JP5105956B2 - Measuring probe and manufacturing method thereof - Google Patents

Description

  The present invention relates to an immunological test probe for immobilizing proteins such as antibodies and antigens on the surface of a support and detecting pathogens, physiologically active substances, chemical substances and the like using the proteins.

  Various substances including biologically related substances, food contents substances, and environment related substances (proteins that may interact with specific substances) are supported on the support. It is often used as a component module of an immunoassay element for detecting. As such a module, for example, a protein that selectively reacts with a specific object such as an antigen or an antibody is immobilized on a support surface such as glass, metal, paper, or resin.

  Usually, as the support of the module, a fine particle support is used in order to increase the reactivity, and a slab (plate) is used in order to facilitate B / F separation (separation between the bound substance and the free substance). ) -Like support is often used.

  On the other hand, in the immunoassay, there are various methods for obtaining a signal related to detection of a target substance, and in general, optical signals such as absorption, fluorescence, and chemiluminescence are often used. Among them, a fluorescence method for detecting fluorescence as an optical signal has been developed for a long time and widely used because various physicochemical properties and emission wavelengths can be adjusted.

  In an immunoassay using such a fluorescent chromophore as a label (hereinafter sometimes referred to as “fluorescent immunoassay”), it is necessary to excite the fluorescent chromophore. In recent years, instead of excitation by ordinary radiation, a technique called evanescent waves, which is excited by light with a limited propagation region, has been used. As a method for generating evanescent waves, the material used as the support is made of an optically transparent material, light is introduced into the support at an angle at which total reflection is made at the interface, and the support is surfaced. There is a technique for generating evanescent waves. In this method, a detection protein having a function of specifically capturing a target substance such as an antigen or an antibody is immobilized on the surface of the support in advance, and the target substance labeled with a fluorescent chromophore is captured. Then, since the evanescent wave is effective only in the region of several hundred nanometers from the support surface, the evanescent wave can be used as the excitation light. By this evanescent wave, only the fluorescent chromophore that is present on the surface of the support and labels the target substance captured by the capture protein is excited. As a result, since the fluorescent chromophore simply floating in the sample solution is not excited, the so-called S / N ratio is improved, and as a result, the measurement sensitivity is improved.

  As a conventional technique configured to generate an evanescent wave for an immunoassay, an optical measurement apparatus disclosed in Patent Document 1 can be cited. In this optical measuring device, the support for immobilizing the molecules that capture the target substance is a slab polymer, and at the same time serves as the wall of the optical cell, so it is convenient and useful for optical detection. is there.

On the other hand, when the shape of the support is a cylindrical optical fiber type, an improvement in sensitivity is expected due to an increase in surface area, and since it can be used not only as a support but also as a probe system, there is a degree of freedom in form. It also has the advantage of spreading. As a form using a cylindrical measuring probe, there is a type in which a part of an optical fiber directly connected to a light source or a detector is used as a sensor. However, as in Patent Documents 2 to 4, a part used as a sensor is physically independent. There is what I did.
JP-A 63-273042 JP-A-5-5742 U.S. Pat. No. 4,582,809 US Pat. No. 6,136,611

  Fluorescence immunization by evanescent wave excitation using a cylindrical measurement probe can show very excellent performance as described above, but since the support is cylindrical, that is, the surface is curved, antibodies, antigens, etc. There was a problem that the captured protein was easily peeled off.

  In order to solve this problem, there has conventionally been a technique for immobilizing a detection protein by covalent bonding with a support surface using a crosslinking treatment. However, the cross-linking treatment between the probe surface and the capture protein has the problem that the capture protein and the probe surface are separated from each other physically and distancely, which reduces the influence of evanescent waves and lowers the detection sensitivity. there were.

  An object of the present invention is to provide a measurement probe for detecting a target substance in a specimen, which includes an optical waveguide having a columnar structure such as a fiber type. An object of the present invention is to provide an excellent measurement probe and a manufacturing method thereof.

A measurement probe according to the present invention is a measurement probe for detecting the presence or absence or concentration of a target substance in a sample by contacting the sample,
A support having an optical waveguide comprising a fiber-shaped part, a flange part, and a convex lens-like part;
A layer formed by cross-linking between proteins, formed in an endless ring shape on the surface of the fiber-shaped part,
Proteins that constitute the layer made by cross-linking of the protein to each other, a measurement probe, characterized in that it is crosslinked by treatment with a crosslinking agent solution molarity of 1 to 5 times with respect to the protein.

