JP2010107502A - Quantitative analyzing method and detecting cartridge - Google Patents

Abstract

A quantitative analysis method is provided that enables analysis of biochemical substances and the like with high sensitivity and with a relatively short analysis time and with reduced background noise.
A flow path to which a test solution containing a detection substance is supplied, a storage section provided in the middle of the flow path, and a downstream side of the storage section in the main flow path. In the quantitative analysis method using the detection cartridge 1 provided with the provided detection unit 18, the storage unit 16 concentrates the association of the molecule recognition reagent labeled with the metal nanoparticles and the detected substance. A step of dissolving the metal nanoparticles bonded to the aggregate to obtain a metal ion solution, a step of transporting the metal ion solution to the detection unit 18, and And a step of measuring an ion concentration and quantifying the concentration of the substance to be detected according to the metal ion concentration.
[Selection] Figure 1

Description

  The present invention relates to a quantitative analysis method used for quantifying various biochemical substances such as antigens, antibodies, allergens, bacterial cells, viruses, drugs, DNA adducts, etc., using an immunoassay. The present invention relates to a quantitative analysis method, a reagent, and a detection cartridge using a detection cartridge having a flow path through which a small amount of test liquid flows.

  Conventionally, when various allergens and biochemical substances are quantified, it is strongly required to quantify these biochemical substances with higher sensitivity. For example, when measuring a substance to be detected from a test solution having a very low concentration such as several tens of fg / mL to several hundreds pg / mL, the following is a conventional method. That is, first, a substance to be detected is subjected to solid phase extraction from a test solution to obtain a concentrate. Next, a method of supplying and quantifying the concentrate to a liquid chromatography apparatus or an electrophoresis apparatus, or a method of quantifying by ELISA using this concentrate is used. However, in these methods, pretreatment for concentration such as solid-phase extraction treatment took time, and the total analysis time required several hours to several days.

  On the other hand, in order to put biomarkers discovered one after another into practical use in medical treatment, it has been demanded to quantitatively analyze a substance to be detected in situ with high sensitivity. In order to meet such needs, simple quantitative inspection chips using immunochromatography and colorimetric methods have been developed. However, the sensitivity of this type of simple quantitative inspection chip is limited to about 0.1 ng / mL. That is, it was impossible to measure a much smaller amount of a substance to be detected with high accuracy.

  The following Patent Document 1 discloses a device and method for detecting a trace component such as heavy metal in a water eluate from soil with high sensitivity using a portable cartridge type detection device. In the cartridge type detection device described in Patent Literature 1, a detection cartridge having a flow path formed therein is used. In this detection cartridge, a reservoir is provided in the middle of the flow path, and a detector is provided downstream of the reservoir. The substance to be detected in the test solution is stored in the storage part, and once concentrated in the storage part. Thereafter, the concentrated substance to be detected is eluted again using the eluent and supplied to the detector. By detecting the metal ion concentration contained in the concentrate of the substance to be detected newly eluted in the detection unit, it is possible to detect trace components such as heavy metals in the water eluate from the soil with a high sensitivity of about 1 ppb. It is possible.

  Note that Patent Document 1 also mentions a configuration in which an antigen or antibody is immobilized in a reservoir, and describes that a reducing substance by a labeling enzyme may be measured by a detector. This is a configuration for realizing a method similar to the conventional ELISA method.

  On the other hand, in Patent Document 2 below, for example, a metal of a chemical species labeled with a metal in a sample such as a biological sample is used as a soluble electrochemically active complex, the complex is dissolved, and a complex ion is electrochemically converted. A method for automatically measuring and measuring the presence or amount of a metal-labeled species is disclosed. In Example 4 of Patent Document 2, an apparatus in which a detection unit having a flow path and electrodes on a polystyrene substrate is used is used. The latex-labeled antibody, the silver-labeled antibody, and the antigen are mixed, and the resulting mixture solution is supplied to the flow path and led to a filter provided in a filter region provided in the middle of the flow path. In this filter, latex labeled antibody-analyte-silver labeled antibody complexes are captured. Unbound silver-labeled antibody also passes through the filter. The captured chemical species are then washed to remove any remaining unbound silver-labeled antibody and the captured chemical species are complexed by adding a complexing agent. The concentration of complex ions obtained in this way is detected by the detector. Here, when the chemical species are captured by the filter, the mixture solution is supplied in the surface direction of the filter.

  On the other hand, Patent Document 3 below discloses a method for quantifying a biological substance bound to Au nanoparticles by immunoassay. Quantification is performed by using a competitive method and using Au nanoparticles in an unreacted biological substance labeled with Au nanoparticles as Au ions and electrochemically measuring the Au ion concentration.

Japanese Patent No. 4053081 WO2005 / 121792 publication WO2002 / 001178

  In the cartridge type detection device disclosed in Patent Document 1, it is stated that the substance to be detected can be quantified by labeling the substance to be detected with an enzyme and measuring the reducing substance by the labeling enzyme as described above. Yes. However, when such an amplification method using an enzyme is used, the reaction by the enzyme is easily affected by the temperature, and the activity of the enzyme is likely to change depending on the storage history. Therefore, the substance to be detected could not be measured with high accuracy due to variations in measurement temperature and storage history.

  In the analysis method described in Patent Document 2, a complexing agent is added to Ag to form an Ag complex, and finally the Ag complex ion concentration is measured. Since Ag complex ions have higher electrical activity than Ag ions, there is a problem that background noise increases. That is, the SN ratio is lowered and the sensitivity cannot be increased. In addition, in the filter region, since the mixture solution is supplied to the upper surface of the strip-shaped filter in the surface direction of the upper surface, unreacted components and impurities adhere to the electrode, and the SN ratio also decreases. There is a problem.

  Further, in the immunoassay described in Patent Document 3, Au is used as colloidal metal particles, Au nanoparticles are dissolved by acid, and Au ions are measured. I can't do it.

  The object of the present invention is to quantitate a very small amount of a substance to be detected with higher sensitivity and in a relatively short time in view of the current state of the prior art described above. It is an object of the present invention to provide a quantitative analysis method, a reagent, and a detection cartridge that are not easily affected.

  According to the present invention, in order to achieve the above-described problem, a flow path to which a test liquid containing a target substance is supplied, a storage section provided in the middle of the flow path, and the storage section in the main flow path A method for quantitative analysis using a detection cartridge provided with a detection unit provided on the downstream side, wherein in the storage unit, molecular recognition labeled with metal nanoparticles mainly composed of CdSe, CdTe, or Ag A step of concentrating a reagent and an association of the substance to be detected, a step of dissolving the metal nanoparticles contained in the association and obtaining a metal ion solution, and a step of transporting the metal ion solution to the detection unit And a step of measuring the metal ion concentration and quantifying the detected substance concentration according to the metal ion concentration in the detection unit.

