 24 with a space above.
  In the probe assemblies 14 and 16, a plurality of probes 30 and 32 are respectively attached to the probe bases 34 and 36 at intervals in the Y direction, and the support member 28 is moved by a moving mechanism (not shown) for each assembly. Is moved three-dimensionally. As such a moving mechanism, a mechanism in which a movable element for each probe assembly 14 and 16 is movably attached to a stator having a common guide rail in the probe assemblies 14 and 16, for example, a linear motor is used. Can do.
  The same number of probes 30 and 32 are provided, and the pitch in the Y direction is the same. The probes 30 and 32 are respectively connected to a suction / discharge device (not shown) for sucking and discharging a liquid from each probe 30 and a discharge device (not shown) for discharging a drying gas from each probe 32.
  The vial rack 18 is a container that can arrange a plurality of vials 38 as containers that contain a liquid sample and a plurality of containers 40 that contain a cleaning liquid in the X and Y directions (matrix). Each vial 38 and each container 40 are arranged in the vial rack 18 with the opening portion facing upward. The liquid sample in each vial 38 contains environmental pollutants such as dioxins.
  In the illustrated example, the vial rack 18 is capable of arranging 8 × 15 vials 38 and containers 40. For this reason, the probe assemblies 14 and 16 include eight probes 30 and 32, respectively. However, the number of vials and containers that can be installed in the vial rack 18 and the number of probes to be provided in the probe assemblies 14 and 16 can be set to an appropriate number.
  The sensor unit 20 includes a plurality of sensor units 42 each including a piezoelectric element, which will be described later, in a matrix, and a detection circuit (not shown) that is electrically connected to the piezoelectric elements.
  The cleaning unit 22 is configured such that a plurality of containers 44 containing cleaning liquid for the probe 30 can be arranged at the same pitch as the probe 30 and spaced in the Y direction.
  As shown in FIG. 3, each sensor portion 42 has a recess 46 that opens upward in a quartz substrate 48 that is common to all sensor portions 42, and electrodes 50 and 52 are respectively formed on the bottom and bottom surfaces of the recess 46. Is arranged. As shown in FIG. 4, the electrodes 50 and 52 are pulled out to the side of the sensor unit 42 and connected to leads 54 and 56, respectively. Leads 54 and 56 are connected to a detection circuit.
  The analysis sensor chip used for the sensor unit 42 is preferably a crystal resonator, a surface acoustic wave element (SAW), a flexural plate wave (FPW) element, an acoustic plate wave (APM) element, or a shear horizonal acoustic plate (SH-APM). ) Measuring the molecular recognition reaction on the sensor using the piezoelectric effect such as the element, the Flexural Love Wave element, the ceramic vibrator, the surface transversal wave (STW) surface shear wave element, and the molecular recognition reaction on the sensor. Sensor elements such as surface plasmon resonance, optical waveguide spectroscopy, and slab optical waveguide spectroscopy can be used, and the current change of molecular recognition reaction on the sensor can be measured. In the present invention, the use of a piezoelectric element such as a crystal resonator is particularly preferable as a sensor element. The operation principle of such a piezoelectric element sensor is described in J.A. W. Grate, S.M. J. et al. Martin, R.M. M.M. White, Anal. Chem. 65, 21 (1993) 940A-948A. And J.A. W. Grate, S.M. J. et al. Martin, R.M. M.M. White, Anal. Chem. 65, 22 (1993) 987A-996A. It is described in the review.
  A resonance characteristic measuring device such as an impedance analyzer for measuring the resonance characteristics of a piezoelectric element (quartz crystal unit, etc.) is connected via a signal switcher or directly to each piezoelectric element (quartz crystal unit, etc.). Can also be used. Furthermore, the method of measuring sound waves generated upon desorption of molecules from a quartz resonator and the response amplification reaction using magnetic particles presented by Cooper et al. Can also be applied to the present invention. Cooper, M.C. et al. , Direct and sensitive detection of a human virus by routine event scanning, Nature Biotechnology, 19, 833-837 (2001).
  As for the array-type crystal resonator serving as a sensor unit, a single crystal plate described in Japanese Patent Application No. 11-89410 and Japanese Patent Application No. 11-514189 (International Application No. PCT / JP98 / 04948) of Koyama and Tachima et al. The multi-channel crystal sensor device can be used in the above.
  The multi-channel crystal resonator sensor device that can be used in the present invention is a multi-channel type in which a plurality of electrodes are adjacent to the surface of a quartz substrate and a counter electrode is formed on the back surface of each electrode. On the other hand, Abe, Esashi et al. Created a crystal resonator array that created multiple crystal resonators and their electrodes by micro-etching on a single crystal plate using micro processing technology for semiconductor processing. ing. For example, Takashi Abe, Masayoshi Esashi, “Production of Multi-Channel Quartz Microbalance and Application to Chemometric Analysis”, Journal of Applied Physics Society of Organic Molecules and Bioelectronics, M & BE, 11, 2 (2000) p127-138. And can be used in the present invention.
  For example, as a sensor unit of an automatic multi-analyte dioxin sensor of the present invention, such a multi-channel crystal resonator sensor device or a microarray crystal resonator may be used. Furthermore, as shown in FIG. 2, 96 9 MHz AT-cut crystal resonator arrays installed immediately above the 96 oscillation circuits may be used. At this time, the measurement data can measure the oscillation frequency of 9 MHz of 96 crystal resonators with an accuracy of 7 digits every second at the same time by using a multiplexer or a PDLS element. For this reason, it is possible to continuously measure multiple samples with high productivity.
  The substance to be measured is measured by a crystal resonator to which a recognition element is fixed. Any crystal unit can be used in the present invention, and examples of the type thereof include AT cut, GT cut, BT cut, ST cut, and the like, and the electrode material is gold. Although silver, platinum, ITO, copper, etc. are suitable, the stability of gold is excellent particularly in a buffer in which an antibody or an antigen is dissolved during an antigen-antibody reaction.
  The oscillation frequency of the crystal resonator is not particularly limited and may be appropriately selected according to the application. However, 1 to 2,000 MHz is preferable, and 5 to 622 MHz is more preferable. If the oscillation frequency is too low, the sensitivity is not sufficient. On the other hand, if the oscillation frequency is too high, it is not practical because it is easily affected by noise from the oscillation circuit. For this reason, it is particularly preferable to use a crystal resonator of 5 to 160 MHz.
  In the form of a sensor array, a 96-well crystal resonator as shown in FIG. 2 is placed on a case made of a metal, resin, or ceramic combined with a crystal oscillation circuit and its oscillation frequency measurement and external output control mechanism. 6 or 8 are prepared on a single crystal plate, or as shown in FIGS. 6 to 8, with individual crystal resonators installed horizontally on the side of the housing, for simultaneous or parallel analysis of 96 samples. Quartz crystal array plates can be used. The apparatus of the present invention is preferably used for analysis of one or more samples to 1 million samples, but most preferably 8 to 96 samples that are easy to manufacture are suitable.
  The crystal resonator measurement method is an apparatus that can use a fundamental frequency of a crystal resonator of 1 to 10,000 MHz and an oscillation frequency measurement method and a resonance frequency measurement method with its overtones. A piezoelectric element such as a surface acoustic wave element, a surface shear wave element, or a ceramic vibrator can be used for automatic chemical analysis in the same manner in place of the quartz oscillator, and the surface plasmon resonance element can also be applied by changing the configuration of the detection site. .
  As shown in FIG. 5, the quartz substrate may be formed with a recess 46 on the lower surface of the common quartz substrate 48 as shown in FIG. 5A, or as shown in FIG. The recess 46 may not be formed on the crystal substrate 48, and the recess 46 may be formed on both the upper and lower surfaces of the crystal substrate 48 as shown in (C), or the depth of the recess 46 as shown in (D). The dimensions may be increased.
  The automatic analysis method and apparatus of the present invention can be used for the analysis of one or more to one million samples, but preferably corresponds to a well plate for analyzing 96, 384 or 1536 samples, most preferably easy to manufacture the apparatus. 1-96 specimens are suitable.
  A preferable probe tip (nozzle) has a cylindrical shape (an example is shown in FIG. 37 (1)), a bevel tip type (an example is shown in 37 (2)), and a spherical shape (an example is shown in FIG. 37 (3)). ), Cone type (example shown in FIGS. 37 (4) to 37 (5)), needle type (example shown in FIG. 37 (6)), etc., more preferably cylindrical type, bevel tip type (triangle) Mold) or conical, most preferably cylindrical or conical. The shape of the probe tip is preferably a needle shape, a conical shape or a cylindrical shape, more preferably a conical shape or a cylindrical shape, and a cylindrical shape is particularly preferable. Further, the inner surface mirror-polished probe having a surface roughness (arithmetic average roughness: Ra) of about 0.005 μm to about 0.05 μm, and a fluororesin-coated probe whose outer surface is coated with a fluororesin (for example, shown in FIG. 37 (7)) ), Deep-drawing probe with an inner diameter of 0.02 to 0.5 mm, a probe containing a fluororesin with a fluororesin tube with an inner diameter of 0.02 to 0.5 mm in a metallic nozzle, and between suction and discharge There is a probe with a heater capable of adjusting the temperature of the sample in the nozzle to 0 to 50 ° C., and a minute inner diameter probe with an inner diameter of 0.01 to 0.05 mm, preferably a fluororesin-coated probe, an inner diameter of the tip of 0.02 to 0.5 mm The deep-drawing probe is preferably up to a deep-drawing probe with a tip inner diameter of 0.02 to 0.5 mm. The material of the probe is preferably a metal such as glass or stainless steel, an artificial plastic, a natural resin, a resin such as a fluororesin, a ceramic such as alumina or silicon nitride, clay, cement, natural stone / artificial stone, crystal, quartz, or wood, In order to prevent the sample from being contaminated, a disposable or metallic resin is more preferable, and in order to increase the processing accuracy and strength of the probe, a metallic one is particularly preferable. As for the positional relationship with the sensor, the distance from the sensor element surface to the probe tip is preferably 0.01 mm to 10 mm, more preferably 0.05 mm to 1.5 mm.
  Next, an analysis method using the apparatus described above will be described.
  Prior to the inspection, the inspection unit 42 in the first row is cleaned. This cleaning is performed as follows.
  First, in a state where the probe 30 is raised, the probe assembly 34 is moved above the container 40, the probe 30 is lowered to a position where the tip of the probe 30 is arranged in the container 40, and the cleaning liquid in the container 40 is transferred to each probe 30. Inhale.
  Next, in a state where the probe 30 is raised, the probe assembly 34 is moved above the inspection unit 42 in the first row, and the tip of the probe 30 is lowered to the position of the inspection unit 42 in the first row and sucked. The cleaning liquid is ejected from each probe 32 to the sensor section 42 in the first row.
  Next, in a state where the probe 30 is raised, while moving the probe assembly 34 toward the cleaning unit 22, the probe assembly 36 is moved above the first row of inspection units 42, and the probe 32 is aligned with its tip in a row. The gas is lowered to a position directly above the eye inspection unit 42 and the drying gas is ejected from each probe 32 to the sensor unit 42 in the first row.
  While the first row of sensor units 42 is being dried, the probe assembly 34 is moved above the cleaning unit 22 to suck and discharge the cleaning liquid in the container 44 disposed in the cleaning unit 22. Each probe 30 is washed by performing at least once.
  When the cleaning of the sensor part 42 in the first row is completed, an inspection is performed next. This inspection is performed as follows.
  First, the probe assembly 34 is moved above the first row of vials 38, the probe 30 is lowered to a position where the tip is in the vial 38, and the liquid sample in the vial 38 is sucked into each probe 30.
  Next, the probe assembly 34 is moved above the first row of inspection units 42, the probe 30 is lowered to the position of the first row of inspection units 42, and the sucked liquid sample is drawn from each probe 32 to the first row. The sensor part 42 is discharged.