A measurement probe manufacturing method according to the present invention is a method of manufacturing a measurement probe for detecting a target substance in a sample by contacting the sample,
A layer formed by cross-linking proteins with each other via a cross-linking agent on the fiber-shaped portion of the support composed of a fiber-shaped portion, a flange portion, and a convex lens-shaped portion, encircles one axis of the support. Forming a portion having
Production of a measurement probe characterized in that cross-linking of proteins constituting a layer formed by cross-linking of the proteins is performed by treatment with a cross-linking agent solution having a molar concentration of 1 to 5 times the protein. Is the method.

  According to the present invention, since the protein in the endless cyclic protein layer formed on the surface of the optical waveguide is cross-linked via the cross-linking agent, the protein layer is prevented from peeling off. Cross-linking treatment to the surface of the optical waveguide is unnecessary, and the detection sensitivity of measurement becomes stable and high sensitivity.

  What is called a measurement probe in the present invention has at least a support and a protein-containing layer formed on the surface of the support in an endless ring shape. The support has at least one shaft, and the preferred shape of the shaft is a substantially columnar columnar structure. It is used to detect the presence or concentration of a target substance in a specimen by contacting the specimen, and has a structure suitable for a form as a small piece. As the support, one having an optical waveguide is preferable.

  In the present invention, at least a part of the measurement probe surface is coated with a protein layer such as an antibody or an antigen, and the coating is intended for a crosslinking method that is easy to manufacture. Therefore, it can be effectively applied when a material that cannot easily form a covalent bond with a substance immobilized on the surface, such as a protein such as an antibody or an antigen, is used as the support for the measurement probe. Is the method. Typical examples of such materials include hydrophobic resins such as polystyrene and polyethylene that are frequently used in containers.

  In one preferred embodiment of the present invention, the measurement probe has a support having a substantially cylindrical columnar structure, and a protein layer is formed on the surface of the support in the immobilization based on the crosslinking method. By forming so as to have, peeling from the support is suppressed. A protein layer formed in an endless ring shape is formed so that a state in which the protein layer is provided around a cross section (for example, orthogonal) in at least one or more axes of the support exists even in a part of the axis. Layer. In a support having a columnar shape, there is always a line segment that surrounds the support. When a protein molecule or a protein and other components are cross-linked to form a two-dimensional protein layer for fixation, the protein layer grips the support due to the presence of a cross-linking complex corresponding to the line segment. An effect occurs.

  As a result, even if a covalent bond is not formed between the support and the protein, separation and loss of the protein layer are unlikely to occur.

  In general, as a method for fixing a protein to a support in order to reduce or prevent peeling of the protein from the support, a specific substituent on the support and a specific substituent on the protein are linked by a covalent bond. There is a method, which results in a relatively strong and stable fixed layer.

  However, when the present inventors examined, depending on the material used for a support body, when it was difficult to introduce such a substituent beforehand, the complexity of the manufacturing process became a subject.

  In the present invention, the molecules (proteins) to be immobilized are subjected to a cross-linking reaction by covalent bond to assist the immobilization (hereinafter referred to as a cross-linking method).

  In this crosslinking method, although there is no bond between the molecule to be fixed and the support, the mobility is lowered by forming a fixed layer in which the molecule to be fixed is integrated, and the adhesion to the support surface is increased. it is conceivable that.

  In addition, the present invention performs crosslinking between an enzyme and another protein in a method for producing a measurement probe having an optical waveguide.

  There is also a two-stage method in which a protein solution is applied and then impregnated with a solution containing a crosslinking agent.

  The crosslinking method used in the present invention is simple in operation, and a measurement probe having preferable physical properties and functions can be prepared by optimizing the constituent weight ratio of the crosslinking agent.

  The protein layer may contain non-protein macromolecules and may be a protein layer formed by crosslinking these.

  As a specific example of the shape of the support, a fiber shape is desirable, and a sword mountain shape, a mesh shape, etc. obtained by processing the fiber can be cited (see FIGS. 1 (1) to (3)). Moreover, the shape which bent these shapes suitably etc. may be sufficient, for example, dendritic shape, ring shape, pile shape, etc. are mentioned. Further, a non-linear shape or a chipped annular (C shape) structure as shown in FIGS.