  In the present invention, the reservoir is used for concentrating and washing the metal nanoparticles contained in the aggregate through a molecular specific binding reaction typified by the immunoassay in order to shorten the molecular specific binding reaction typified by an immunoassay. Gives a foothold to do.

  The molecular-specific binding reaction can be shortened because the molecular-specific binding reaction represented by immunoassay is diffusion-controlled, and the reaction field is limited to the microspace in the cartridge reservoir, and the diffusion distance is long. Due to the shortness. In the diffusion controlled reaction, the time required for the reaction to saturate is inversely proportional to the square of the reaction space distance. For example, in a 4 mm well and a 40 μm space, the reaction is about 10,000 times faster.

  In the storage part, the aggregate of the substance to be detected and the molecular recognition reagent is concentrated on the porous material having a large surface area. Furthermore, a metal ion solution is obtained by dissolving the metal nanoparticles labeled with the molecular recognition reagent in the concentrated aggregate. This metal ion solution has the effect of concentrating aggregates and the effect of generating tens of thousands of ions from a single metal nanoparticle, resulting in the concentration of the substance to be detected in the original test solution. In comparison, a metal ion solution converted to a concentration of 100 times to 10000 times is obtained. Therefore, even at an extremely low concentration of the substance to be detected, metal ions converted to a high concentration derived from the labeled metal nanoparticles concentrated in the reservoir are quantified in the detector. The metal ion concentration measured by the detection unit is proportional to the concentration of the substance to be detected in the test liquid. By converting the metal ion concentration to the concentration of the substance to be detected, the substance to be detected can be quantified.

  In a specific aspect of the present invention, a specific binding molecule that specifically binds to the substance to be detected is fixed in advance in the reservoir, and a test liquid containing the substance to be detected is stored in the reservoir. By introducing, the specific binding molecules associate with the substance to be detected, the substance to be detected is concentrated in the reservoir, and the molecular recognition reagent labeled with the metal nanoparticles is introduced into the reservoir By doing so, a step of concentrating and storing the association body of the molecule recognition reagent labeled with the metal nanoparticles and the substance to be detected is included in the storage section. In this case, since the specific binding molecule is immobilized in advance in the reservoir, when a test liquid containing the substance to be detected is introduced into the reservoir, an association of the substance to be detected and the specific binding molecule is formed. It will be fixed to the reservoir. Accordingly, the substance to be detected is reliably concentrated in the reservoir. Therefore, when a molecular recognition reagent labeled with metal nanoparticles is introduced into the reservoir, an association between the molecule recognition reagent labeled with metal nanoparticles and the substance to be detected is concentrated and stored in the reservoir. .

  In another specific aspect of the quantitative analysis method of the present invention, a porous body is previously disposed in the reservoir, and the sample liquid labeled with the test solution containing the target substance and the metal nanoparticles at the time of the concentrated storage. A suspension containing an aggregate obtained by mixing a molecular recognition reagent and beads to which specific binding molecules that specifically bind to the substance to be detected are mixed is stored through the main channel. And a step of concentrating and storing the aggregate containing the association of the molecular recognition reagent labeled with metal nanoparticles and the substance to be detected in the storage part. In this case, a mixture of the test solution containing the detection substance, the molecular recognition reagent labeled with the metal nanoparticles, and the beads to which the specific binding molecule is immobilized is introduced as a suspension to the reservoir. As a result, each component diffuses quickly in the suspension, and the diffusion-controlled reaction is quickly and concise. Therefore, the analysis time can be further shortened.

  In still another aspect of the quantitative analysis method according to the present invention, the method further includes a step of introducing a cleaning liquid to the storage section after the concentrated storage. By washing by introducing the washing liquid into the reservoir, the unreacted molecular recognition reagent that has not been incorporated into the aggregate is removed, and impurities that interfere with detection in the subsequent detection of metal ions are removed from the reservoir. It can be easily removed. Thereby, the measurement background can be lowered, and the S / N ratio, which is the ratio of the effective detection signal and noise, can be increased.

  In still another specific aspect of the quantitative analysis method of the present invention, the step of dissolving the metal nanoparticles bound to the aggregate and obtaining a metal ion solution includes a step of adding an acid as an eluent. . In this case, by introducing an acid, the metal nanoparticles incorporated in the aggregate are quickly dissolved, and metal ions corresponding to the concentration of the substance to be detected are eluted, and the metal ion solution is promptly supplied to the detection unit. Can be conveyed.

  In still another specific aspect of the quantitative analysis method according to the present invention, polymer particles are used as the beads. As the polymer particles, polymer particles larger than the porous mesh fixed to the reservoir are used, or one polymer particle is labeled with a substance to be detected and metal nanoparticles through a plurality of specific binding molecules. By forming a network with the molecular recognition reagent, a filterable aggregate or aggregate can be provided. That is, filtration can be efficiently performed by using polymer particles as described above.

  However, in the present invention, particles having at least the outermost surface metal may be used as the beads. In this case, in addition to the molecular recognition reagent labeled with metal nanoparticles, the metal component will be present in the measurement system, so a larger signal amplification factor can be obtained when the same reservoir is used. Can do. When particles having a surface area larger than that of the metal nanoparticles and the outermost surface being a metal are used for the reservoir, a larger signal amplification factor can be obtained. The metal species of the particles whose outermost surface is a metal may not necessarily be the same as the metal species constituting the metal nanoparticles. Even if the reduction potential is close, each can be quantified by using a method such as anodic stripping voltammetry at the detection section.

In still another specific aspect of the quantitative analysis method according to the present invention, metal nanoparticles made of cadmium selenium (CdSe), cadmium tellurium (CdTe), or silver (Ag) are used as the metal nanoparticles. Nanoscale CdSe, CdTe, and Ag dissolve rapidly with an acid to give cadmium ions (Cd ²⁺ ) and silver ions (Ag ⁺ ). Cadmium ions and silver ions can be quantified relatively easily and quickly.

  In still another specific aspect of the quantitative analysis method according to the present invention, an electrochemical electrode for detecting the metal ion is disposed in the detection unit, and the electrochemical ion is detected when detecting the metal ion concentration. A metal is electrochemically deposited on the electrode, and then the stripping current is measured by sweeping the potential. In the case of a detection unit including such an electrochemical electrode, since a bulky optical system is not necessary, the measurement apparatus can be reduced in size. In addition, the electrochemical electrode may be any of a tripolar, quadrupolar, or bipolar electrode, but cleaning with the metal deposited on the anode can reduce the influence of impurities. it can. Further, since the current is measured while sweeping the potential, the signal of the impurity ion species and the signal of the target metal ion can be distinguished by the peak position of the current. Therefore, the S / N ratio, which is a ratio of a significant measurement signal to noise, can be further increased.