  After the sample is discharged to the sensor unit 42, re-aspiration (B) and re-discharge (A) of the sample 60 with respect to the sensor unit 42 are repeated a plurality of times as shown in FIG. As a result, a stirring action occurs in the liquid sample re-discharged to the sensor unit 42.
  Next, a voltage is applied to the electrodes 50 and 52 of the inspection unit 42 in the first row to inspect.
  When the re-suction and re-discharge of the sample with respect to the sensor unit 42 are repeated a plurality of times, environmental pollutants and the like are uniformly distributed in the discharged liquid sample due to the stirring action of the liquid sample, and the degree of reaction of the sample is uniform. Therefore, an accurate test result can be obtained.
  While inspecting the sample in the vial 38 in the first row, the probe 30 is washed, the second row inspection unit 42 is washed, the sample is discharged to the second row inspection unit 42, and the second row inspection unit 42 is scanned. The sample is re-sucked and re-discharged.
  As described above, the sample in the last row vial 38 is inspected from the sample in the first row vial 38. The liquid sample in the vial 38 may be the same sample, the same type of sample, or a different sample.
  Cleaning of the sensor unit after the measurement may be performed by using another probe assembly with another probe assembly, or by using the probe 32.
  The present invention is not limited to the above example, and various modifications can be made without departing from the spirit of the present invention.
  Fields of application of the present invention include the use of molecular recognition reactions such as immune reactions and molecular recognition functions of receptors, DNA and DNA aptamer hybridization, lectin sugar chain recognition, peptide arrays synthesized by peptides and combinatorials, etc. Specific chemical substances that interact with the molecules immobilized on these, viruses, bacteria, and the like that are related to new rapid, simple, and highly sensitive measurement methods.
  Probes equipped with machine-controllable sample dispensing with precise positioning in the XYZ axis direction, which is a three-dimensional space, use microcomputer-controlled precise probe movement and a micropump connected to the probe. Strict suction and discharge with controlled volume of analysis sample. The number of samples that can be handled at the same time is also used for analysis of one to more than one million samples, but preferably corresponds to a probe for analyzing 96, 384 or 1536 samples, and most preferably for 1 to 96 samples that are easy to manufacture. The probe is suitable.
  The amount of liquid injected using the above probe onto a piezoelectric element (such as a quartz crystal resonator) can be 0.001 to 10,000 μL, but preferably has a small sample variation, 0.01 to 1,000 μL. 1 to 50 μL is optimal, and most preferably, sample fluctuation is slight. Of course, simultaneous injection of one sample or more to one million samples or injection with individual liquid amounts for each sample is also possible.
  The amount of pure water or buffer injection in the washing step on a piezoelectric element (such as a quartz crystal resonator) using a mechanically controlled micropump can be 0.1 to 1,000 μL at the same time for eight samples, but preferably Is suitable for 50 to 100 μL of 8 samples at the same time. Individual injections for each specimen are also possible.
  Specific means of the analysis method using the apparatus of the present invention are shown in FIGS. 9 to 12 and FIGS.
  FIG. 9 (a) shows an overall view of an automated antigen-antibody reaction performed by subtracting a crystal resonator in each step. First, the oscillation frequency F of the eight crystal units₁Measure. Next, in step 1, antibody-immobilized active groups are introduced into all the eight crystal resonators. After that, two of them were washed with water, dried with nitrogen gas, and the amount of active group introduced onto the crystal resonator was changed to F.₂Measure with Next, as step 2, antibody immobilization, blocking, and introduction of a stabilizing agent are applied to the remaining six crystal resonators. Thereby, an antibody chip is formed on the crystal resonator. After that, two of them were washed with water and dried with nitrogen gas, and the amount of antibody immobilized on the crystal resonator, the amount of blocking agent, and the amount of stabilizer were set to F.₃Measure with Next, as step 3, antigen-antibody reaction is performed in the remaining four crystal resonators, the four crystal resonators are washed with water, dried with nitrogen gas, and then the oscillation frequency F₄And measure and calculate the amount of antigen-antibody reaction. Details of this are shown in FIGS.
  FIG. 9B shows an overall view of an antigen-antibody reaction automation operation in which measurement is performed by all crystal resonators. First, the oscillation frequency F of the eight crystal units₁Measure. Next, in step 1, antibody-immobilized active groups are introduced into all the eight crystal resonators. Thereafter, all the eight crystal resonators are washed with water and dried with nitrogen gas, and the amount of active group introduced onto the crystal resonator is set to F.₂Measure with Next, as step 2, antibody immobilization, blocking, and introduction of a stabilizing agent are performed on all the eight crystal resonators. Thereby, an antibody chip is formed on the crystal resonator. Thereafter, all the eight crystal resonators are washed with water and dried with nitrogen gas, and the amount of antibody immobilized on the crystal resonator, the amount of blocking agent, and the amount of stabilizer are set to F.₃Measure with Next, as step 3, an antigen-antibody reaction is performed in all the eight crystal resonators, and after completion of the reaction, all the eight crystal resonators are washed with water, dried with nitrogen gas, and an oscillation frequency F₄And measure and calculate the amount of antigen-antibody reaction. This detail is also shown in FIGS.
  FIG. 10 shows the activation process of the surface of the crystal electrode for antigen immobilization. Steps 1- (1) and 1- (2) are performed as shown, and after removing the solution by suction, cleaning the probe, and cleaning the crystal resonator (QCM) with pure water, two crystal resonators (QCM) After drying in nitrogen gas, the active groups are quantified. ΔF₁= F₂-F₁
  FIG. 11 shows the steps of antibody immobilization, blocking and introduction of a stabilizing agent. Steps 2- (1) to 2- (3) are performed as shown in the figure, and after the suction and removal of the solution, the probe, and the pure water, the two crystal resonators (QCM) are dried in nitrogen gas, Measure the amount of antibody immobilized (F₃). ΔF₂= F₃-F₁
  FIG. 12 shows the measurement process of the antigen-antibody reaction amount.
  In the present invention, any reaction of the substance to be measured can be used on the piezoelectric element as long as it can cause a mass change so that an electrical change can be detected and quantified on the piezoelectric element. For example, a labeled second antibody may be used in the antigen-antibody reaction, or a non-labeled second antibody may be used, and then a polymer or metal fine particles may be further reacted and immobilized on the non-second antibody. Similarly, a labeled second probe DNA may be used, or a non-labeled second probe DNA may be used and then a polymer, metal fine particles, or the like may be further reacted and immobilized on the non-second probe DNA.
  The reaction / measurement method preferably used in the present invention will be described more specifically below.
1-1 Direct reaction method (flow chart of experimental operation is shown in FIG. 13)
  First, the oscillation frequency (F₁) Is measured (FIG. 13A). Next, an aldehyde group is introduced to immobilize the anti-CRP antibody on the gold electrode of the crystal resonator (activation treatment of the surface of the crystal resonator gold electrode). The quartz crystal was immersed in a 0.01M cysteamine hydrochloride solution and a 1% glutaraldehyde solution in that order for 60 minutes, respectively, washed with 3 mL (1 mL × 3 times) of ultrapure water, and a flow rate of 2.5 L. After drying for 90 minutes under a nitrogen flow of / min, the oscillation frequency (F₂) Is measured (FIG. 13B). F of each crystal unit₁The amount of the surface modification film introduced by the activation treatment is calculated from the average value (ΔF₁= F₁-F₂).
  Next, 30 μL of anti-CRP antibody (first antibody) is dropped on the activated crystal resonator electrode, and the anti-CRP antibody is immobilized in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. Do. After 90 minutes, the substrate was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F₃) Is measured (FIG. 13C). The difference between the oscillation frequencies of the individual crystal resonators is obtained and the average value is calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF₂= (F₁-F₃-ΔF₁). Subsequently, 30 μL of a 0.02 M glycine solution is dropped on the quartz resonator to block unreacted aldehyde groups, and the reaction is performed in an air thermostat at 25.0 ± 0.1 ° C. for 20 minutes. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₄) Is measured (FIG. 13C). The amount of antibody introduced onto the crystal resonator electrode by CRP antibody immobilization and glycine treatment is calculated from the average value of the oscillation frequency change of the crystal resonator (ΔF₃= (F₁-F₄-ΔF₂-ΔF₁). After immobilizing the anti-CRP antibody, 30 μL of CRP is dropped on each glycine-blocked crystal resonator, and an antigen-antibody reaction is carried out for 90 minutes in an air thermostat at 25.0 ± 0.1 ° C. Then, after washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₅) Is measured (FIG. 13D). The amount of antigen-antibody reaction with each concentration of CRP is calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value is obtained (ΔF₄= (F₁-F₅-ΔF₃-ΔF₂-ΔF₁).
1-2 Chemical amplification reaction of sensor response by sandwich reaction after direct reaction method (flow chart of experiment is shown in FIG. 14)
  30 μL of an anti-CRP antibody-immobilized latex (second antibody-bound particle) suspension in which an anti-CRP monoclonal antibody is immobilized on a quartz crystal resonator after direct reaction of CRP by 1-1 is added to 25.0 ± 0.1 ° C. The antigen-antibody reaction is performed for 60 minutes in an air constant temperature bath. Polystyrene is used as the latex. After that, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured.₆(FIG. 14E). ΔF₅The value is calculated from the following equation (ΔF₅= (F₁-F₆-ΔF₄-ΔF₃-ΔF₂-ΔF₁).
2-1 Competitive reaction method (flow chart of experiment is shown in FIG. 15)
  First, the oscillation frequency (F₁) Is measured (FIG. 15A). Next, an aldehyde group is introduced to immobilize the anti-DNP (dinitrophenol) antibody on the gold electrode of the crystal resonator (activation treatment on the surface of the crystal resonator gold electrode). The quartz crystal was immersed in a 0.01M cysteamine hydrochloride solution and a 1% glutaraldehyde solution in that order for 60 minutes, respectively, washed with 3 mL (1 mL × 3 times) of ultrapure water, and a flow rate of 2.5 L. After drying for 90 minutes under a nitrogen flow of / min, the oscillation frequency (F₂) Is measured (FIG. 15B). F of each crystal unit₁The amount of the surface modification film introduced by the activation treatment is calculated from the average value (ΔF₁= F₁-F₂).
  Next, 30 μL of anti-DNP antibody (first antibody) is dropped on the activated crystal resonator electrode, and the anti-DNP antibody immobilization reaction is carried out in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. Do. After 90 minutes, the substrate was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F₃) Is measured (FIG. 15C). The difference between the oscillation frequencies of the individual crystal resonators is obtained and the average value is calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF₂= (F₁-F₃-ΔF₁). Subsequently, 30 μL of a 0.02 M glycine solution is dropped on the quartz resonator in order to block unreacted aldehyde groups, and the reaction is performed in an air thermostat at 25.0 ± 0.1 ° C. for 20 minutes. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₄) Is measured (FIG. 15C). The amount of antibody introduced onto the crystal resonator electrode by DNP antibody immobilization and glycine treatment is calculated from the average value of the oscillation frequency change of the crystal resonator (ΔF₃= (F₁-F₄-ΔF₂-ΔF₁). A competitive reaction solution is prepared by mixing equal amounts of a DNP-bound albumin solution, which is a competitive molecule of DNP, and a DNP antigen solution, and used for measurement. 30 μL of this competitive reaction solution was sampled and dropped on an anti-DNP antibody-immobilized crystal resonator (each glycine-blocked crystal resonator), and the antigen-antibody for 90 minutes in an air thermostat at 25.0 ± 0.1 ° C. Perform the reaction. Then, after washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₅) Is measured (FIG. 15D). The amount of antigen-antibody reaction with each concentration of DNP is calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value is obtained (ΔF₄= (F₁-F₅-ΔF₃-ΔF₂-ΔF₁).