  In general, protein immobilization by such a crosslinking method uses an enzyme (particularly an oxidoreductase) as the type of protein and an electrode material as the support. That is, most cases are used for applications in which an enzymatic reaction (redox reaction) is detected electrochemically.

  On the other hand, in the present invention, proteins are antibodies and antigens, and the support has a fiber optical waveguide for optical detection. Furthermore, the present invention provides a measurement probe particularly effective for fluorescence immunization by evanescent wave excitation using a cylindrical measurement probe.

  In the measurement probe of the present invention, an antibody is often used as a protein for detection, and the shape of the support is cylindrical. In general, when an antibody is immobilized while reducing its exfoliation, a method of immobilizing the antibody via a covalent bond is usually used. However, in such a method, the surface of the support becomes non-uniform and the uniformity is further controlled. Things were difficult.

  On the other hand, in the present invention, since the cross-linking method using a general cross-linking agent is usually applied as a method for immobilizing the enzyme to the electrode as described above, the uniformity of the solid surface can be controlled.

  The uniformity of the solid surface is very important for the fluorescence immunization method using the evanescent wave excitation of the present invention, and the non-uniformity of the solid surface greatly affects the signal variation.

  By using the measurement probe of the present invention, the uniformity of the solid phase surface can be ensured and a stable signal can be obtained.

  In the measurement probe of the present invention, it is more desirable to improve the strength of the protein layer by crosslinking while securing the function of the immobilized protein when it is desired to further improve the performance of the fluorescence immunization method by evanescent wave excitation. For this purpose, the molar ratio of the protein to the cross-linking agent is preferably about 1: 1 after the reaction, and a charge ratio that can realize this is desirable. That is, as the conditions, it is preferable to act a cross-linking agent solution having a molar concentration (mol / L) of 1 to 5 times the molar concentration (mol / L) of the protein existing on the support surface before the cross-linking reaction. I found out. The range of 1 to 5 times is a magnification in consideration of a decrease in the amount of reaction that may occur due to deactivation of the crosslinking agent in the solution, steric hindrance of the protein, and the like, and is set according to the material and reaction conditions used. In addition, the molar concentration of the protein on the surface of the support mentioned here is the number of moles of the protein contained in the layer formed on the surface of the support divided by the volume of the layer. The said volume is a volume calculated | required from the coating area of a support body, and the thickness of substantial layer, and the volume which components other than a space | gap and the said protein occupy is also included.

  Even in the above concentration range, the cross-linking agent may affect the function of the protein, but the amount is substantially negligible in terms of detection function. When the molar concentration of the cross-linking agent is 1 or more times the protein molar concentration on the surface of the support, sufficient cross-linking can be performed. Particularly, the strength of the protein chain formed by cross-linking goes around the columnar portion of the support. This is preferable because the above effect can be easily obtained. Moreover, it is preferable that the molar concentration of the crosslinking agent is 5 times or less the protein molar concentration on the surface of the support because the protein function is easily maintained.

  The measurement probe of the present invention can be configured to include a protein serving as a component for detecting a target substance (hereinafter referred to as a detection protein) as a protein constituting the protein layer. Here, the detection component is a component having a function of detecting a target substance, and can be made of biopolymers such as proteins, nucleic acids, sugar chains, and various synthetic polymers. Here, having a target substance detection function means a function of specifically capturing a target substance. By holding the detection component on the support surface of the measurement probe of the present invention, the detection component is The target substance can be captured and the target substance can be held on the support surface. Examples of the detection protein include antibodies, antigens (antigen proteins), enzymes, and receptors. The protein layer includes at least one selected from these. In the present invention, if necessary, two or more kinds of detection proteins selected from these can be used in combination. Compared to enzymes and the like, the recognition portion of the antibody is exposed at the terminal, and the possibility of damage to the crosslinking reaction is higher. Therefore, it is more preferable to apply the above-described composition ratio.

  As a form different from the form in which the detection component is included in the protein layer, the above-mentioned cross-linked protein layer is formed on a predetermined surface of the support as a base, and the detection protein as the detection component of the target substance is connected thereon It is also possible to take a scheme. Here, the term “base” is used because the cross-linked protein layer serves as a base for immobilizing the detection protein when the detection protein is held on the support. This method is useful when it is desired to reduce damage to the detection protein due to the crosslinking reaction or when the direction of the detection protein is very important. Examples of the base protein suitable for such purposes include avidin and protein G. Avidin can be selectively connected via biotin. Protein G is useful for directing the Fc site of an antibody to a support.