  In still another specific aspect of the quantitative analysis method according to the present invention, a suspension of a water-insoluble sulfide salt generated by adding sulfide ions to a metal ion solution in which the metal nanoparticles are dissolved is detected. A method of quantifying the metal ion concentration by optical detection at the unit is used. In this case, the metal ion concentration can be easily and quickly quantified by optical means without contact. Therefore, the metal ion concentration can be detected promptly and with high accuracy by applying a known absorbance measurement method, reflected light measurement method, measurement method using an optical waveguide, or the like. In particular, in the case of heavy metal ions such as cadmium ions and silver ions, a sulfurizing agent such as Lawson's reagent and Ravesson's reagent should be added as sulfide ions, and precipitated as a water-insoluble sulfide salt to form a suspension. Therefore, the metal ion concentration can be easily measured by an optical method, which is preferable.

  In the quantitative analysis method according to the present invention, in the cartridge, the concentration of the labeled substance according to the concentration of the detected substance is rapidly performed through the immune reaction between the molecular recognition reagent labeled with the metal nanoparticles and the detected substance, The metal ion concentration in the metal ion solution from which the metal nanoparticles as the labeling substance are eluted correlates with the detected substance concentration in the test solution, and the metal ion is converted to a high concentration with respect to the detected substance. However, it enables signal detection at the detection part promptly and with very high sensitivity. That is, highly sensitive quantitative analysis of about 10 pg / mL can be completed in a relatively short time, for example, within 20 minutes.

  In addition, the quantitative analysis method of the present invention does not use amplification or the like utilizing an enzyme reaction, and the amplification magnification is clear depending on the metal nanoparticles used, so that there is little influence of the measurement temperature, and storage of analysis reagents and the like Insensitive to history. Therefore, the reliability of measurement can be improved.

(A) is a perspective view showing the appearance of a detection cartridge according to an embodiment of the present invention, and (b) is a schematic perspective view showing the relationship between a flow path, a storage part, and a detection part formed in the cartridge body. FIG. In one Embodiment of this invention, it is front sectional drawing which shows typically the relationship between the flow path comprised in the cartridge for a detection, a 1st storage part, a detection part, etc. FIG. It is a disassembled perspective view of the cartridge for detection of one embodiment of the present invention. It is a disassembled perspective view for demonstrating the structure of the cartridge for detection concerning other embodiment of this invention. It is a schematic diagram for demonstrating the flow-path connection switching mechanism in the cartridge for detection concerning other embodiment of this invention. It is a schematic diagram for demonstrating the flow-path connection switching mechanism in the detection cartridge which concerns on other embodiment of this invention. (A) And (b) is each schematic diagram for demonstrating the flow-path connection switching mechanism of the cartridge for a detection in further another embodiment of this invention. It is a figure which shows the current density measurement result in Example 1 of this invention. It is a figure which shows the case of Example 1 using a CdSe nanoparticle, and the measurement result of Example 2 using Ag nanoparticle. It is a figure which shows the to-be-detected substance measurement result in Example 2 using Ag nanoparticle, and the comparative example using Au nanoparticle. In Example 3, it is a figure which shows the relationship between the nitric acid density | concentration and the peak electric current value derived from Ag ion which melt | dissolved and produced | generated Ag nanoparticle at the time of changing nitric acid concentration. It is a figure which shows the to-be-detected substance measurement result in Example 4. FIG.

  Hereinafter, details of the present invention will be clarified while describing specific embodiments of the present invention.

  In the quantitative analysis method of the present invention, a quantitative analysis cartridge including a flow path, a storage section provided in the middle of the flow path, and a detection section provided downstream of the storage section in the flow path is used. Then, at least a step of concentrating and storing the association body of the molecular recognition reagent labeled with the metal nanoparticles and the substance to be detected in the storage section, and dissolving the metal nanoparticles contained in the association body as a metal ion solution And a step of conveying to the detection unit.

  The main flow channel means a flow channel that realizes a series of analysis methods for flowing and quantifying a test solution containing a substance to be detected, that is, a flow channel for analysis. It should be pointed out that the expression “main flow path” is intended to distinguish it from the waste liquid flow path shown in a modified example described later.

  The reservoir provides a scaffold for concentrating and washing while shortening the molecular-specific binding reaction typified by immunoassay. Since the reaction field of the molecule-specific binding reaction, which is diffusion-limited, is a micro space, the time required for the reaction equilibrium is shortened. In addition, the aggregate of the substance to be detected and the molecular recognition reagent is concentrated on a porous material having a large surface disposed in the reservoir. The metal nanoparticles contained in the conjugate of the substance to be detected and the molecular recognition reagent concentrated and washed in the reservoir are dissolved by introducing an appropriate eluent such as an acid to give a metal ion solution. Since the metal ion becomes several thousand to several tens of thousands of metal ions with respect to one original metal nanoparticle, the concentration is 100 times to 10000 times the concentration of the substance to be detected in the test liquid. . The eluted metal ions are transported to the detection unit and quantified. Even if the concentration of the substance to be detected in the test solution is extremely low, the detection unit measures the metal ions converted to a high concentration proportional to the concentration of the substance to be detected in the test solution. Quantification of the metal ion concentration at is easy.

  The measured metal ion concentration is converted into the concentration of the substance to be detected. In this way, the concentration and analysis, which has been complicated and took several hours, can be completed in a relatively short time, for example, within 20 minutes. In addition, since no enzyme is used for signal amplification, it is difficult to be affected by measurement temperature and storage history. In the present invention, the term “detection” broadly means collecting information for quantifying the metal ion concentration according to the concentration of the substance to be detected. The test solution is not particularly limited as long as it may contain a detection target substance to be detected. For example, a biological sample [for example, an animal body fluid (for example, blood, serum, Plasma, cerebrospinal fluid, ascites, tears, sweat, pus, sputum, or throat swab) or excrement (eg, urine, feces), organs, tissues, or animals or plants themselves or their dry bodies], or the environment Samples derived from the source (for example, river water, lake water, seawater, or soil), food pulverized products, combustion residues, and extracts from these can be mentioned.

The flow path in the detection cartridge has a shape, length, and size as long as the flow of various reagents such as a test solution, a masking agent, a washing solution, and an eluent can be secured and the liquid can be transferred and / or stored. The number and the like are not particularly limited. For example, it is suitable that the groove has a width on the order of several tens of μm to several mm, and is formed by a groove having a depth of several tens of μm to several hundreds of μm. For example, the cross-sectional area of the flow path is about 100 μm ^{2 to 1} mm ² .

  The molecular recognition reagent labeled with metal nanoparticles used in the present invention has a molecular recognition portion, a linker portion, and metal nanoparticles.

  As the molecular recognition moiety, an antibody used in an immunoassay (immunoassay) is preferably used. In particular, many monoclonal antibodies are excellent in specificity. The molecular recognition moiety is not limited to an antibody, and has a molecule-specific binding ability such as sugar chain, lectin, selectin, DNA, RNA, peptide nucleic acid, aptamer, antibody fragment Fab, antibody recognition site Fv, crown ether, etc. Molecular recognition reagents can be used.

  Examples of the linker moiety include at least one selected from the group consisting of protein G, protein A, protein L, biotin-avidin conjugate, derivatives thereof, and modifications.