2-2 Chemical amplification reaction of sensor response by sandwich reaction after competitive reaction method (flow chart of experiment is shown in FIG. 16)
  30. Add 30 μL of an anti-DNP antibody-immobilized latex (anti-DNP antibody-bound particle, second antibody-bound particle) suspension in which an anti-DNP monoclonal antibody is immobilized on the quartz crystal resonator after the competitive reaction of DNP by 2-1. The antigen-antibody reaction is performed for 60 minutes in a 0 ± 0.1 ° C. air constant temperature bath. After that, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured.₆(FIG. 16E). ΔF₅The value is calculated from the following equation (ΔF₅= (F₁-F₆-ΔF₄-ΔF₃-ΔF₂-ΔF₁).
3 Amplification reaction of sensor response by an antibody immobilized on a polymer with a clear molecular structure (for example, polyacrylic acid) (The flow chart of the experiment is shown in FIG. 17. In this document, it is abbreviated as ATRP method.)
3-1 Coating of plasma polymerized film (FIG. 17A)
  A plasma-polymerized allyl alcohol film (pp-allyl alcohol film) is polymerized on an At-cut 9 MHz crystal resonator using allyl alcohol as a monomer. Before the polymerization, etching is performed on the surface of the crystal resonator in helium gas for 2 minutes under the conditions of a discharge output of 100 W and a reaction pressure of 100 Pa. As the conditions for plasma polymerization, a discharge power of 40 W and a monomer pressure of 100 Pa, which are conditions in which the proportion of hydroxyl groups is high and the durability of the polymerized film to the solvent is used, are used. The polymerization time is 1 minute.
  After polymerization, the oscillation frequency of the quartz crystal resonator before and after coating the plasma polymerized film is measured, and the amount of the polymer film on the crystal resonator electrode is measured.
3-2 Functional group conversion on plasma polymerized film surface (Fig. 17 (B))
  2-Bromoisobutyryl bromide is bonded to the hydroxyl group on the surface of the obtained crystal resonator, and the functional group on the surface is converted from a hydroxyl group to a bromine group. A quartz oscillator is immersed in dichloromethane, equimolar tritylamine and 2-bromoisobutyryl bromide are added, and the mixture is reacted at 0 ° C. for 1 hour in a nitrogen atmosphere.
3-3 Atom transfer radical polymerization (ATRP) (FIG. 17C)
  The obtained crystal moving element is put, and the inside of the screw-mouth test tube containing the stirrer bar is replaced with nitrogen gas. A powder of copper (I) bromide serving as a catalyst is placed in a test tube and replaced with nitrogen gas. Thereafter, each time the lid is opened to add the reagent, the inside of the test tube is filled with nitrogen gas. Next, monomer t-butyl acrylate is added. Add acetone as a solvent and stir with a stirrer. Add N, N, N ′, N ′, N ″ -pentamethyldiethylenetriamine (PMDETA) as a catalyst activator and stir. Add ethyl 2-bromoisobutyrate as a reaction initiator and tighten the stopper tightly. Keep in an oil bath at 60 ° C. Based on the number of moles of monomeric tert-butyl acrylate, the number of moles divided by the desired degree of polymerization is the required amount of reagents other than the monomer. Regardless of the ratio, the amount of acetone is adjusted by the viscosity of the solution after polymerization, and 1 mL of acetone is added to 5 mL of monomer.
3-4 Hydrolysis (Fig. 17D)
  A crystal resonator is immersed in a solution containing 10% trifluoroacetic acid in dichloromethane, and is hydrolyzed by shaking with a shaker for 15 hours.
3-5 Functional group activation (FIG. 17E)
  1- [3- (Dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed in a petri dish containing 10 ml of distilled water, and the hydrolysis was performed on the solution. The crystal unit attached to is immersed and activated for 1 hour.
3-6 Immobilization of antibody (FIG. 17 (F)) and antigen-antibody reaction (FIG. 17 (G))
  First, the oscillation frequency (F₁). 30 μL of anti-CRP antibody is dropped on the activated crystal resonator electrode, and the anti-CRP antibody is immobilized in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. After 90 minutes, the substrate was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F₂). The difference between the oscillation frequencies of the individual crystal resonators is obtained and the average value is calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF₁= F₂-F₁). Subsequently, 30 μL of a 0.02 M glycine solution is dropped on the quartz resonator to block unreacted aldehyde groups, and the reaction is performed in an air thermostat at 25.0 ± 0.1 ° C. for 20 minutes. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₃). The amount of antibody introduced onto the crystal resonator electrode by CRP antibody immobilization and glycine treatment is calculated from the average value of the oscillation frequency change of the crystal resonator (ΔF₂= (F₁-F₃-ΔF₁). After immobilizing the anti-CRP antibody, 30 μL of CRP is dropped on each glycine-blocked crystal resonator, and an antigen-antibody reaction is carried out for 90 minutes in an air thermostat at 25.0 ± 0.1 ° C. Then, after washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F₄). The amount of antigen-antibody reaction with each concentration of CRP is calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value (ΔF₃) (ΔF₃= (F₁-F₄-ΔF₂-ΔF₁).
  Thereafter, as described in the above 1-2 and 2-2, the sensor response may be subjected to a chemical amplification reaction by a sandwich reaction.
4. Measurement method of DNA hybridization (Experimental flow diagram is shown in FIG. 18)
  First, the oscillation frequency (F₁). A sample to be measured (for example, E. coli in the embodiment shown in FIG. 40) is treated with a surfactant (SDS) and proteinase K, and the protein is removed with phenol. Thereafter, a DNA solution designed and synthesized for each measurement target is denatured with 0.5 M sodium hydroxide to form a single strand. Thereafter, 30 μL of the single-stranded DNA-containing solution returned to neutrality is dropped on a crystal resonator, and DNA is immobilized under reduced pressure at 80 ° C. for 2 hours (FIG. 18A). Next, in order to suppress non-specific adsorption, a solution obtained by removing the probe from a hybridization solution (DNA probe containing NaCl, sodium citrate, Denhardt's solution, 0.1% SDS, 50% formaldehyde) is used. Pre-hybridization is performed (FIG. 18 (b)). F of each crystal unit₁The amount of DNA immobilized on the crystal resonator by DNA immobilization is calculated from the average value (ΔF).₁= F₁-F₂). Next, 30 μL of a solution containing a biotin-immobilized DNA probe (first probe) is dropped onto the crystal resonator (FIG. 18 (c)), and it is kept in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. Hybridization is performed (FIG. 18 (d)). After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water (FIG. 18 (e)), dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of the remaining crystal unit. (F₃). The difference between the oscillation frequencies of the individual crystal resonators is obtained, the average value thereof is calculated, and the amount of hybridization with the DNA on the crystal resonator is calculated (ΔF₂= (F₁-F₃-ΔF₁).
  30 μL of a solution containing avidin-bonded nanoparticles (second probe-bonded particles) is dropped on the crystal resonator after the hybridization reaction, and avidin for 60 minutes in an air thermostat at 25.0 ± 0.1 ° C. Perform biotin binding reaction. After that, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured.₄And ΔF₃The value is calculated from the following equation (ΔF₃= (F₁-F₄-ΔF₂-ΔF₁).
  Thereafter, as described in the above 1-2 and 2-2, the sensor response may be subjected to a chemical amplification reaction by a sandwich reaction.
  In the above items 1-1 to 4, the compound (reagent) to be used, its amount, conditions such as temperature and time for various reactions and operations, etc. have been specifically described with examples. Therefore, the present invention is not limited to these, and various modifications are possible.
  2,3,7,8-Tetrachlorodibenzopararadixin (2,3,7,8-TCDD) has a toxicity equivalent factor of 1 and may have a higher toxicity equivalent factor than other dioxins in the environment. Are known. 2,3,7,8-TCDD is known to have a high concentration in the environment among dioxins. Therefore, by measuring the concentration of 2,3,7,8-TCDD by the method of the present invention, the toxic equivalent amount of the whole dioxins in the sample can be calculated from the calibration curve prepared in advance using the GC / MS method. It becomes possible to measure. This toxic equivalent measurement method can be applied to environment-derived samples or biological samples.
  The apparatus of the present invention can be combined with an automatic processing apparatus for extraction of dioxins from a collected sample, pretreatment, and automatic analysis operation of a multi-component sample. Since general-purpose liquid operation is possible, it can also be used to automate the extraction, purification, and pretreatment of analytical samples. Features of the device of the present invention include automation, labor saving, elimination of the necessity of skilled workers, improvement of productivity (throughput), improvement of accuracy (reproducibility), prevention of chemical exposure of workers, and analysis Automation, time control accuracy, reaction time reduction, simultaneous analysis of multiple samples, and measurement space scale down (downsizing).
  In the apparatus of the present invention, an extraction operation (or supercritical extraction, micro extraction, etc.) of dioxins using high temperature and high pressure by a high-speed solvent extraction method from a sample containing dioxins collected from the environment can be used. It can be used when offline batch processing can improve productivity), cleanup operation by micro column processing, and concentration operation are fully automated as a series of online processing. This increases productivity and shortens the time required for the entire analysis process for obtaining analysis results from sample collection, and enables continuous management of dioxin emission concentrations at incineration facilities, which can be used immediately for dioxin emission reduction measures.
  One of the excellent advantages provided by the method and apparatus of the present invention is as follows.
  A conventional stirring technique in a quartz crystal immunosensor in a salt-containing solution such as a buffer (buffer solution) is disclosed in, for example, Japanese Patent Application Laid-Open No. 2001-083154 ("Disease marker substance simple and compact detection device", Shigeru Kurosawa). In order to prevent an electrical short circuit of the crystal unit, one side of the crystal unit is sealed through an air layer on the electrode with silicon adhesive and a crystal plate, and only one side of the crystal unit is brought into contact with the measurement solution. In addition, in order to make the measurement sample concentration of the entire solution uniform, stirring was performed with a stirrer chip or the like. The volume of the measurement solution in this method usually requires about 1 mL, and the solution is gently stirred using a very small stirrer so as not to affect the quartz vibrator due to vibrations caused by stirring. is there. The amount of solution required is large (usually about 10 μL in the method of the present invention, but about 1 mL in the conventional method), and the stirring speed is 100 rev / min slower than the present invention. The reaction time was longer, and the effect of promoting the immune response was small. Further, in the method in which the reaction solution is artificially dropped only on the quartz resonator electrode by a dispensing pipette, the amount of the reaction solution is small (20 μL), but it is difficult to stir the solution with a stirrer or the like. It takes a very long time to complete the immune reaction between the antibody of the antibody and the antigen in the dropped solution, and the dropped solution evaporates due to changes in temperature and humidity during the measurement. There was a problem of large fluctuations. In the present invention, it is possible to extremely reduce the influence of stirring and solution evaporation on the vibrator by a method of repeating rapid and highly accurate solution suction and discharge with a dispensing probe even in a very small amount of reaction solution. In addition, by accelerating the immune reaction, it is possible to perform a quicker and more accurate measurement than the conventional method.
(Detection target using the automatic analysis method and apparatus of the present invention)
  Immunoassays for suspected environmental hormones, immunoassays for sex hormones, immunoassays for detergents, immunoassays for pesticides, immunoassays for toxins, immunocolumns for toxins, immunoassays for environmental hormone biomarkers, screening for environmental hormone action, food Allergen immunoassay, recombinant protein immunoassay, antibiotic synthesis immunoassay immunoassay, etc. are described below, but can also be used to detect other measurement targets if antibodies or complementary DNA probes or receptors against the measurement target can be produced. it can.
  Diagnosis of hepatitis B virus (HBs antigen / antibody), diagnosis of hepatitis C virus using disease marker substances (specific proteins produced when a person suffers from illness) contained in human blood, sweat, feces, urine, saliva (HCV antibody), C-reactive protein test (CRP), rheumatic diagnosis (RA), streptococcal infection diagnosis (ASO), syphilis diagnosis (STS), AIDS diagnosis (HIV), and tumor marker diagnosis include carcinoembryonic antigen (CEA) ), Α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), ferritin, prostatic acid phosphatase (PAP), tissue polypeptide antigen (TPA), and other allergic diseases A diagnostic (IgE) reagent or the like is a detection target.