  In addition, the underlying protein may be able to prevent nonspecific adsorption, and may be more useful than a synthetic polymer depending on the measurement target. Examples of such a base protein include albumin and casein.

  As a component which comprises a protein layer, 2 or more types of protein and the component which is not protein may be contained. For example, in order to provide a void convenient for detection reaction around the detection protein, or to adjust the strength of the protein layer, other crosslinkable polymers can coexist. Examples of such a polymer include cellulose and polyvinyl alcohol.

  Various known crosslinking agents can be used as the crosslinking agent, but those that are less likely to impair the function of the protein are preferable. That is, a crosslinking agent having a reactive terminal (functional group for crosslinking) capable of sufficiently reacting at around room temperature is preferable. Examples of such reactive terminal include aldehyde, carboxylic acid, and carboxylic acid ester. Bivalent crosslinking agents having these reactive ends can be suitably used, and glutaraldehyde is preferably used as both reactive ends are aldehydes. Carboxylic acid must be activated with a carbodiimide reagent. As the carboxylic acid ester, for example, N-hydroxysuccinimide ester is preferable. The span of the cross-linking agent (chain length between reactive ends) is not uniquely determined, but it depends on the system in consideration of the flexibility of the protein layer (related to adhesion), the efficiency of the cross-linking reaction, water solubility, etc. Need to select.

  It is also effective to stop the reaction with a suitable terminator in order to control the crosslinking reaction. For example, in the case of a reaction that crosslinks to the amino group of a protein, an amine derivative may be added, for example, tris (hydroxymethyl) aminomethane.

  In the measurement probe of the present invention, any molecule can be used as an antibody used as a detection protein as long as it contains a region having a so-called immunoglobulin fold structure. Usually, antibody bodies such as IgG, IgM, IgA, IgE, IgD and their complexes are used, but Fab'2, Fab, Fv, which are fragments, and VH, VL alone can also be used. It is also possible to use antibody fragment complexes such as scFv, diabody, triabodies, tetrabodies, etc. in which these are genetically engineered and made into a single chain with a peptide linker.

  Target substances that can be detected by an immunoassay using the measurement probe of the present invention are roughly classified into non-biological substances and biological substances.

  Industrially valuable non-biological substances include PCBs with different chlorine substitution numbers / positions as environmental pollutants, dioxins with different chlorine substitution numbers / positions, and so-called endocrine disrupting substances called environmental hormones. It is done. Examples of endocrine disruptors include hexachlorobenzene, pentachlorophenol, 2,4,5-trichloroacetic acid, 2,4-dichlorophenoxyacetic acid, amitrol, atrazine, alachlor, hexachlorocyclohexane, ethyl parathion, chlordane, oxychlordane, nonachlor 1,2-dibromo-3-chloropropane, DDT, Kelsen, Aldrin, Endrin, Dildoline, Endosulfan (Benzoepine), Heptachlor, Heptachlor Epoxide, Malathion, Mesomil, Methoxychlor, Milex, Nitrophen, Toxaphene, Trifluralin, Alkylphenol 5-9), nonylphenol, octynonylphenol, 4-octylphenol, bisphenol A, di-2-ethylhexyl phthalate, butylbenthyl phthalate , Di-n-butyl phthalate, dicyclohexyl phthalate, diethyl phthalate, benzo (a) pyrene, 2,4-dichlorophenol, di-2-ethylhexyl adipate, benzophenone, 4-nitrotoluene, octachlorostyrene, aldi Curve, Benomyl, Kypon (Chlordecone), Manzeb (Mancozeb), Mannebu, Methylam, Metribuzin, Cipermethrin, Esfenvalerate, Fenvalerate, Permethrin, Vinclozoline, Dinebu, Diram, Dipentyl phthalate, Dihexyl phthalate, Dipropyl phthalate, etc. Is mentioned.

  Biological materials include biological materials selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof, and more specifically, biomolecules selected from nucleic acids, proteins, sugar chains, and lipids. Specifically, selected from DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosugar, sphingolipid The present invention can be applied to any substance as long as it contains any other substance. Furthermore, bacteria and cells themselves that produce the above-mentioned “biological substances” can also be target substances as “biological substances” targeted by the present invention.