  As the metal nanoparticles used for labeling, cadmium selenium (CdSe) nanoparticles, cadmium tellurium (CdTe) nanoparticles, and silver (Ag) nanoparticles having a diameter of 3 nm to 30 nm are preferably used. Nanoparticles smaller than this range are unlikely to exist stably. If the nanoparticles are too large, they may settle and become unsuitable as a label for the antibody reagent.

  In the present invention, the reservoir has a function of associating the substance to be detected contained in the test solution with the molecular recognition reagent labeled with metal nanoparticles, concentrating and holding the generated aggregate. On the other hand, a dispersion medium, a masking agent, a cleaning solution, or the like including impurities including metal nanoparticles that have not been incorporated into the aggregate passes through the reservoir. The storage section preferably includes a supporting means for supporting the concentrate for increasing the reaction field for generating the aggregate and storing / holding the substance to be detected.

  Therefore, preferably, a porous material, a mesh-like material, or a material having a finely processed surface is suitably used as a supporting means in the storage portion. Since porous materials and mesh-like materials have a large surface area, they can provide more reaction fields than the filters and membrane filters described in Patent Document 2, and can carry more aggregates. it can. The porous material is not limited to the material itself having a large number of pores, but also includes those in which fine particles are aggregated and large pores are formed between the particles.

  The material constituting the supporting means is preferably the porous material or mesh-like material, but more specifically, sponge, woven fabric, non-woven fabric, filled fine particles, fiber, sintered body, monolithic porous body Any mode or a mode consisting of a combination thereof is adopted. As the finely processed surface, a structural surface by a nanoimprinting method or a spray deposition method is preferably used. As a material constituting the supporting means, for example, ceramic, glass, synthetic fiber, plant fiber, animal fiber, synthetic resin, cellulosic material, metal, or the like may be used. In addition, a surface of the base material that has been treated with a molecule or a functional group that performs a chemical reaction with a substance to be detected or has a specific binding ability may be used.

  As a method of eluting metal ions from the molecular recognition reagent labeled with the metal nanoparticles, a method of adding an acid is used. Here, 0.5 to 3N nitric acid can be suitably used as a suitable acid. Hydrochloric acid and sulfuric acid are undesirable because they produce poorly water soluble chlorides and sulfides.

  The eluted metal ions are transported to the detection unit and quantified. The detection mechanism is not particularly limited, and any mechanism that can realize a method capable of detecting a target substance can be used. Examples of the method include electrochemical analysis, optical analysis, and analysis using other principles. The mechanism is not particularly limited. For example, an electrode for electrochemical analysis and means for applying or reading current / voltage, an optical cell in an optical analysis method, a light source, a spectroscope, etc., or a part thereof are detected. The detection section in the cartridge is configured and mounted.

  The electrochemical analysis method used in the detection method of the present invention will be described more specifically. For example, linear sweep voltammetry (LSV), couloamperometry (CA), coulochronometry (CC), cyclic volta Metering (CV) or anodic stripping voltammetry (ASV). As the electrode configuration, any of a three-pole type, a four-pole type, and a two-pole type can be adopted. However, considering the balance between accuracy and usability, the three-pole type is desirable.

  In the present invention, the detection unit is arranged on the downstream side of the storage unit. However, the detection unit may be provided on the downstream side of the storage unit, and the two may be integrated.

  In the detection unit, the metal ion concentration corresponding to the concentration of the substance to be detected is detected. In this case, the detection means is not necessarily provided in the cartridge or on the cartridge. However, when at least a part of the detection means is present in or on the cartridge, the size can be reduced.

  Further, only a part of the detection means may be provided in the cartridge. For example, when an optical detection means is used, an optical cell is arranged as a part of the detection unit in the cartridge or on the cartridge, and the measurement part including the light receiving means for receiving the light transmitted through the optical cell is measured outside the cartridge. It may be provided in the apparatus.

  Preferably, among the members constituting the detection means, a member that is difficult to regenerate, such as a concentration column, or a member that requires complicated regeneration processing before measurement, such as an electrochemical electrode, is preferably provided on the cartridge side. In addition, since the electrode used for electrochemical electrode analysis must be polished before measurement, it is desirable to be provided on the cartridge side that is discarded every time or every certain number of analysis times.

  On the other hand, since the optical cell can be used again by a simple cleaning operation before measurement, it may be arranged in the cartridge.

  More specifically, when an electrochemical analysis means is used as the detection means, for example, an electrode such as a working electrode, a counter electrode, and a reference electrode is arranged in the detection portion in the cartridge, and an electric current or a current is supplied in the processing unit. A means for applying or reading a voltage may be provided. When using an optical analysis means, an optical cell may be provided in the cartridge, and a light source or a light receiving means may be provided outside the cartridge.

  One preferable aspect of the detection means in the detection unit is to use an eluate from the reservoir that may contain eluted metal ions and an eluate that does not contain metal ions to compare electrochemical responses. Analytical means to do this.

  Another preferred detection means is a means for optically detecting the concentration of a collision suspension produced by adding sulfide ions. Cd ions and Ag ions precipitate and disperse in an aqueous solution as water-insoluble sulfide ions, suspend the solution, and exhibit a specific color. This can be easily measured by optical means. A method for measuring absorbance by transmitted light may be used, a measurement method using reflected light may be used, and various optical measurement methods such as a measurement method using an optical waveguide may be used.

  When it is necessary to transport the liquid between the cartridge and the measuring device outside the cartridge, a plurality of ports for supplying and discharging liquid may be provided on the surface of the cartridge. The plurality of ports are used for introducing or discharging a test solution or a reagent, and the size and position are not particularly limited. For example, the plurality of ports supply the test liquid upstream of the storage unit, and therefore supply the solution and cleaning liquid used for detection between the storage unit and the detection mechanism, and so on. It can arrange | position in the appropriate position. The selection and switching of the ports may be performed using an appropriate valve mechanism using an electromagnetic valve or the like.

  Next, a specific structural example of the cartridge used in the present invention will be described with reference to the drawings.

  FIG. 1A is a perspective view illustrating an appearance of a detection cartridge according to an embodiment of the present invention, and FIG. 1B is a schematic perspective view illustrating a relationship between a flow path formed in the cartridge and a storage portion. FIG. 2 is a schematic diagram showing the relationship between a flow path, a storage part, and a detection part provided in the cartridge. FIG. 3 is an exploded perspective view showing the internal structure of the detection cartridge of this embodiment.

  As shown in FIG. 1, the detection cartridge 1 of the present embodiment has a cartridge body 2 formed by laminating first to third plates 11 to 13 from below. The plates 11 to 13 are made of, for example, a synthetic resin. Each of the plates 11 to 13 typically has a rectangular planar shape of 35 mm × 50 mm, and the thickness of one plate is about 1 mm. Although omitted in FIG. 1A, as shown in FIG. 3, the first to third plates 11 to 13 are stacked via adhesive sheets 14 and 15. Accordingly, the cartridge body 2 has a thickness of about 4 mm.