  In the detection of stress markers, for example, 8-hydroxydeoxyguanosine, 4-hydroxynonenal, 4-hydroxyhexenal, acrolein, methylglyoxal, malondialdehyde, crotonaldehyde, 7-ketocholesterol, thioredoxin, chromogranin A, oxidized α1 antitrypsin Cortisol and the like are detection targets. In addition, detection of abnormal prion protein of influenza, influenza virus, mad cow disease (detection of abnormal prion protein is possible in combination with gene amplification reaction by anti-BSA antibody or PCR method), detection of severe acute respiratory syndrome (SARS) (A new human coronavirus can be detected in combination with a gene amplification reaction using an anti-SARS antibody or PCR method).
  In addition to this, a multi-sample sample to be detected according to the following (1) to (19), which is a measurement target of conventional ELISA method, immunochromatography method, DNA probe method, ELISA method microorganism, ELISA method microorganism toxin, is analyzed It is possible to make an automatic analyzer for this purpose.
  (1) DNA / immunoassay application for detection of microorganisms / microbe toxins, for general live bacteria, coliforms, fungi, Staphylococcus aureus, salmonella, E. coli coli. O157: For H7, Salmonella, Listeria, Salmonella, Listeria, Monocytogenes, Escherichia coli, Staphylococcus aureus, Campylobacter, Verotoxin (VT1 / VT2), Staphylococcus aureus enterotoxin, etc. It is.
  (2) Immunoassay applications for detecting environmental microorganisms / natural toxins, Cryptosporidium giardia, PSP (paralytic shellfish poison), microcystin, and the like are detection targets.
  (3) Immunoassay for freshness / vitamin detection, histamine, folic acid, vitamin B12, biotin and the like are the detection targets.
  (4) BSE measures DNA or immunoassay for detecting mad cow disease specific risk sites, cerebrospinal tissue (for raw meat / wiping quantification), cerebrospinal tissue content test (for meat product quantification), processed meat species discrimination (animals) For tissue detection) for cattle, pigs, sheep, birds, mixed samples, meat-and-bone meal appropriate heat processing management (high-temperature high-pressure processed animal meat species discrimination (animal muscle detection)), processed by DNA probe method Animal meat species discrimination (animal tissue detection) for cattle, pigs, sheep, goats, chickens, ducks, turkeys, ducks complex (cow, sheep, goats), composites (chicken, ducks, turkeys) , Ducks), composites (cow, pig), composites (chicken, turkey), and the like.
  (5) Genetically modified crops Use of DNA assay for genetically modified corn testing, Bt9 corn, Bt1 corn, genetically modified round-up ready soybean test (soy kit), genetically modified corn test (corn bulk test), gene set Replacement round-up ready soybean test (RUR soybean bulk test, RUR heated soybean test) and the like are detection targets.
  (6) Use for immunoassay for detection of allergic specific raw materials, allergy series, gluten, gliadin, β-lactoglobulin, peanut, walnut, etc.
  (7) Mycotoxin Mycotoxin Immunoassay application for detection of mycotoxins, aflatoxin, aflatoxin M1, ochratoxin A, DON, fumonisin, zearalenone, T-2 toxin, citrinin, aflatoxin B1, aflatoxin total, aflatoxin M1, ochratoxin A, DON ( Deoxynivalenol), fumonisin, zearalenone, T-2 toxin and the like are detection targets.
  (8) Immunoassay for detection of residual animal drugs, sulfa drugs (synthetic antibacterial agents sulfamethazine, tetracycline, chloramphenicol, streptomycin), hormone agents (acetyl getagen, melengestrol acetate, 17β estradiol, ethinyl estradiol, diethyl Stilbestrol, zeranol, testosterone, 19 nortestosterone, methyltestosterone, trenbolone, Fast clenbuterol, clenbuterol, dexamethasone), for milk (delvotest), pesticide residue (alachlor, aldicarb, atrazine, triazine, benomyl, captan, carbaryl, carbofuran, Chlordane, chlorosalonyl, chlorpyrifos, cyanazine, cyclodiene, DDT, diazinon, Endosulfan, endosal, fluridone, fenitrothion, isoproturon, linden, mesomil, metolachlor, metribudine, metsulfuron, molinate, picloram, paraquat, parathion, pyrimifosmethyl, prosimidone, dilbex (2,4,5-T), simazine, spinosad, thiabendazole, toxaphene , Trisulfuron, trichloropyridinol, triclopyr, urea herbicide, chlorotolulone, diuron, isoproturon, mecoprop, pentachlorophenol, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2, 4-D), atrazine, alachlor, triazine, simazine, parathion, carbaryl, cyclodiene, chlordane, endosulfan, toxaphene, meso Somil, Aldicarb, Benomyl, Benomyl / Carbendazim, Metribuzin, Chlorosaronyl (TPN), Procymidone, Carbofuran, Carbaryl, Chlorpyrifos, Picloram, Cyanazine, Thiabendazole, Metrachlor, Triasulfuron, Metosulfuron, Pirimiphos, Pirimiphosmethyl, Molinate, γBHC , Captan, spinosad, trichloropyridinol, paraquat, sylvex, spinosad, triclopyr, metribuzin, carbamate) and the like.
  (9) Immunoassay applications using environmental hormone acting receptors, Estrogen-R (α), Estrogen Receptor β, Androgen Receptor, and the like are detection targets.
  (10) Immunoassay for detection of pollutant chemicals, detection of dioxins, PCBs, petroleum fuel BTEX, petroleum fuel TPH, PAH (polycyclic aromatics), PCB, PCP, PETRO (petroleum fuel), etc. It is a target.
  (11) Immunoassay for detecting environmental hormones, bisphenol A, alkylphenols, phthalates, 2,4-DCP, benzo [α] pyrene, dioxins, PCBs, and the like are detection targets.
  (12) Immunoassay applications for detecting microbial toxins, verotoxin, verotoxin (VT1 / VT2), Staphylococcus aureus enterotoxin, Staphylococcus aureus toxin and the like are detection targets.
  (13) Immunoassay for detecting food allergy, egg (ovalbumin), milk (components such as β-lactoglobulin, whey protein, casein, α-Lactalbumin, β-lactoglobulin), peanut (peanut protein, soy protein) , Gluten), wheat (gliadin), buckwheat (buckwheat protein), sesame (sesame protein), bovine serum (bovine serum protein), hazelnut (hazelnut protein), and the like.
  (14) Immunoassay for detecting recombinant protein in foods, corn (Bt, Cry9C, Bt1 corn, Bt9 corn, Bt11 Corn, Bt176 Corn, Mon810 Corn), soybean (GMO soybean, Trait RUR heated soybean, HIFI GMO), cauliflower (cauliflower 35-S Gene) and the like are detection targets.
  (15) Immunoassay applications for detecting antibiotics / synthetic antibacterial agents, sulfamethazine, chloramphenicol, tetracycline, streptomycin and the like are detection targets.
  (16) Immunoassay for detecting sex hormones, 17β-estradiol, estrone, estriol, estrogen, ethinylestradiol, diethylstilbestrol, androstenedione, testosterone, 19-nortestosterone, methyltestosterone, and the like are detection targets.
  (17) Immunoassay application for detection of chemical agents (poisonous gases), nerve agents (tabun, sarin, soman, VX, cyclohexylmethylphosphonofluoridate), glazes (mustards, sulfur mustard, nitrogen mustard, leucite, phosgene oxime , Phenyldichloroarsine (PD), ethyldichloroarsine (ED), suffocating agent (phosgene, chlorine, chloropicrin, diphosgene, perfluoroisobutylene), cyanide (hydrogen cyanide, cyanogen chloride), disabling agent (3-quinucli Dinyl benzylate, lysergic acid diethylamide (LSD)), riot depressant (stimulant, tear agent: chlorobenzylidenemalononitrile, dibenzo-1,4-oxazepine, chloroacetophenone, emetic: adamsite, diphenylchloroarsine, diphenylsi Noarushin) and the like to be detected.
  (18) Immunoassay for detection of biological weapons and terrorist weapons, viruses, fungi, toxins such as anthrax (lung anthrax, skin anthrax, intestinal anthrax), smallpox virus, plague and botulinum toxin is there.
  (19) Immunoassay applications for detection of animal-derived infections, viruses (rabies, Japanese encephalitis, influenza, etc.), rickettsia chlamydia (Q fever, erythema fever, parrot disease), bacteria (pest, salmonellosis, leptospirosis, anthrax) Anthrax by fungi), fungi (dermatophytosis, cryptococcosis, pasteurellosis, cat scratch disease, toxoplasmosis, tuberculosis, cryptosporidiosis, recurrent fever), protozoa (babesiosis, toxoplasmosis, amebiasis, Japan dwelling) Blood flukes), parasites (canine / cat feline asymptosis, echinococcosis, fluke, scabies, salmonellosis, anisakiasis) and the like are detection targets.
  ADVANTAGE OF THE INVENTION According to this invention, the method and apparatus used for it which can analyze a trace amount substance simply and rapidly using piezoelectric elements, such as a crystal oscillator, can be provided.
  According to the present invention, a trace chemical component to be analyzed contained in a multi-component contaminant such as an environment-derived sample or a biological sample can be measured quickly and with high sensitivity by a very simple operation. For example, trace components can be detected using biological samples such as human serum, urine, body fluids, or environmental samples such as river / lake water, sludge, incinerator dust, etc. as analysis samples. In particular, environmental pollutants such as so-called environmental hormones designated as endocrine disrupting substances such as dioxins and trace hormones in the human body can be measured with high sensitivity.
  The sample re-discharged to the sensor unit is agitated by performing re-suction and re-discharge of the sample by the probe at least once with respect to the sensor unit including a piezoelectric element such as a crystal resonator. Thereby, the concentration of environmental pollutants and the like is uniformly distributed quickly and with high accuracy in the discharged sample, and an accurate inspection result can be obtained.
  The analysis method according to the present invention includes suction of the sample in the container to the probe, discharge of the sample from the probe to the sensor unit, re-suction of the discharged sample to the probe, and the Re-ejection of the re-aspirated sample can be performed by an automatic analyzer including a plurality of the containers, a plurality of the probes, and a plurality of the sensor units. On the other hand, in the analyzer according to the present invention, the container placement unit is capable of placing a plurality of containers, and the plurality of probes are provided so as to be selectively movable to the first and second positions. And a plurality of the sensor units can be provided. By doing so, a plurality of samples can be analyzed simultaneously and accurately.
  The analysis method according to the present invention may further include washing the probe with a washing solution. On the other hand, the analyzer according to the present invention further includes a second container placement portion for placing a second container containing a cleaning liquid, and the plurality of probes further wash the probe with the cleaning liquid. It is possible to move to the third position. By doing so, since the probe can be washed, it is possible to perform inspections one after another in a large number of sensor units.
  The analysis method according to the present invention may further include cleaning the sensor unit by spraying a cleaning liquid or gas from the probe or another probe to the sensor unit. On the other hand, the analyzer according to the present invention can further include a cleaning device that sprays a cleaning liquid or gas onto the sensor unit to clean the sensor unit. By doing so, since the sensor part can be cleaned, the same sample or the same kind of sample can be repeatedly inspected by the common sensor part, and the time required for the repeated inspection is shortened.
  The analysis apparatus according to the present invention further includes a plurality of second probes that spray a cleaning liquid or a drying gas onto the sensor unit, and the second probe sprays the gas onto the sensor unit. 3 and a fourth position spaced from the third position may be selectively movable.