  Specific proteins that serve as target substances include so-called disease markers.

Examples of disease markers are acidic glycoproteins produced in hepatocytes in the fetal period and present in fetal blood, and markers for hepatocellular carcinoma (primary liver cancer), hepatoblastoma, metastatic liver cancer, Yorksack tumor and Α-fetoprotein (AFP), an abnormal prothrombin that appears in liver parenchymal disorders, PIVKA-II, which is confirmed to appear specifically in hepatocellular carcinoma, and a glycoprotein that is immunohistochemically a breast cancer-specific antigen BCA225, a marker of primary advanced breast cancer, recurrent / metastatic breast cancer, a basic fetal protein found in human fetal serum, intestinal and brain tissue extracts, ovarian cancer, testicular tumor, prostate cancer, pancreatic cancer, biliary tract It is a carbohydrate antigen that is a marker for basic fetoprotein (BFP), a marker for cancer, hepatocellular carcinoma, kidney cancer, lung cancer, stomach cancer, bladder cancer, colon cancer, advanced breast cancer, recurrent breast cancer, primary breast cancer, and ovarian cancer. CA15-3, pancreatic cancer, gallbladder CA19-9, a sugar chain antigen that serves as a marker for cancer, stomach cancer, liver cancer, colon cancer, ovarian cancer, CA72-4, a sugar chain antigen that serves as a marker for ovarian cancer, breast cancer, colorectal cancer, stomach cancer, pancreatic cancer, CA125, epithelial ovarian cancer, egg, which is a glycan antigen marker for ovarian cancer (especially serous cystadenocarcinoma), endometrial adenocarcinoma, fallopian tube cancer, cervical adenocarcinoma, pancreatic cancer, lung cancer, colon cancer CA130, a glycoprotein that serves as a marker for duct cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, core protein antigen that serves as a marker for ovarian cancer (especially serous cystadenocarcinoma), endometrial adenocarcinoma, cervical adenocarcinoma CA602, ovarian cancer (especially mucinous cystadenocarcinoma), cervical adenocarcinoma, CA54 / 61 (CA546), a nucleoside sugar-related antigen that is a marker for endometrial adenocarcinoma, colon cancer, stomach cancer, rectal cancer, Currently, it is the most widely used to assist cancer diagnosis as a tumor-related marker antigen for biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, urinary tract cancer, etc. Carcinoembryonic antigen (CEA), pancreatic cancer, biliary tract cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, sugar chain antigen that is a marker for colon cancer, DUPAN-2,
Elastase 1, a pancreatic exocrine proteolytic enzyme that exists in the pancreas and specifically hydrolyzes the elastic fibers of the connective tissue, elastin (which constitutes arterial walls and tendons), and serves as a marker for pancreatic cancer, pancreatic sac cancer, and biliary tract cancer.
Glycoprotein present in high concentrations in ascites and serum of human cancer patients, lung cancer, leukemia, esophageal cancer, pancreatic cancer, ovarian cancer, renal cancer, bile duct cancer, stomach cancer, bladder cancer, colon cancer, thyroid cancer, malignant lymphoma Immunosuppressive acidic protein (IAP), which is a marker of pancreatic cancer, biliary tract cancer, breast cancer, colon cancer, hepatocellular carcinoma, lung adenocarcinoma, gastric cancer, NCC-ST-439, a sugar chain antigen that is a marker for prostate cancer, and a marker for prostate cancer Γ-seminoprotein (γ-Sm), a glycoprotein extracted from human prostate tissue, and only present in prostate tissue, and therefore a prostate-specific antigen (PSA) that is a marker for prostate cancer , An enzyme that hydrolyzes phosphate esters under acidic pH secreted from the prostate, and is a prostatic acid phosphatase (PAP) used as a tumor marker for prostate cancer, a glycolytic system that exists specifically in nerve tissue and neuroendocrine cells Enzyme and lung (Especially small cell lung cancer), neuroblastoma, nervous system tumor, pancreatic islet cancer, small cell carcinoma of the esophagus, gastric cancer, kidney cancer, neuronal specific enolase (NSE) as a marker for breast cancer, cervical squamous cell carcinoma Protein extracted and purified from liver metastases, squamous cell carcinoma associated antigen (SCC antigen) and lung gland that is a marker of uterine cancer (cervical squamous cell carcinoma), lung cancer, esophageal cancer, head and neck cancer, skin cancer Sialyl Le ^X- i antigen (SLX), a sugar chain antigen that is a marker for cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, colon cancer SPan-1, a glycan antigen that is a marker of esophageal cancer, gastric cancer, colorectal cancer, breast cancer, hepatocellular carcinoma, biliary tract cancer, pancreatic cancer, lung cancer, uterine cancer, especially in combination with other tumor markers Single-chain polypeptide useful for predicting advanced cancer and predicting recurrence and monitoring treatment Tissue polypeptide antigen (TPA), ovarian cancer, metastatic ovarian cancer, gastric cancer, colorectal cancer, biliary tract cancer, pancreatic cancer, lung cancer, sialyl Tn antigen (STN), which is a nucleoside sugar chain antigen marker, non-pulmonary Two types (PG I and PG II) of pephsin (cytokeratin; CYFRA), a tumor marker effective in the detection of small cell carcinoma, especially lung squamous cell carcinoma, and a protein digestive enzyme secreted in gastric juice Active precursor, gastric ulcer (especially lower gastric ulcer), duodenal ulcer (especially relapsed or refractory), Brunnell's adenoma, Zollinger-Ellison syndrome, pepsinogen (PG) as a marker of acute gastritis, plasma caused by tissue damage or infection C-reactive protein (CRP) that shows high levels when necrosis occurs in the myocardium due to acute myocardial infarction, etc. Acute phase reaction protein that changes in plasma due to tissue damage or infection Serum Amyloid A protein (SAA), a heme protein with a molecular weight of approximately 17500, mainly present in the heart muscle and skeletal muscle. Creatin kinase (CK) (skeletal skeleton) is an enzyme that exists mainly in the art and is released into the blood by cell damage. It is a marker for acute myocardial infarction, hypothyroidism, progressive muscular dystrophy, and polymyositis. CK-MM type derived from muscle, CK-BB type derived from brain and smooth muscle, CK-MB type derived from cardiac muscle, and CK (macro CK) combined with mitochondrial isozymes and immunoglobulins), horizontal It is a protein with a molecular weight of 39,000 that forms a troponin complex with troponin I and C on thin filaments of striated muscle, and is involved in the regulation of muscle contraction. It is associated with rhabdomyolysis, myocarditis, myocardial infarction, and renal failure. Ma -Troponin T, a protein contained in both skeletal muscle and myocardial cells, and an increase in the measurement result means skeletal muscle, myocardial damage or necrosis. And ventricular myosin light chain I.