  In FIG. 3, for ease of illustration and easy understanding, the orientation of the first plate 11 and the adhesive sheet 14 and the remaining second and third plates 12, 13 and the adhesive sheet are shown. The direction of 15 is changed and shown.

  First to fifth ports 3 to 7 are formed on the lower surface of the cartridge body 2. The first to fifth ports 3 to 7 are connected to a flow path 8 provided in the cartridge body 2. The flow path 8 is a groove formed between the first plates 11 and 12 and between the second plate 12 and the third plate 13 and / or a through groove provided in the second plate 12. It is formed by. In addition, the flow path 8 includes a first flow path portion 9 and a second flow path portion 10. The first flow path portion 9 connects the first and second ports 3 and 4. A reservoir 16 is provided in the middle of the first flow path portion 9. Further, the second flow path portion 10 connects the third port 5 to the fifth port 7. A detector 18 is provided in the middle of the second flow path portion 10.

  Further, the downstream side of the second flow path part 10 is connected to the waste liquid storage part 19 in a part not shown in FIG. As shown in FIG. 3, the waste liquid storage unit 19 includes a recess 19 a that opens upward on the upper surface of the first plate 11, a large opening 19 b provided in the second plate 12, and the third plate 13. A concave portion 19c that opens downward on the lower surface is formed in combination.

  On the other hand, the storage portion 16 is formed by laminating a concave portion 16 a opened upward on the upper surface of the first plate 11 and a radial opening portion 16 b provided in the second plate 12. Note that the second port 4 described above is formed in the first plate 11 so as to be connected to the storage portion 16. As shown schematically in FIG. 2, a concentration carrier 21 as a concentration carrier means is accommodated in the storage unit 16. The concentrated carrier 21 is formed of the appropriate carrier material described above.

  In this case, as shown in FIG. 2, since the reservoir 16 is provided in the portion where the flow path 9 extends in the vertical direction, the concentrated carrier 21 in the reservoir 16 is covered from above. The test solution is supplied, and the liquid flowing through the flow path passes through the concentration carrier 21 in the vertical direction. Therefore, when the concentrated carrier 21 is made of the porous material or mesh-like material described above, a reaction for generating an association of the molecular recognition reagent labeled with the metal nanoparticles and the substance to be detected in the test liquid. The field can be increased. In Patent Document 2 described above, since the test liquid is supplied in the surface direction of the strip-shaped filter, the reaction field is small. In this embodiment, the test is performed so as to pass through the concentrated carrier 21 in the vertical direction. Since the liquid is supplied, the reaction field can be significantly increased. Therefore, it is possible to increase the detection sensitivity.

  In the present embodiment, the test solution is supplied to the storage unit 16 from the flow path. In this reservoir 16, an association of the molecular recognition reagent labeled with the metal nanoparticles and the substance to be detected in the test liquid is generated and concentrated. In this case, a mixed solution containing the test solution, the molecular recognition reagent, and an appropriate liquid medium may be supplied to the flow path and guided to the storage unit 16 or the test solution may be supplied to the storage unit first. Then, the molecular recognition reagent may be guided to the storage unit 16 after that. In the reservoir 16, the association body of the molecular recognition reagent and the substance to be detected is concentrated.

  On the other hand, in the detection part 18, the reference electrode R, the working electrode W, and the counter electrode C are arrange | positioned as a chemical electrode. Here, based on the voltage between the counter electrode C and the reference electrode R and the voltage between the working electrode W and the counter electrode C, metal ion deposition and potential sweep amperometry are performed, and the metal ion concentration is measured. . That is, the metal nanoparticle taken in the aggregate in the reservoir 16 is caused to flow through an eluent that dissolves the metal nanoparticle in the flow path, and the metal nanoparticle in the aggregate is dissolved, whereby the metal ion solution is stored. It is discharged from the part 16 to the downstream side. The metal ion concentration in the metal ion solution is detected by the detection unit 18.

  In addition, as shown in FIG. 3, the 1st-3rd plates 11-13 are bonded together and laminated | stacked through the adhesive sheets 14 and 15. As shown in FIG. The pressure-sensitive adhesive sheets 14 and 15 have through holes to form the above-described waste liquid storage portion 19 and the like. It also has a plurality of small through holes for connecting the flow paths.

  As the working electrode W, for example, a plate-like carbon electrode of about 3.5 mm × 8.4 mm × 0.5 mm can be suitably used. As for the counter electrode C and the reference electrode R, a plate-like electrode having the same size can be used, and the counter electrode C can be produced using a plate-like carbon electrode similarly to the working electrode W. For the reference electrode R, an optimal electrode is appropriately selected depending on the metal ion species. For example, when the metal ion species is cadmium ion, an electrode in which a silver paste is coated on an alumina substrate can be used as a reference electrode. When the metal ion species is silver ion, an electrode in which ferrocene is fixed to a gold base can be used.

  However, the dimensions and structure of the chemical electrode are not particularly limited, and may be formed by printing an appropriate electrode material on the first plate 11.

  In this embodiment, a concave portion is formed on the upper surface of the first plate 11 so that the upper surface of the plate 11 and the surface of the chemical electrode are flush with each other, and an electrode material is filled so as to fill the concave portion.

  Therefore, the upper surfaces of the reference electrode R, the working electrode W, and the counter electrode C and the upper surface of the first plate 11 are flush with each other. Therefore, the first plate 11 and the second plate 12 can be easily stacked without generating a gap.

  The adhesive sheet 14 is formed with through holes 14a to 14c for exposing the reference electrode R, the working electrode W, and the counter electrode C, respectively.

  The liquid comes into contact with the reference electrode R, the working electrode W, and the counter electrode C through the through holes 14a to 14c.

  Among the first to fifth ports 3 to 7, the first port 3 is a port for introducing a test liquid or an eluent, and is formed as a through hole in the first plate 11. . The second plate 12 may be provided with an electrolyte solution chamber in which an electrolyte solution as an activation liquid for activating the reference electrode R is stored, although not particularly illustrated. By connecting such an electrolyte solution to the second flow path portion 10 and supplying it to the second flow path portion, the reference electrode R can be easily activated.

  It is desirable to use a chemical electrode as the detection means in the cartridge. More desirably, as in the above-described embodiment, the cartridge body 2 is provided with a detection section in which a reference electrode R as a chemical electrode, a working electrode W, and the like are arranged. By using the chemical electrode, it is possible to reduce the size of the detection means, thereby reducing the size of the entire cartridge.

  The structure of the cartridge body 2 in the detection cartridge 1 is not limited to the structure shown in FIGS. 1 to 3 as long as the storage part and the detection part and the flow path connecting them are formed.

  FIG. 4 is an exploded perspective view for explaining a detection cartridge according to another embodiment of the present invention. The detection cartridge 51 is configured by bonding two plates 52 and 53 using an adhesive or the like. That is, the cartridge main body is configured by bonding the plates 52 and 53 together.