表１から明らかなように、本発明では０．００１ｎｇ／ｍＬ（１ｐｇ／ｍＬ）〜１００ｎｇ／ｍＬの微小濃度の２，３，７，８−ＴＣＤＤを測定することができる。
図１９に、ダイオキシン濃度（２，３，７，８−ＴＣＤＤ）の測定結果の一例を示す。各２，３，７，８−ＴＣＤＤ濃度は、独立した６個の抗２，３，７，８−ＴＣＤＤモノクローナル抗体固定化水晶振動子を用いて測定し、実験誤差を標準偏差として記載した。人の手（手動）で測定を行ったものを（ａ）に、本発明の自動化装置で測定を行った結果を（ｂ）にそれぞれ示す。人の手で行うと、各濃度の実験誤差（標準偏差）は、１０〜２０％と大きな値を示すが、自動化装置を用いると僅か１〜２％と極めて小さな値となる。このようにエラーバーが著しく減少したことは、分析精度の信頼性が著しく向上したことを意味する。また、ダイオキシン測定時間も、人の手では６０分が必要であるが、自動化装置では僅か１０分で測定が完了する。
表１には、自動化装置と手動による抗原抗体反応時間と発振周波数変化の比較を示す。自動化装置での１分間での吸引・吐出の繰り返し回数を増やすことで、抗原抗体反応が促進され水晶振動子の発振周波数変化が大きくなり、１分の反応時間で３００回の吸引・吐出の繰り返しが、手動の２０分の反応と略等しいが、その標準偏差には顕著な差があった。以上、測定誤差の最小化と測定時間の短縮に、本発明は著しく効果があることが明らかである。
他の実施例
各実施例での試験方法を以下に述べる。
１−１ 直接反応法（実験操作の流れ図を図１３に示す。）
はじめに実験に使用する水晶振動子の発振周波数（Ｆ_１）を測定した（図１３（Ａ））。次に水晶振動子の金電極上に抗ＣＲＰ抗体を固定化するためアルデヒド基を導入した（水晶振動子金電極表面の活性化処理）。水晶振動子を、０．０１Ｍのシステアミン塩酸塩溶液、１％のグルタルアルデヒド溶液の順にそれぞれ６０分間ずつ浸漬させた後、３ｍＬ（１ｍＬ×３回）の超純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後に、水晶振動子の発振周波数（Ｆ_２）を測定した（図１３（Ｂ））。各水晶振動子のＦ_１からの差を求めその平均値から活性化処理で導入した表面修飾膜量を算出した（ΔＦ_１＝Ｆ_１−Ｆ_２）。
次に、活性化した水晶振動子電極上に抗ＣＲＰ抗体（第一抗体）を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で９０分間、抗ＣＲＰ抗体の固定化反応を行った。９０分経過後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、残った水晶振動子の発振周波数（Ｆ_３）を測定した（図１３（Ｃ））。個々の水晶振動子の発振周波数差を求めてその平均値を算出し、水晶振動子上に固定化した抗体量を得た（ΔＦ_２＝（Ｆ_１−Ｆ_３）−ΔＦ_１）。続いて、未反応のアルデヒド基をブロッキング処理するために水晶振動子上に０．０２Ｍのグリシン溶液を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で２０分間反応させた。３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分の間乾燥後、水晶振動子の発振周波数（Ｆ_４）を測定した（図１３（Ｃ））。水晶振動子の発振周波数変化の平均値からＣＲＰ抗体固定化及びグリシン処理で水晶振動子電極上に導入した抗体量を算出した（ΔＦ_３＝（Ｆ_１−Ｆ_４）−ΔＦ_２−ΔＦ_１）。抗ＣＲＰ抗体を固定化した後、グリシンブロッキングした各水晶振動子上にＣＲＰを３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で９０分間の抗原抗体反応を行った。その後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間の乾燥後に、各水晶振動子の発振周波数（Ｆ_５）を測定した（図１３（Ｄ））。各水晶振動子での発振周波数変化の平均値から各濃度のＣＲＰとの抗原抗体反応量を算出し、その平均値を求めた（ΔＦ_４＝（Ｆ_１−Ｆ_５）−ΔＦ_３−ΔＦ_２−ΔＦ_１）。
１−２ 直接反応法後のサンドイッチ反応によるセンサー応答の化学的な増幅反応（実験の流れ図を図１４に示す。）
前記１−１によるＣＲＰの直接反応後の水晶振動子に抗ＣＲＰモノクローナル抗体を固定化した抗ＣＲＰ抗体固定化ラテックス（第二抗体結合粒子）懸濁液を３０μＬ加え２５．０±０．１℃の空気恒温槽中で６０分間の抗原抗体反応を行った。前記ラテックスとしては、ポリスチレンを用いた。その後３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、各水晶振動子の発振周波数測定を行いその測定値をＦ_６とした（図１４（Ｅ））。ΔＦ_５値を次式から算出した（ΔＦ_５＝（Ｆ_１−Ｆ_６）−ΔＦ_４−ΔＦ_３−ΔＦ_２−ΔＦ_１）。
２−１ 競争反応法（実験の流れ図を図１５に示す。）
はじめに実験に使用する水晶振動子の発振周波数（Ｆ_１）を測定した（図１５（Ａ））。次に水晶振動子の金電極上に抗ＤＮＰ（ジニトロフェノール）抗体を固定化するためアルデヒド基を導入した（水晶振動子金電極表面の活性化処理）。水晶振動子を、０．０１Ｍのシステアミン塩酸塩溶液、１％のグルタルアルデヒド溶液の順にそれぞれ６０分間ずつ浸漬させた後、３ｍＬ（１ｍＬ×３回）の超純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後に、水晶振動子の発振周波数（Ｆ_２）を測定した（図１５（Ｂ））。各水晶振動子のＦ_１からの差を求めその平均値から活性化処理で導入した表面修飾膜量を算出した（ΔＦ_１＝Ｆ_１−Ｆ_２）。
次に、活性化した水晶振動子電極上に抗ＤＮＰ抗体（第一抗体）を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で９０分間、抗ＤＮＰ抗体の固定化反応を行った。９０分経過後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、残った水晶振動子の発振周波数（Ｆ_３）を測定した（図１５（Ｃ））。個々の水晶振動子の発振周波数差を求めてその平均値を算出し、水晶振動子上に固定化した抗体量を得た（ΔＦ_２＝（Ｆ_１−Ｆ_３）−ΔＦ_１）。続いて、未反応のアルデヒド基をブロッキング処理するために水晶振動子上に０．０２Ｍのグリシン溶液を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で２０分間反応させた。３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分の間乾燥後、水晶振動子の発振周波数（Ｆ_４）を測定した（図１５（Ｃ））。水晶振動子の発振周波数変化の平均値からＤＮＰ抗体固定化及びグリシン処理で水晶振動子電極上に導入した抗体量を算出した（ΔＦ_３＝（Ｆ_１−Ｆ_４）−ΔＦ_２−ΔＦ_１）。ＤＮＰの競争分子となるＤＮＰ結合アルブミン溶液とＤＮＰ抗原溶液を等量混合した競争反応の溶液を作成し、測定に用いた。この競争反応溶液を３０μＬ採取し、抗ＤＮＰ抗体固定化水晶振動子（グリシンブロッキングした各水晶振動子）上に滴下し、２５．０±０．１℃の空気恒温槽中で９０分間の抗原抗体反応を行った。その後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間の乾燥後に、各水晶振動子の発振周波数（Ｆ_５）を測定した（図１５（Ｄ））。各水晶振動子での発振周波数変化の平均値から各濃度のＤＮＰとの抗原抗体反応量を算出し、その平均値を求めた（ΔＦ_４＝（Ｆ_１−Ｆ_５）−ΔＦ_３−ΔＦ_２−ΔＦ_１）。
２−２ 競争反応法後のサンドイッチ反応によるセンサー応答の化学的な増幅反応（実験の流れ図を図１６に示す。）
前記２−１によるＤＮＰの競争反応後の水晶振動子に抗ＤＮＰモノクローナル抗体を固定化した抗ＤＮＰ抗体固定化ラテックス（抗ＤＮＰ抗体結合粒子、第二抗体結合粒子）懸濁液を３μＬ加え２５．０±０．１℃の空気恒温槽中で６０分間の抗原抗体反応を行った。その後３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、各水晶振動子の発振周波数測定を行いその測定値をＦ_６とした（図１６（Ｅ））。ΔＦ_５値を次式から算出した（ΔＦ_５＝（Ｆ_１−Ｆ_６）−ΔＦ_４−ΔＦ_３−ΔＦ_２−ΔＦ_１）。
３ 分子構造の明確なポリマー（例えばポリアクリル酸）上に固定化した抗体によるセンサー応答の増幅反応（実験の流れ図を図１７に示す。）（ＡＴＲＰ法）
３−１ プラズマ重合膜の被覆（図１７（Ａ））
Ａｔ−ｃｕｔ ９ＭＨｚ水晶振動子上にアリルアルコールをモノマーとしてプラズマ重合アリルアルコール膜（ｐｐ−アリルアルコール膜）を重合した。重合前に水晶振動子表面をヘリウムガス中で、放電出力１００Ｗ、反応圧力１００Ｐａの条件で２分間エッチングを行った。プラズマ重合の条件は、水酸基の割合が高くかつ重合膜の溶媒への耐久性が高い条件である、放電出力４０Ｗ、モノマー圧力１００Ｐａを用いた。重合時間は、１分間とした。
重合後にプラズマ重合膜被覆前後の水晶振動子の発振周波数を計測し、水晶振動子電極上の重合膜の量を測定した。
３−２ プラズマ重合膜表面の官能基変換（図１７（Ｂ））
得られた水晶振動子の表面の水酸基に臭化２−ブロモイソブチリルを結合し、表面の官能基を水酸基から臭素基に変換した。水晶振動子をジクロロメタンに浸し、等モルのトリチルアミンと臭化２−ブロモイソブチリルを加えて、窒素雰囲下、０℃で１時間反応させた。
３−３ 原子移動ラジカル重合（ＡＴＲＰ）（図１７（Ｃ））
得られた水晶動子を入れ、スターラーバーを入れたねじ口試験管内を窒素ガスで置換した。触媒となる臭化銅（Ｉ）の粉末を試験管にいれ、窒素ガスで置換した。以後、試薬を加えるためにふたを開けるたびに窒素ガスで試験管内を充填した。次にモノマーである、アクリル酸ｔ−ブチルを加えた。溶媒としてアセトンを加えて、スターラーで攪拌した。触媒の活性化剤として、Ｎ，Ｎ，Ｎ’，Ｎ’，Ｎ”−ペンタメチルジエチレントリアミン（ＰＭＤＥＴＡ）を加えて攪拌した。反応開始剤として２−ブロモイソ酪酸エチルを加えてしっかりと栓を締めて、オイルバスで６０℃に保った。モノマーのアクリル酸ｔｅｒｔ−ブチルのモル数を基準とし、このモル数を目的の重合度で割ったモル数をモノマー以外の試薬の必要量とした。ただし、溶媒のアセトンはその比率に関係なく、重合後の溶液の粘度により調整し、モノマー５ｍＬに対してアセトン１ｍＬを加えた。
３−４ 加水分解（図１７（Ｄ））
ジクロロメタンに対し、１０％トリフロオロ酢酸を含む溶液に水晶振動子を浸漬し、振盪機で１５時間振盪して加水分解した。
３−５ 官能基の活性化（図１７（Ｅ））
１−［３−（ジメチルアミノ）プロピル］−３−エチルカルボジイミド塩酸塩（ＥＤＣ）、Ｎ−ヒドロキシこはく酸イミド（ＮＨＳ）を１０ｍｌの蒸留水が入ったシャーレに混ぜ入れ、その溶液に前記加水分解に付した水晶振動子を浸漬し、１時間活性化処理を行った。
３−６ 抗体の固定化（図１７（Ｆ））と抗原抗体反応（図１７（Ｇ））
はじめに前記活性基導入後の水晶振動子の発振周波数（Ｆ_１）を測定した。活性化した水晶振動子電極上に抗ＣＲＰ抗体を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で９０分間、抗ＣＲＰ抗体の固定化反応を行った。９０分経過後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、残った水晶振動子の発振周波数（Ｆ_２）を測定した。個々の水晶振動子の発振周波数差を求めてその平均値を算出し、水晶振動子上に固定化した抗体量を得た（ΔＦ_１＝Ｆ_２−Ｆ_１）。続いて、未反応のアルデヒド基をブロッキング処理するために水晶振動子上に０．０２Ｍのグリシン溶液を３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で２０分間反応させた。３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分の間乾燥後、水晶振動子の発振周波数（Ｆ_３）を測定した。水晶振動子の発振周波数変化の平均値からＣＲＰ抗体固定化及びグリシン処理で水晶振動子電極上に導入した抗体量を算出した（ΔＦ_２＝（Ｆ_１−Ｆ_３）−ΔＦ_１）。抗ＣＲＰ抗体を固定化した後、グリシンブロッキングした各水晶振動子上にＣＲＰを３０μＬ滴下し、２５．０±０．１℃の空気恒温槽中で９０分間の抗原抗体反応を行った。その後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間の乾燥後に、各水晶振動子の発振周波数（Ｆ_４）を測定した。各水晶振動子での発振周波数変化の平均値から各濃度のＣＲＰとの抗原抗体反応量を算出し、その平均値（ΔＦ_３）を求めた（ΔＦ_３＝（Ｆ_１−Ｆ_４）−ΔＦ_２−ΔＦ１）。
４ ＤＮＡハイブリダイゼーションの測定法（実験の流れ図を図１８に示す）