  Examples of the bacteria contained in the target substance include bacteria to be subjected to cytomicrobiological examination. Examples include bacteria such as Escherichia coli, Salmonella and Legionella, and bacteria that cause problems in public health.

  Further, viruses contained in the target substance include hepatitis virus antigens such as antigens of C and B hepatitis viruses, HIV virus p24 protein antigen, CMV (cytomegalovirus) pp65 protein antigen, HPV (papillomavirus) E6. Or E7 protein etc. are mentioned.

  The detection of the target substance by the measurement probe of the present invention can be performed by measuring a signal based on the binding between the detection component held on the support and the target substance. The signal based on the binding between the detection component and the target substance is a signal that can be detected by the measurement probe while the target substance is held on the support surface by binding to the detection component held on the support surface. Therefore, it can be detected as a signal by an optical detection means, an electrical detection means or a magnetic detection means in which the support surface is set as a detection region. When the chromophore is retained by the target substance, the target substance may be directly labeled with the chromophore, or separately from the target substance, the chromophore is labeled with a substance that specifically binds to the target substance. It can also be held indirectly by the target substance. In the form in which the chromophore is indirectly held by the target substance, the same substance as the component for detection held on the support may be used as the substance that specifically binds to the target substance. For example, a method in which an antibody having a target substance as an antigen is held on a support and the same antibody is labeled with a chromophore to form a secondary antibody can be achieved by using a polyclonal antibody as the antibody. Since polyclonal antibodies include antibodies that recognize different regions of the same antigen, it is possible to bind the same antibody as a secondary antibody to the target substance captured by the antibody held as a detection component on the support. it can. As a chromophore that transmits a signal used for detection, a fluorescent chromophore can be preferably used. In the configuration using the optical waveguide as the support for the measurement probe, the fluorescent chromophore is excited by the excitation light emitted so as to be introduced into the optical waveguide from the light source being led to the surface of the optical waveguide.