  The plates 52 and 53 are not limited to being bonded by an adhesive, but can be bonded by an appropriate bonding method such as diffusion bonding, anodic bonding, or laser bonding.

  A porous concave portion indicated by a broken line is provided on the lower surface of the plate 52, and the first flow path portion 9 is formed by the meandering concave portion. Further, the plate 52 is formed with through holes for constituting the first to fourth ports 3 to 6. Note that the fifth port 7 in FIG. 2 is not described.

  On the upper surface of the plate 53, a meandering concave portion that overlaps the meandering concave portion provided on the lower surface of the plate 52 is provided in a portion where the first flow path portion 9 is formed. The serpentine concave portions of the plates 52 and 53 are combined to form the first flow path portion 9. In addition, a first reservoir 16 including a recess including the concentrated carrier 21 is provided on the upper surface of the plate 53. One end of the first flow path portion 9 is connected to the first port 3, and the other end is connected to the second port 4.

  Further, the second flow path portion 10 is formed on the upper surface of the plate 53, and the second storage portion 17 formed of a concave portion is provided in the middle of the second flow path portion 10. A detection unit 18 is provided on the downstream side of the second storage unit 17. The detection unit 18 is configured in the same manner as in the first embodiment.

  FIG. 5 is a schematic diagram for explaining a flow path connection switching mechanism used in the detection cartridge shown in FIGS. 1 and 4.

  In FIG. 1 and FIG. 4, the downstream end of the first flow path portion 9 is connected to the second port 4, and the upstream end of the second flow path portion 10 is connected to the third port 5. It is connected. As a switching mechanism for connecting the flow path between the second port 4 and the third port 5, for example, a structure using a flow path switching device shown in FIG. Here, a three-way cock 31 is prepared as a flow path switching device outside the cartridge body 2.

  The three-way cock 31 is a known three-way cock having first to third ports 31a to 31c. A flow path 32 is connected to the second port 4 and the three-way cock 31, and a flow path 33 connects the three-way cock, 31 and the third port 5.

  In the state shown in FIG. 5, the first port 31 a is connected to the flow path 32, and the second and third ports 31 b and 31 c are not connected to the flow path 33. Therefore, the liquid that has flowed into the flow path 32 from the first flow path portion 9 through the second port 4 passes through the three-way cock 31 from the first port 31a and is discharged from the second port 31b or 31c. Is done. Accordingly, the residual liquid flowing from the first flow path portion 9 can be discarded, and unreacted reagents or the like do not reach the second flow path portion 10. Therefore, in this state, a buffer solution or the like is supplied as a cleaning solution from the first port 31a, and the concentrated carrier 21 and the first flow path portion 9 are cleaned. The cleaning liquid can also be discharged from the port 31b or 31c of the three-way cock 31.

  Next, in measurement, the three-way cock 31 is rotated, and the port 31 b is connected to the flow path 32 and the port 31 c is connected to the flow path 33. In that state, the eluate is injected from the first port 31a, and the eluate in which the metal nanoparticles are dissolved is guided to the second flow path portion 10 via the flow path 32, the three-way cock 31 and the flow path 33, Detection is performed by the detection unit 18. As described above, by providing a flow path connection switching mechanism such as the three-way cock 31 outside the cartridge main body 2, quantitative determination can be performed using the detection cartridge 1.

  FIG. 6 is a schematic view for explaining a flow path connection switching mechanism in a detection cartridge according to still another embodiment of the present invention. This flow path connection switching mechanism can be configured in the cartridge body 2. In the present embodiment, the first flow path portion 9 and the second flow path portion 10 are connected at the connection portion X in the cartridge body 2. That is, the downstream end of the first flow path portion 9 reaches the connection portion X. Further, a waste liquid channel 41 is connected between the connection portion X and the second port 4.

  In other words, the configuration shown in FIG. 2 corresponds to a configuration in which the connection portion X is provided at the downstream end of the first flow path portion 9 and the waste liquid flow path 41 is provided between the connection portion X and the first port 4. To do. Since the upstream end of the second flow path portion 10 is connected to the connection portion X as described above, the third port 5 in the structure shown in FIG. 2 is omitted. On the other hand, the detection part 18 is provided in the middle of the 2nd flow-path part 10, the port 6 is provided in the downstream end side, and the port 7 of FIG. 2 is abbreviate | omitted. Port 6 is open to the outside.

  In use, the test solution and the reagent are introduced from the first port 3 with the port 6 closed. The aggregate is concentrated in the concentrated carrier 21. Excess liquid is discharged from the second port 4 via the waste liquid channel 41. Since the port 6 is closed, there is no air passage in the second flow path portion 10, so that no liquid enters the second flow path portion 10.

  Next, the cleaning liquid is introduced from the first port 3, and the cleaned cleaning liquid is similarly discharged from the second port 4.

  After the cleaning is completed, the entire first flow path portion 9, the second flow path portion 10, and the waste liquid flow path 41 are occupied by air.

  Next, the second port 4 is closed and the port 6 is opened. In this state, the eluate is introduced from the first port 3. As a result, the metal nanoparticles contained in the aggregate are dissolved, the solution containing the metal nano ions flows into the second flow path portion 10, and the ion concentration can be quantified by the detection unit 18. In this case, since the second port 4 is closed, the solution selectively flows into the second flow path portion 10.

  FIGS. 7A and 7B are schematic views for explaining each flow path connection switching mechanism in the detection cartridge according to still another embodiment of the present invention.

  In FIG. 7A, the first flow path portion 9, the second flow path portion 10, and the waste liquid flow path 41 are connected at the connection portion X as in the case of FIG. 6. The difference is that a valve 42 that opens and closes the flow path is provided on the second flow path portion 10 on the upstream side of the detection section 18 and on the downstream side of the connection section X. In addition, the flow resistance increasing portion 41 a is provided in the waste liquid flow path 41 so that the flow resistance of the fluid in the waste liquid flow path 41 is larger than the flow resistance of the second flow path portion 10.

  In a state where the valve 42 is closed, the test solution and the reagent are introduced from the first port 3, and the immune aggregate is concentrated by the concentration carrier 21. The remaining liquid is discharged from the second port 4 because the valve 42 is closed. In the cleaning, the cleaning liquid is similarly introduced from the first port 3 and discharged from the second port 4. Also in this case, since the valve 42 is closed, the cleaning liquid does not reach the downstream side of the valve 42 of the second flow path portion 10.

  For the determination, the valve 42 may be opened. In this state, the eluate is introduced from the first port 3. Since the flow resistance of the second flow path portion 10 is lower than the flow resistance in the waste liquid flow path 41, the eluate in which the metal nano ions are dissolved does not reach the waste liquid flow path 41 and passes through the valve 42. It reaches the second flow path portion 10. Therefore, the metal nanoion concentration can be measured by the detection unit 18.