はじめに実験に使用する水晶振動子の発振周波数（Ｆ_１）を測定した。測定対象となる試料（図４０に示した実施例では大腸菌を用いた）を界面活性剤（ＳＤＳ）とプロテイナーゼＫで処理し、フェノールでタンパクを除去した。その後、測定対象ごとに設計、合成によって作成したＤＮＡの溶液を、０．５Ｍの水酸化ナトリウムで変性させて１本鎖とした。その後、中性に戻した前記１本鎖ＤＮＡ含有溶液を３０μＬ、水晶振動子上に滴下し、８０℃２時間、減圧下でＤＮＡの固定化を行った（図１８（ａ））。次に非特異吸着を抑制するために、ハイブリダイゼーション溶液（ＤＮＡプローブにＮａＣｌ、クエン酸ナトリウム、Ｄｅｎｈａｒｄｔ’ｓ溶液、０．１％ＳＤＳ、５０％ホルムアルデヒドを含む）からプローブを除去した溶液を用いてプレハイブリダイゼーションを行った（図１８（ｂ））。各水晶振動子のＦ_１からの差を求めその平均値からＤＮＡ固定化で水晶振動子上に固定化されたＤＮＡの量を算出した（ΔＦ_１＝Ｆ_１−Ｆ_２）。次に、ビオチン固定化のＤＮＡプローブ（第一プローブ）を含む溶液３０μＬを水晶振動子上に滴下し（図１８（ｃ））、２５．０±０．１℃の空気恒温槽中で９０分間ハイブリダイゼーションを行った（図１８（ｄ））。９０分経過後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し（図１８（ｅ））、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、残った水晶振動子の発振周波数（Ｆ_３）を測定した。個々の水晶振動子の発振周波数差を求めてその平均値を算出し、水晶振動子上のＤＮＡとのハイブリダイゼーションの量を算出した（ΔＦ_２＝（Ｆ_１−Ｆ_３）−ΔＦ_１）。
前記ハイブリダイゼーション反応後の水晶振動子に、アビジン結合したナノ粒子（第二プローブ結合粒子）を含む溶液３０μＬを滴下し、２５．０±０．１℃の空気恒温槽中で６０分間のアビジン−ビオチン結合反応を行った。その後３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、各水晶振動子の発振周波数測定を行いその測定値をＦ_４とした。ΔＦ_３値を次式から算出した（ΔＦ_３＝（Ｆ_１−Ｆ_４）−ΔＦ_２−ΔＦ_１）。
以上で説明した本発明の各方法に従って、各種被検物質について試験を行った。試験では、各測定対象物質に対して、前述の抗体（ＣＰＲ抗体など）を適宜変更して、同様の操作を行った。
環境汚染物質（ダイオキシン類やＰＣＢ類以外のものである）については、競争反応法とその後のサンドイッチ反応による増幅反応に従って測定した結果を図２０〜２９に、疾病マーカー物質については、直接反応法とその後のサンドイッチ反応による増幅反応に従って測定した結果を図３０〜３６に、食品検査における微生物については、直接反応法とその後のサンドイッチ反応による増幅反応に従って測定した結果を図３７〜３８に、それぞれ示す。
また、ダイオキシン類の例として２，３，７，８−ＴＣＤＤについて周波数変化を測定した結果を図３９に示す。測定は以下のように行った。１回目の抗原抗体反応では、前記２−１競争反応法に従って行った。すなわち、ＤＮＰの競争分子となるＤＮＰ結合アルブミン溶液と各ＤＮＰ抗原溶液を等量混合した競争反応の溶液を作成し、測定に用いた。この競争反応溶液を３０μＬ採取し、抗ＤＮＰ抗体固定化水晶振動子上に滴下し、摂氏２５．０±０．１度の空気恒温槽中で９０分間の抗原抗体反応を行った。その後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間の乾燥後に、各水晶振動子の発振周波数（Ｆ_５）を測定した。各水晶振動子での発振周波数変化の平均値から各濃度のＤＮＰとの抗原抗体反応量を算出した。２回目の抗原抗体反応では、前記２−２競争反応法後のサンドイッチ反応によるセンサー応答の化学的な増幅反応に従って行なった。すなわち、ＤＮＰの競争反応後の水晶振動子に抗ＤＮＰモノクローナル抗体を固定化した抗ＤＮＰ抗体固定化ラテックス懸濁液を３０μＬ加え摂氏２５．０±０．１度の空気恒温槽中で６０分間の抗原抗体反応を行った。その後３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、各水晶振動子の発振周波数測定を行い、ラテックス懸濁液添加前後における周波数の変化量を算出した。
また、大腸菌について周波数変化を測定した結果を図４０に示す。測定は以下のように行った。従来法では、ビオチン固定化のＤＮＡプローブ（第一プローブ）を含む溶液３μＬを水晶振動子上に滴下し、摂氏２５．０±０．１度の空気恒温槽中で９０分間ハイブリダイゼーションを行った（図１８（ｄ）参照）。９０分経過後、３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後、残った水晶振動子の発振周波数（Ｆ_３）を測定した。個々の水晶振動子の発振周波数差を求めてその平均値を算出し、水晶振動子上のＤＮＡとのハイブリダイゼーションの量を算出した。第二プローブ結合粒子を用いる方法では、ハイブリダイゼーション反応後の水晶振動子にアビジン結合したナノ粒子（第二プローブ結合粒子）を含む溶液３０μＬを滴下し、摂氏２５．０±０．１度の空気恒温槽中で６０分間のアビジン−ビオチン結合反応を行った（図１８（ｅ）の後工程参照）。その後３ｍＬ（１ｍＬ×３回）の純水で洗浄し、流速２．５Ｌ／ｍｉｎの窒素流通下で９０分間乾燥後各水晶振動子の発振周波数測定を行いその測定値をＦ_４とした。ΔＦ_３値を次式から算出した。
また、癌マーカータンパク質と環境汚染物質の例として、人血清中のα−フェトプロテイン濃度について、直接反応法とその後のサンドイッチ反応による増幅反応に従って測定した結果を図４１に、環境水中のアトラジン濃度については競争反応による測定結果を図４２に、それぞれ示す。図４１及び図４２において、従来法（図中「○」で示した）では、共有結合を用いて水晶振動子上に抗体を固定化する二次元的な固定化法であった。一方、本発明に従って、原子移動ラジカル重合法（Ａｔｏｍ ｔｒａｎｓｆｅｒ ｒａｄｉｃａｌ ｐｏｌｙｍｅｒｉｚａｔｉｏｎ：ＡＴＲＰ）により分子量と鎖長の揃った高分子鎖上に抗体を三次元的に固定化することで（図中「□」で示した）、抗原の検出感度を著しく増幅することが可能であった。
各図において、（ａ）は熟練者の手作業による測定結果であり、（ｂ）は本発明の自動分析装置での測定結果を示す。各測定点は６個の独立した水晶振動子を用いた際の平均値であり、縦軸のエラーバーは標準偏差（誤差）をそれぞれ示す。
なお、以上の各センサー感度増幅実験の対象になった各物質の環境基準値や正常値などを以下の各表に示した。
以上の各実施例によっても、前記実施例１〜８と同様の優れた分析が可能であった。

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
[Examples 1-8, Comparative Examples 1-5]
<Anti-2,3,7,8-TCDD antibody immobilization and stabilization treatment of quartz crystal>
The test was conducted using 2,3,7,8-TCDD, which is a low molecular compound, as a substance to be measured.
The crystal resonator used was AT cut (9 MHz). First, the gold electrode surface of the crystal unit was washed with a sulfuric acid / hydrogen peroxide mixed solution. After washing with pure water, nitrogen was circulated over the quartz vibrator at a flow rate of 2.5 L / min for drying. Next, the gold electrode surface of the quartz crystal was treated with 0.01 M cysteamine, followed by treatment with 1% glutaraldehyde solution to activate the gold electrode surface for antibody binding, and then with 1 mL of pure water. Washed 3 times.
Next, 30 μL of 50 μg / mL anti-2,3,7,8-TCDD monoclonal antibody in PBS (pH 7.4) was added onto the crystal resonator, and suction / discharge was performed at 300 ° C. for 10 minutes at 25 ° C. The antibody was immobilized on the quartz resonator by reacting under repeated times. After washing three times with 1 mL of pure water, 30 μL of 0.02 M glycine solution is added onto the crystal unit to cap the unreacted aldehyde groups remaining on the crystal unit, and suction is performed at 25 ° C. for 10 minutes. -The discharge was repeated under 300 times per minute. After washing 3 times with 1 mL of pure water, 30 μL of a 0.2% -PBS solution of stabilizer is added onto the crystal resonator, and the reaction is repeated under suction and discharge 300 times per minute at 25 ° C. for 10 minutes. It was. Lipidure-HM (trade name, manufactured by NOF Corporation) was used as a stabilizer. After washing with 1 mL of pure water three times, nitrogen is circulated on the crystal unit at a flow rate of 2.5 L / min for drying, and after 10 minutes, the oscillation frequency of the antibody-immobilized crystal unit is measured. F1.
<Antibody-antibody reaction I>
1: 3 ratio of 2,3,7,8-TCDD-conjugated ovalbumin (RDI) in 10 mM PBS solution (pH 7.4) to 2,3,7,8-TCDD in DMSO To make a 25% DMSO solution. Antigen solutions for competitive reaction were prepared by dissolving the 2,3,7,8-TCDD concentrations to 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL, respectively.
10 μL of this antigen solution is added to the anti-2,3,7,8-TCDD antibody-immobilized crystal resonator prepared above, and the mixture is reacted at 25 ° C. for 10 minutes, with suction and discharge repeated 300 times per minute. Then, an antigen-antibody reaction I was performed. The antigen-antibody reaction is as large as about 200 Hz. After completion of the reaction, it was washed 3 times with 1 mL of pure water, and nitrogen was circulated over the crystal resonator at a flow rate of 2.5 L / min for drying. After 10 minutes, the oscillation frequency of the antibody-immobilized crystal resonator was measured, The measured value was F2.
A value obtained by subtracting F2 from F1 is defined as a change in oscillation frequency (ΔF1 (Hz)), which is shown in Table 1. Moreover, the relationship with 2,3,7,8-TCDD density | concentration is shown in FIG. 13, and it used as a calibration curve in the case of the dioxin density | concentration measurement contained in the sample extracted from the fly ash of the garbage incineration site.

As is clear from Table 1, in the present invention, 2,3,7,8-TCDD having a minute concentration of 0.001 ng / mL (1 pg / mL) to 100 ng / mL can be measured.
FIG. 19 shows an example of the measurement result of dioxin concentration (2, 3, 7, 8-TCDD). Each 2,3,7,8-TCDD concentration was measured using 6 independent anti-2,3,7,8-TCDD monoclonal antibody immobilized crystal resonators, and the experimental error was described as the standard deviation. (A) shows the result of measurement by human hand (manual), and (b) shows the result of measurement by the automated apparatus of the present invention. When performed manually, the experimental error (standard deviation) of each concentration shows a large value of 10 to 20%, but becomes an extremely small value of only 1 to 2% when an automated apparatus is used. Thus, the fact that the error bars are remarkably reduced means that the reliability of the analysis accuracy is remarkably improved. Also, the dioxin measurement time needs 60 minutes by human hand, but the measurement is completed in only 10 minutes by an automated apparatus.
Table 1 shows a comparison between the antigen-antibody reaction time and the oscillation frequency change by the automated apparatus and the manual operation. By increasing the number of repetitions of suction / discharge per minute in an automated device, the antigen-antibody reaction is promoted and the oscillation frequency change of the crystal resonator increases, and 300 times of suction / discharge are repeated in one minute reaction time. However, there is a significant difference in its standard deviation, although it is roughly equivalent to a manual 20 minute response. As described above, it is apparent that the present invention is extremely effective in minimizing measurement errors and shortening measurement time.
Other Examples Test methods in each example are described below.
1-1 Direct reaction method (flow chart of experimental operation is shown in FIG. 13)
_First , the oscillation frequency (F ₁ ) of the crystal resonator used in the experiment was measured (FIG. 13A). Next, an aldehyde group was introduced to immobilize the anti-CRP antibody on the gold electrode of the crystal resonator (activation treatment of the crystal electrode gold electrode surface). The quartz crystal was immersed in a 0.01M cysteamine hydrochloride solution and a 1% glutaraldehyde solution in that order for 60 minutes, respectively, washed with 3 mL (1 mL × 3 times) of ultrapure water, and a flow rate of 2.5 L. After drying for 90 minutes under a nitrogen flow of / min, the oscillation frequency (F ₂ ) of the crystal resonator was measured (FIG. 13B). The difference from F _{1 of} each crystal resonator was obtained, and the surface modification film amount introduced by the activation treatment was calculated from the average value (ΔF ₁ = F ₁ −F ₂ ).
Next, 30 μL of anti-CRP antibody (first antibody) is dropped on the activated crystal resonator electrode, and the anti-CRP antibody is immobilized in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. went. After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and the oscillation frequency (F ₃ ) of the remaining crystal unit was measured. (FIG. 13C). The difference between the oscillation frequencies of the individual crystal resonators was obtained and the average value thereof was calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF ₂ = (F ₁ −F ₃ ) −ΔF ₁ ). Subsequently, 30 μL of a 0.02 M glycine solution was dropped on the quartz resonator in order to perform blocking treatment of unreacted aldehyde groups, and reacted for 20 minutes in an air constant temperature bath at 25.0 ± 0.1 ° C. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F ₄ ) of the crystal unit was measured (FIG. 13C )). The amount of antibody introduced onto the crystal resonator electrode by CRP antibody immobilization and glycine treatment was calculated from the average value of the oscillation frequency change of the crystal resonator (ΔF ₃ = (F ₁ −F ₄ ) −ΔF ₂ −ΔF ₁ ). . After immobilizing the anti-CRP antibody, 30 μL of CRP was dropped on each glycine-blocked crystal resonator, and an antigen-antibody reaction was carried out for 90 minutes in an air constant temperature bath at 25.0 ± 0.1 ° C. Thereafter, the crystal was washed with 3 mL (1 mL × 3 times) of pure water and dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F ₅ ) of each crystal resonator was measured (FIG. 13). (D)). The amount of antigen-antibody reaction with each concentration of CRP was calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value was obtained (ΔF ₄ = (F ₁ -F ₅ ) −ΔF ₃ −ΔF ₂ −ΔF ₁ ).
1-2 Chemical amplification reaction of sensor response by sandwich reaction after direct reaction method (flow chart of experiment is shown in FIG. 14)
30 μL of an anti-CRP antibody-immobilized latex (second antibody-bound particle) suspension in which an anti-CRP monoclonal antibody is immobilized on a quartz crystal resonator after direct reaction of CRP by 1-1 is added to 25.0 ± 0.1 ° C. The antigen-antibody reaction was performed for 60 minutes in an air constant temperature bath. As the latex, polystyrene was used. Thereafter, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow rate of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured, and the measured value was defined as F ₆ ( FIG. 14 (E)). The ΔF ₅ value was calculated from the following equation (ΔF ₅ = (F ₁ −F ₆ ) −ΔF ₄ −ΔF ₃ −ΔF ₂ −ΔF ₁ ).
2-1 Competitive reaction method (flow chart of experiment is shown in FIG. 15)
_First , the oscillation frequency (F ₁ ) of the crystal resonator used in the experiment was measured (FIG. 15A). Next, an aldehyde group was introduced to immobilize the anti-DNP (dinitrophenol) antibody on the gold electrode of the crystal resonator (activation treatment of the surface of the crystal resonator gold electrode). The quartz crystal was immersed in a 0.01M cysteamine hydrochloride solution and a 1% glutaraldehyde solution in that order for 60 minutes, respectively, washed with 3 mL (1 mL × 3 times) of ultrapure water, and a flow rate of 2.5 L. After drying for 90 minutes under a nitrogen flow of / min, the oscillation frequency (F ₂ ) of the crystal resonator was measured (FIG. 15B). The difference from F _{1 of} each crystal resonator was obtained, and the surface modification film amount introduced by the activation treatment was calculated from the average value (ΔF ₁ = F ₁ −F ₂ ).
Next, 30 μL of anti-DNP antibody (first antibody) is dropped on the activated crystal resonator electrode, and the anti-DNP antibody immobilization reaction is carried out in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. went. After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and the oscillation frequency (F ₃ ) of the remaining crystal unit was measured. (FIG. 15C). The difference between the oscillation frequencies of the individual crystal resonators was obtained and the average value thereof was calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF ₂ = (F ₁ −F ₃ ) −ΔF ₁ ). Subsequently, 30 μL of a 0.02 M glycine solution was dropped on the quartz resonator in order to perform blocking treatment of unreacted aldehyde groups, and reacted for 20 minutes in an air constant temperature bath at 25.0 ± 0.1 ° C. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F ₄ ) of the crystal resonator was measured (FIG. 15C )). The amount of antibody introduced onto the crystal resonator electrode by DNP antibody immobilization and glycine treatment was calculated from the average value of the change in the oscillation frequency of the crystal resonator (ΔF ₃ = (F ₁ −F ₄ ) −ΔF ₂ −ΔF ₁ ). . A competitive reaction solution was prepared by mixing equal amounts of a DNP-bound albumin solution, which is a competitive molecule of DNP, and a DNP antigen solution, and used for measurement. 30 μL of this competitive reaction solution was sampled and dropped on an anti-DNP antibody-immobilized crystal resonator (each glycine-blocked crystal resonator), and the antigen-antibody for 90 minutes in an air thermostat at 25.0 ± 0.1 ° C. Reaction was performed. Thereafter, the crystal was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F ₅ ) of each crystal resonator was measured (FIG. 15). (D)). The antigen-antibody reaction amount with each concentration of DNP was calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value was obtained (ΔF ₄ = (F ₁ -F ₅ ) −ΔF ₃ −ΔF ₂ −ΔF ₁ ).
2-2 Chemical amplification reaction of sensor response by sandwich reaction after competitive reaction method (flow chart of experiment is shown in FIG. 16)
25. Add 3 μL of an anti-DNP antibody-immobilized latex (anti-DNP antibody-bound particle, second antibody-bound particle) suspension in which an anti-DNP monoclonal antibody is immobilized on the quartz crystal resonator after the competitive reaction of DNP by 2-1. The antigen-antibody reaction was performed for 60 minutes in an air constant temperature bath of 0 ± 0.1 ° C. Thereafter, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow rate of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured, and the measured value was defined as F ₆ ( FIG. 16 (E)). The ΔF ₅ value was calculated from the following equation (ΔF ₅ = (F ₁ −F ₆ ) −ΔF ₄ −ΔF ₃ −ΔF ₂ −ΔF ₁ ).
3 Amplification reaction of sensor response by antibody immobilized on polymer (for example, polyacrylic acid) having clear molecular structure (flow chart of experiment is shown in FIG. 17) (ATRP method)
3-1 Coating of plasma polymerized film (FIG. 17A)
A plasma-polymerized allyl alcohol film (pp-allyl alcohol film) was polymerized on an At-cut 9 MHz crystal resonator using allyl alcohol as a monomer. Prior to polymerization, the surface of the crystal resonator was etched in helium gas for 2 minutes under conditions of a discharge output of 100 W and a reaction pressure of 100 Pa. As the conditions for plasma polymerization, a discharge power of 40 W and a monomer pressure of 100 Pa were used, which are conditions in which the proportion of hydroxyl groups is high and the durability of the polymerized film to the solvent is high. The polymerization time was 1 minute.
After polymerization, the oscillation frequency of the quartz crystal resonator before and after coating with the plasma polymerized film was measured, and the amount of polymerized film on the crystal resonator electrode was measured.
3-2 Functional group conversion on plasma polymerized film surface (Fig. 17 (B))
2-Bromoisobutyryl bromide was bonded to the hydroxyl group on the surface of the obtained crystal resonator, and the functional group on the surface was converted from a hydroxyl group to a bromine group. A quartz oscillator was immersed in dichloromethane, equimolar tritylamine and 2-bromoisobutyryl bromide were added, and the mixture was reacted at 0 ° C. for 1 hour in a nitrogen atmosphere.
3-3 Atom transfer radical polymerization (ATRP) (FIG. 17C)
The crystal crystal obtained was put, and the inside of the screw-mouth test tube containing the stirrer bar was replaced with nitrogen gas. A powder of copper (I) bromide serving as a catalyst was placed in a test tube and replaced with nitrogen gas. Thereafter, each time the lid was opened to add the reagent, the inside of the test tube was filled with nitrogen gas. Next, the monomer t-butyl acrylate was added. Acetone was added as a solvent and stirred with a stirrer. N, N, N ′, N ′, N ″ -pentamethyldiethylenetriamine (PMDETA) was added as a catalyst activator and stirred. Ethyl 2-bromoisobutyrate was added as a reaction initiator and the stopper was tightly closed. The temperature was kept in an oil bath at 60 ° C. Based on the number of moles of monomeric tert-butyl acrylate, the number of moles divided by the desired degree of polymerization was taken as the required amount of reagents other than the monomer. Regardless of the ratio, the solvent acetone was adjusted by the viscosity of the solution after polymerization, and 1 mL of acetone was added to 5 mL of the monomer.
3-4 Hydrolysis (Fig. 17D)
A crystal resonator was immersed in a solution containing 10% trifluoroacetic acid in dichloromethane, and hydrolyzed by shaking with a shaker for 15 hours.
3-5 Functional group activation (FIG. 17E)
1- [3- (Dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were mixed in a petri dish containing 10 ml of distilled water, and the hydrolysis was performed on the solution. The quartz crystal attached to was immersed and activated for 1 hour.