  Note that, as shown in FIG. 7B, the valve 42 may be provided in the second flow path portion 10 on the downstream side of the detection unit 18.

  The method for detecting a substance to be detected of the present invention includes various allergens such as egg yolk, egg white, milk, peanut, shrimp, crab, fish, shellfish, soybean, mango and other foods, dust mites, feathers, pollen, fungi, bacteria, Cockroaches, dog or cat hair. In addition, endocrine disrupting substances, agricultural chemicals, immunoglobulins such as IgE or IgG, histamine, genes (RNA), stress markers, various proteins, antigens or antibodies contained in human or animal blood, blood components, urine, saliva or body fluids, It can be used for detection of a substance that can be detected by a component indicating a specific disease, asbestos, a blood drug, or other antibodies.

  Next, specific examples of measuring a substance to be detected using the detection cartridge of the present invention will be described more specifically.

Example 1
First, C-reactive protein (hereinafter referred to as CRP) was selected as an analysis target, and two types of antibodies capable of sandwich assay, namely, a first anti-CRPIgG and a second anti-CRPIgG were prepared. CdSe nanoparticles were bound to the second anti-CRP IgG via protein G as an adapter to obtain a CdSe nanoparticle-labeled antibody reagent.

  The first anti-CRP IgG was physically sensitized to a nitrocellulose membrane, blocked with albumin, dried, punched into a disk shape having a diameter of 4 mm, and used as a concentrated carrier for CRP.

  Next, the plates 11 to 13 shown in FIG. 1 were prepared, and the concentrated carrier was set in the storage part. Furthermore, a chemical electrode was arranged for the detection unit according to the embodiment described above.

  As the 1st-3rd plates 11-13, the resin plate which consists of a polymethyl methacryl plate of 50 mm x 35 mm x thickness 1mm was used, and the double-sided adhesive tape of thickness 20 micrometers was used as the adhesive sheets 14 and 15.

  Using the detection cartridge 1 obtained as described above, a standard sample having a CRP concentration of 1 μg / mL is prepared, and sample solutions having respective concentrations of 10 pg / mL, 50 pg / mL, and 100 pg / mL are prepared. A calibration curve was prepared using the cartridge 1. The analysis procedure for one sample solution was as follows.

  First, 1 mL of the sample solution was introduced from the first port 3 into the first flow path portion 9 of the flow path 8 using a microsyringe and led to the storage section 16.

  Next, the cartridge main body was set in a measuring apparatus, and the following cleaning solution, antibody reagent and cleaning solution (second time), and eluate were sequentially guided to the storage unit by a liquid feeding system controlled by a microcomputer. That is, first, 4 mL of a phosphate buffer solution as a cleaning solution is supplied from the port 3 to the storage portion 16 through the first flow path portion 9 at 1 mL / min. Introduced at the speed of Next, the antibody reagent is supplied from the first port 3 to 1.0 mL, 200 μL / min. Was led to the reservoir 16 at a flow rate of. Thereafter, a phosphate buffer solution as a washing solution is supplied from the first port 3 to 2 mL, 1 mL / min. The label solution is introduced from the first port 3 at a rate of 0.2 mL and 50 μL / min. Was led to the reservoir 16 at a flow rate of.

  Next, an eluate in which the eluted metal ions may be dissolved was sent to the inspection unit. In the inspection part, the measurement was performed by linear sweep voltammetry (LSV). The sweep rate was 5 mV / second, the current was measured by sweeping from 200 mV to 700 mV. The results are shown in FIG. In FIG. 8, the horizontal axis represents the sweep potential, and the vertical axis represents the current value. The area of the hatched portion in the vicinity of −0.8 V to −0.6 V is the detected amount of the metal ion derived from the label. Contaminant ion species have peaks at different potentials, so they can be clearly distinguished from the target metal ion species. A small peak of silver derived from Ag / AgCl used for the reference electrode is in the vicinity of 0 V, which can also be distinguished. Therefore, it can be seen that the concentration of the substance to be detected can be detected with high accuracy by using the linear sweep voltammetry.

(Example 2 using silver nanoparticles)
In the same manner as in Example 1 using the CdSe particles, except that Ag nanoparticles were used instead of CdSe nanoparticles, detection of the concentration of the detection substance was attempted in the same manner as in Example 1. The results are shown in FIG. In FIG. 9, in order to facilitate the comparison, the result in the case of Example 1 using the CdSe nanoparticles is also shown. As is apparent from FIG. 9, it can be seen that the concentration of the substance to be detected can be detected with higher sensitivity when Ag nanoparticles are used than when CdSe nanoparticles are used. In addition, the small silver peak derived from Ag / AgCl used for the reference electrode seen in Example 1 is used for correction as a background of signals derived from Ag nanoparticles.

(Comparative example)
For comparison, the concentration of the substance to be detected was measured in the same manner as in Example 2 except that Au particles were used instead of Ag particles. FIG. 10 shows the results of measurement by linear sweep voltammetry in Example 2 and the comparative example. As can be seen from FIG. 10, when Ag nanoparticles are used, the peak value of the current is significantly higher than that of the comparative example using Au ions.

(Example 3 for preferred nitric acid concentration)
In Example 1 and Example 2 above, nitric acid was used as the eluent when ionizing the metal nanoparticles, but the preferred range of nitric acid concentration was studied. Ag nanoparticles having a particle diameter of 20 nm were dissolved in 0.01 N to 5 N nitric acid to prepare a 10 μm Ag ion solution. The current value was measured by linear sweep voltammetry (LSV) with an insertion speed of 50 mV / sec. From the results, the relationship between the peak current value and the nitric acid concentration was determined. The results are shown in FIG.

  As is apparent from FIG. 11, it is understood that Ag ions can be measured with high sensitivity if the nitric acid concentration is 0.5N to 3N, more preferably 1N to 2N. When the concentration of nitric acid was 5N, the silver / silver chloride portion of the reference electrode was discolored, and the current was not stable and measurement could not be performed.

Example 4
As a substance to be detected, a brain natriuretic peptide (hereinafter referred to as BNP), which is a heart disease marker, is selected, an anti-BNP antibody that recognizes BNP as a primary antibody, an anti-BNP antibody and BNP Detection was performed by the following procedure using an anti-Fab24C5 / BNP antibody that recognizes the complex.

(1) Preparation of water-soluble silver nanoparticles 15 mg of hydrophobic silver nanoparticles (Daiken Chemical Co., Ltd.) were dispersed in 800 μL of ethanol. 25 mg of N-[(S) -3-mercapto-2-methylpropionyl] -L-proline (manufactured by Sigma-Aldrich) dissolved in ethanol was added, and an aqueous NaOH solution was added to prepare a dispersion.

  Using “HPB-450” manufactured by AKICO, which is a high-pressure liquid reaction device equipped with a pinch-cocked capillary channel (automatic adjustment device for pinch-cock width), the dispersion is passed through at a pressure of 35 MPa and a flow rate of 2.6 mL / min. Liquid. The time required for processing the whole amount was about 10 minutes. This treatment replaced the surface modifier of the hydrophobic silver nanoparticles and produced water-soluble silver nanoparticles. After the passed liquid was washed with chloroform, it was purified using an ultrafiltration membrane (Millipore, “Amicon Ultra-4”) and a Sephadex column (Amersham Biosciences, “MicroSpin G-25 Columns”). And concentrated to obtain purified aqueous silver nanoparticles.

  The resulting silver nanoparticles are dispersed in water to give an orange dispersion. As a result of measuring the particle size distribution using a particle size measuring device by a dynamic light scattering method (“ZETASIZER Nano Series Nano-ZS” manufactured by Malvern), the peak particle size was 3.6 nm.

(2) Preparation of water-soluble silver nanoparticle labeled antibody The above silver nanoparticle aqueous solution was added to 100 mM cysteine solution of the same volume with 100 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and The mixture was mixed at 4 ° C. for 1 hour in the presence of a hydroxysuccinimide (NHS) coupling agent (both manufactured by Pierce Biotechnology). After removing free amino acids using a Nap-5 column (Amersham Biosciences), the secondary antibody was bound to silver nanoparticles using a sulfo-SMCC coupling agent (Pierce Biotechnology) at 4 ° C. Ultrasonic separation was performed for 2 hours. The product is purified using a Sephadex column (Amersham Biosciences), concentrated after ultrafiltration (NMWL 100 kDa, manufactured by Millipore), and water-soluble silver nanoparticles with anti-BNP antibody bound to the surface of the silver nanoparticles A labeled antibody reagent was prepared.

(3) Preparation of latex antibody reagent To 1 volume of polystyrene latex (solid content 10% (W / V)) having an average particle size of 0.087 μm, 9 volumes of latex dilution buffer was added to dilute the latex. A 0% (W / V) latex solution was obtained. The primary antibody was diluted with an antibody dilution buffer to obtain a sensitive antibody solution. While stirring 600 μL of 1.0% (W / V) latex solution with a magnetic stirrer in an incubator at 25 ° C., 1200 μL of the antibody solution was quickly added thereto and stirred at 25 ° C. for 1 hour. Thereafter, 3.0 mL of blocking buffer was added, and the mixture was stirred at 25 ° C. for 2 hours. Then, it centrifuged for 15 minutes at 15 degreeC and 15,000 rpm. 4.0 mL of blocking buffer was added to the resulting precipitate, and the precipitate was washed in the same manner by centrifugation. The washing operation was performed 3 times. To this precipitate, 1.8 mL of blocking buffer was added and stirred well, followed by dispersion treatment with an ultrasonic crusher. Further, 1.8 mL of blocking buffer was added thereto to obtain a latex antibody reagent.

(4) Analysis example using water-soluble silver nanoparticle-labeled antibody A standard sample having a BNP concentration of 2 μg / mL is prepared, and 0.01 pg / mL, 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL. Sample solutions of various concentrations were prepared. To each 1 mL of these standard sample solutions, 20 μL of the latex antibody reagent (3) was added and stirred. 30 μL of the water-soluble silver nanoparticle-labeled antibody reagent (2) was added thereto and further stirred. This solution was introduced into the detection cartridge of FIG. In the following operation, the peak area was measured in the same manner as in Example 2. The results are shown in FIG.

  As is apparent from FIG. 12, it can be seen that the concentration of the detection object can be detected with high accuracy by using linear sweep voltammetry as in the first embodiment.

DESCRIPTION OF SYMBOLS 1 ... Detection cartridge 2 ... Cartridge main body 3-7 ... 1st-5th port 8 ... Main flow path 9 ... 1st flow path part 10 ... 2nd flow path part 11-13 ... 1st-3rd Plate 14 ... Adhesive sheet 14a-14c ... Through-hole 15 ... Adhesive sheet 16, 17 ... 1st, 2nd storage part 16a ... Recessed part 16b ... Opening part 18 ... Detection part 19 ... Waste liquid storage part 19a ... Recessed part 19b ... Opening part 19c ... Concave part 21 ... Concentration carrier 31a-31c ... 1st-3rd port 32, 33 ... Channel 41 ... Waste liquid channel 41a ... Flow resistance increase part 42 ... Valve 51 ... Detection cartridge 52, 53 ... Plate

Claims (8)

A main channel to which a test solution containing a substance to be detected is supplied, a reservoir provided in the middle of the main channel,
A quantitative analysis method using a detection cartridge including a detection unit provided downstream of the storage unit in the main flow path,
A step of concentrating an association of a molecule recognition reagent labeled with metal nanoparticles mainly composed of CdSe, CdTe, or Ag and the substance to be detected in the reservoir;
Dissolving the metal nanoparticles bound to the aggregate to obtain a metal ion solution;
Transporting the metal ion solution to the detection unit;
And a step of measuring the metal ion concentration in the detection unit and quantifying the concentration of the substance to be detected according to the metal ion concentration. In the reservoir, a specific binding molecule that specifically binds to the substance to be detected is fixed in advance,
By introducing a test solution containing the substance to be detected into the reservoir, the substance to be detected and the specific binding molecule are associated, and the substance to be detected is concentrated in the reservoir,
When the molecular recognition reagent labeled with the metal nanoparticles is introduced into the reservoir, the association of the molecule recognition reagent labeled with metal nanoparticles and the substance to be detected is concentrated in the reservoir. The quantitative analysis method according to claim 1, wherein the method is stored. A porous body is previously disposed in the storage part,
During the concentration and storage, a test solution containing the substance to be detected, a molecular recognition reagent labeled with the metal nanoparticles, and a bead on which a specific binding molecule that specifically binds to the substance to be detected is fixed The suspension containing the aggregate obtained by mixing the mixture is introduced into the reservoir through the main channel, and the aggregate includes the association of the molecular recognition reagent labeled with metal nanoparticles and the substance to be detected. The quantitative analysis method according to claim 1, wherein an object is concentrated and stored in the storage unit.   The quantitative analysis method according to claim 1, wherein the step of dissolving the metal nanoparticles to obtain a metal ion solution includes a step of adding an acid as an eluent.   The quantitative analysis method according to claim 3, wherein polymer particles are used as the beads.   The quantitative analysis method according to claim 3 or 5, wherein particles comprising at least a part of the outermost surface made of metal are used as the beads.   An electrochemical electrode for detecting the metal ion is disposed in the detection unit, and when quantifying the metal ion concentration, after depositing the metal electrochemically on the electrochemical electrode, the potential is applied. The quantitative analysis method according to claim 1, wherein the current is measured while sweeping.   The metal ion concentration is obtained by optically detecting, in the detection unit, a suspension in which a water-insoluble sulfide salt generated by adding sulfide ions to the metal ion solution in which the metal nanoparticles are dissolved is dispersed. The quantitative analysis method according to any one of claims 1 to 6, wherein

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