3-6 Immobilization of antibody (FIG. 17 (F)) and antigen-antibody reaction (FIG. 17 (G))
_First, the oscillation frequency (F ₁ ) of the crystal resonator after the introduction of the active group was measured. 30 μL of anti-CRP antibody was dropped on the activated crystal resonator electrode, and the anti-CRP antibody was immobilized in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and then the oscillation frequency (F ₂ ) of the remaining crystal unit was measured. . The difference between the oscillation frequencies of the individual crystal resonators was obtained and the average value was calculated to obtain the amount of antibody immobilized on the crystal resonator (ΔF ₁ = F ₂ −F ₁ ). Subsequently, 30 μL of a 0.02 M glycine solution was dropped on the quartz resonator in order to perform blocking treatment of unreacted aldehyde groups, and reacted for 20 minutes in an air constant temperature bath at 25.0 ± 0.1 ° C. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, the oscillation frequency (F ₃ ) of the crystal unit was measured. The amount of antibody introduced onto the crystal resonator electrode by CRP antibody immobilization and glycine treatment was calculated from the average value of the change in the oscillation frequency of the crystal resonator (ΔF ₂ = (F ₁ -F ₃ ) −ΔF ₁ ). After immobilizing the anti-CRP antibody, 30 μL of CRP was dropped on each glycine-blocked crystal resonator, and an antigen-antibody reaction was carried out for 90 minutes in an air constant temperature bath at 25.0 ± 0.1 ° C. Thereafter, the crystal was washed with 3 mL (1 mL × 3 times) of pure water, and after drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F ₄ ) of each crystal resonator was measured. The amount of antigen-antibody reaction with each concentration of CRP was calculated from the average value of the oscillation frequency change in each crystal resonator, and the average value (ΔF ₃ ) was obtained (ΔF ₃ = (F ₁ -F ₄ ) −ΔF _2- ΔF1).
4. Measurement method of DNA hybridization (Experimental flow diagram is shown in FIG. 18)
_First, the oscillation frequency (F ₁ ) of the crystal resonator used in the experiment was measured. A sample to be measured (E. coli was used in the example shown in FIG. 40) was treated with a surfactant (SDS) and proteinase K, and the protein was removed with phenol. Thereafter, a DNA solution designed and synthesized for each measurement target was denatured with 0.5 M sodium hydroxide to form a single strand. Thereafter, 30 μL of the single-stranded DNA-containing solution returned to neutrality was dropped onto a crystal resonator, and DNA was immobilized under reduced pressure at 80 ° C. for 2 hours (FIG. 18A). Next, in order to suppress non-specific adsorption, a solution obtained by removing the probe from a hybridization solution (DNA probe containing NaCl, sodium citrate, Denhardt's solution, 0.1% SDS, 50% formaldehyde) is used. Prehybridization was performed (FIG. 18 (b)). The difference from F _{1 of} each crystal resonator was obtained, and the amount of DNA immobilized on the crystal resonator by DNA immobilization was calculated from the average value (ΔF ₁ = F ₁ −F ₂ ). Next, 30 μL of a solution containing a biotin-immobilized DNA probe (first probe) is dropped onto the crystal resonator (FIG. 18 (c)), and it is kept in an air constant temperature bath at 25.0 ± 0.1 ° C. for 90 minutes. Hybridization was performed (FIG. 18 (d)). After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water (FIG. 18 (e)), dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of the remaining crystal unit. (F ₃ ) was measured. The difference between the oscillation frequencies of the individual crystal resonators was calculated and the average value thereof was calculated, and the amount of hybridization with DNA on the crystal resonator was calculated (ΔF ₂ = (F ₁ -F ₃ ) −ΔF ₁ ).
30 μL of a solution containing avidin-bonded nanoparticles (second probe-bonded particles) is dropped on the crystal resonator after the hybridization reaction, and avidin for 60 minutes in an air thermostat at 25.0 ± 0.1 ° C. A biotin binding reaction was performed. Thereafter, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a flow of nitrogen at a flow rate of 2.5 L / min, and then the oscillation frequency of each crystal resonator was measured, and the measured value was defined as F ₄ . The ΔF ₃ value was calculated from the following equation (ΔF ₃ = (F ₁ −F ₄ ) −ΔF ₂ −ΔF ₁ ).
In accordance with each method of the present invention described above, various test substances were tested. In the test, the same operation was performed by appropriately changing the above-described antibody (CPR antibody or the like) for each measurement target substance.
For environmental pollutants (other than dioxins and PCBs), the results of measurement according to the competitive reaction method and the subsequent amplification reaction by sandwich reaction are shown in FIGS. The results measured according to the subsequent amplification reaction by the sandwich reaction are shown in FIGS. 30 to 36, and the microorganisms in the food inspection are measured according to the direct reaction method and the subsequent amplification reaction by the sandwich reaction are shown in FIGS.
Moreover, the result of having measured the frequency change about 2,3,7,8-TCDD as an example of dioxins is shown in FIG. The measurement was performed as follows. The first antigen-antibody reaction was performed according to the 2-1 competition reaction method. That is, a competitive reaction solution was prepared by mixing equal amounts of a DNP-bound albumin solution serving as a competitive molecule of DNP and each DNP antigen solution, and used for measurement. 30 μL of this competitive reaction solution was collected and dropped onto an anti-DNP antibody-immobilized quartz crystal resonator, and an antigen-antibody reaction was performed for 90 minutes in an air constant temperature bath at 25.0 ± 0.1 degrees Celsius. Thereafter, the crystal was washed with 3 mL (1 mL × 3 times) of pure water, and after drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency (F ₅ ) of each crystal resonator was measured. The amount of antigen-antibody reaction with each concentration of DNP was calculated from the average value of the change in oscillation frequency in each crystal resonator. The second antigen-antibody reaction was performed according to the chemical amplification reaction of the sensor response by the sandwich reaction after the 2-2 competitive reaction method. That is, 30 μL of an anti-DNP antibody-immobilized latex suspension in which an anti-DNP monoclonal antibody is immobilized on a quartz crystal resonator after a competitive reaction of DNP is added for 60 minutes in an air thermostat at 25.0 ± 0.1 degrees Celsius. Antigen-antibody reaction was performed. After washing with 3 mL (1 mL × 3 times) of pure water and drying for 90 minutes under a nitrogen flow rate of 2.5 L / min, the oscillation frequency of each crystal resonator was measured, and the frequency before and after addition of the latex suspension. The amount of change was calculated.
Moreover, the result of having measured the frequency change about colon_bacillus | E._coli is shown in FIG. The measurement was performed as follows. In the conventional method, 3 μL of a solution containing a biotin-immobilized DNA probe (first probe) was dropped on a crystal resonator, and hybridization was performed in an air thermostat at 25.0 ± 0.1 degrees Celsius for 90 minutes. (See FIG. 18D). After 90 minutes, it was washed with 3 mL (1 mL × 3 times) of pure water, dried for 90 minutes under a nitrogen flow rate of 2.5 L / min, and the oscillation frequency (F ₃ ) of the remaining crystal unit was measured. . The difference between the oscillation frequencies of the individual crystal resonators was calculated and the average value was calculated, and the amount of hybridization with DNA on the crystal resonator was calculated. In the method using the second probe-bonded particles, 30 μL of a solution containing nanoparticles (second probe-bonded particles) bonded to avidin is dropped on the crystal resonator after the hybridization reaction, and air at 25.0 ± 0.1 degrees Celsius The avidin-biotin binding reaction was performed for 60 minutes in a thermostatic chamber (see the post-process of FIG. 18 (e)). Then washed with pure water 3 mL (1 mL × 3 times), dried over a nitrogen stream under a flow rate of 2.5L / min 90 minutes the measured value until oscillation frequency measurement of each crystal oscillator was set to _{F 4.} ΔF ₃ value was calculated from the following equation.
In addition, as an example of cancer marker protein and environmental pollutants, the results of measuring the α-fetoprotein concentration in human serum according to the direct reaction method and the subsequent amplification reaction by sandwich reaction are shown in FIG. The measurement results by competitive reaction are shown in FIG. 41 and 42, the conventional method (indicated by “◯” in the figure) is a two-dimensional immobilization method in which an antibody is immobilized on a crystal resonator using a covalent bond. On the other hand, according to the present invention, an antibody is three-dimensionally immobilized on a polymer chain having a uniform molecular weight and chain length by an atom transfer radical polymerization (ATRP) (“□” in the figure). It was possible to significantly amplify the antigen detection sensitivity.
In each figure, (a) is a measurement result by an expert's manual work, and (b) shows a measurement result by the automatic analyzer of the present invention. Each measurement point is an average value when six independent crystal resonators are used, and an error bar on the vertical axis indicates a standard deviation (error).
The environmental reference values and normal values of the substances subjected to the above-described sensor sensitivity amplification experiments are shown in the following tables.
According to each of the above examples, the same excellent analysis as in Examples 1 to 8 was possible.

The method of the present invention is suitable as a method for analyzing trace substances simply and quickly.
Moreover, the apparatus of the present invention is suitable as an apparatus used for a simple and rapid analysis method for trace substances.
While this invention has been described in conjunction with its embodiments, we do not intend to limit our invention in any detail of the description unless otherwise specified and are contrary to the spirit and scope of the invention as set forth in the appended claims. I think it should be interpreted widely.

Claims (8)

In a method for analyzing a substance to be measured by converting a mass change due to a substance to be measured bonded to a piezoelectric element sensor having a recognition element fixed on an electrode into a frequency change and quantifying it,
(I) The recognition element sample of the substance to be measured in the container is sucked by using a solution suction / discharge probe, the sucked sample is discharged onto the electrode of the piezoelectric element, and the discharged recognition element sample is sucked again by the probe. Forming a recognition element for the substance to be measured on the piezoelectric element electrode by performing at least one time to re-discharge the recognition element sample that has been re-sucked onto the piezoelectric element electrode;
(Ii) The sample of the substance to be measured in the container is sucked by the solution suction / discharge probe, the sucked sample is discharged onto the fixed recognition element, and the discharged sample of the measured substance is re-sucked by the probe. And bonding the substance to be measured onto the immobilized recognition element by re-ejecting the re-aspirated measurement substance sample onto the recognition element at least once;
A method for analyzing a substance to be measured, comprising at least two steps.   Aspiration of the recognition element sample of the substance to be measured in the container by the solution suction / discharge probe, discharge of the sample onto the recognition element from the probe, re-suction of the discharged sample to the probe, and re-suction of the re-suctioned sample The analysis method according to claim 1, wherein the re-ejection is executed by an automatic analyzer including a plurality of containers, a plurality of probes, and a plurality of piezoelectric element sensors. Aspiration of a sample to be measured in a container by a solution aspiration / ejection probe, ejection of a sample from a probe onto a piezoelectric element, re-aspiration of the ejected sample into the probe, and re-ejection of the re-aspirated sample The analysis method according to claim 2, wherein the analysis method is executed by an automatic analyzer including a plurality of containers, a plurality of probes, and a plurality of piezoelectric element sensors. The number of suction / discharge is 1 to 1,000 times / minute, the repetition time of suction / discharge is 0.1 to 100 minutes, and the amount of suction / discharge is 0.01 to 100 microliters. 4. The analysis method according to any one of 3. The analysis method according to claim 1, comprising washing the inside and outside of the probe with a washing liquid. The analysis method according to claim 5, comprising spraying a cleaning liquid or gas from the probe or another probe to the sensor unit to clean the sensor unit. The nozzle shape of the probe tip is cylindrical, bevel tip, sphere, cone, needle, or polyhedron, and the positional relationship (distance between the top and bottom) between the noise level and the piezoelectric element sensor during suction / discharge 0.01mm ~ 10mm The analysis method according to any one of claims 1 to 6, wherein an angle between the dispensing pipette used for detection by the piezoelectric element and the piezoelectric element is in a range of 10 degrees to 360 degrees. The shape of the piezoelectric element is such that the crystal base plate is processed into a concave shape on both sides or one side by chemical treatment or machining, or a flat shape such as # 4000 finish, mirror finish, etc., and electrodes of platinum, gold, silver, copper, etc. Platinum in a groove in which a plurality of vibrators created by vapor deposition are integrated, or a quartz plate is processed into a flat shape such as concave or # 4000 finish, mirror finish, etc. on both sides or only one side by chemical treatment or machining The analysis method according to any one of claims 1 to 7, which is a single element prepared by vapor deposition of electrodes such as gold, silver and copper.

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