  The measurement probe shown in the present embodiment can increase the strength against peeling of the protein layer, and can be suitably used under measurement conditions in which the flow rate of the liquid brought into contact with the probe, such as a specimen, a reagent, or a washing solution, is relatively large.

(Measurement probe manufacturing method)
Next, a method for manufacturing a measurement probe according to the present invention will be described. The method for producing a measurement probe of the present invention includes a step of cross-linking proteins constituting the protein-containing layer so that the protein-containing layer covers the support in an endless ring shape. In other words, this step is a step of forming, on the support, a layer containing proteins cross-linked with each other via a cross-linking agent so as to have a part surrounding the axis of the support. That is, an antibody against the target substance is immobilized on the side surface of the optical waveguide 3 of the measurement probe shown in FIGS. The fixing method is basically performed by immersing the measurement probe in a buffer solution in which the antibody is dissolved.

  The immobilization procedure may be a procedure in which a solution in which a protein and a cross-linking agent are mixed in advance is brought into contact with the support. The procedure of contacting the solution is preferred.

  The latter may have a speed disadvantage due to the solid-liquid reaction, but conversely, the crosslinking reaction is easy to control. Further, it is useful when the cross-linking reaction is difficult to be performed in the liquid phase due to the pH and concentration of the antibody solution as a raw material. There is also an advantage that removal of unreacted reagents and by-products is easy.

  In the case of the procedure of mixing in advance, the concentration of the crosslinking agent may be set so that a desired molar concentration ratio is obtained in the solution. In the case of the procedure of bringing the crosslinking agent solution into contact after physical adsorption, the protein concentration on the surface of the support (optical waveguide 3) after physical adsorption is calculated in advance, and a desired molar concentration ratio is set for this. Good. The protein concentration on the surface of the support (optical waveguide 3) can be calculated from, for example, the mass and absorbance of the adsorbed protein and the volume of the adsorption layer. The volume of the adsorption layer can be calculated from the covering area of the support (optical waveguide 3) and the thickness of the adsorption layer. The thickness of the adsorption layer can be determined by actual measurement using an observation means such as an electron microscope or by estimation from the theoretical size of the protein molecule. The optimum molar concentration ratio between these various amounts and the crosslinking agent is preferably confirmed in advance by experiments.

  If the antibody buffer concentration at the time of antibody immobilization is too high, antibody aggregation will occur, and if it is too low, the amount of antibody immobilized on the probe surface will be limited. Therefore, it is usually prepared in the range of 1 μg / mL to 100 μg / mL. Things are desirable. In addition, the immersion conditions are such that the temperature is low enough not to denature the antibody as a protein, that is, about 1 ° C. to 10 ° C., usually about 4 ° C. In this case, the immersion time is 15 to 30 hours, usually about 24 hours.

  The amount of antibody immobilized on the probe can be estimated by studying the difference in antibody concentration in the antibody buffer before and after immersion. For the antibody concentration in the antibody buffer, a protein quantification method such as the normal Lowry method or BCA method is used.

  The surface antibody concentration is calculated from the amount of the fixed antibody thus obtained. In this case, since the size of one molecule of antibody (immunoglobulin G; IgG) is usually about 10 nm, the volume is calculated based on the assumption that a 10 nm layer covers the periphery of the probe.

  A crosslinking agent is added at an appropriate ratio to the surface antibody concentration determined by the above conversion. The addition method is performed by immersing in a buffer solution in which the crosslinking agent is dissolved. In this case, the solid-phase antibody may be denatured when immersed for a long time. On the other hand, there is a possibility that sufficient addition of the crosslinking agent may not be performed in a short time, so the immersion time is about 10 to 60 minutes. The temperature is preferably about 20 ° C to 30 ° C. More preferably, the crosslinking reaction is carried out so that the molar concentration of the crosslinking agent is 1 to 5 times the molar concentration of the surface antibody. A layer containing a protein containing at least one selected from the group consisting of an antibody, an antigenic protein, an enzyme or a receptor, which is a component for detecting the target substance, as at least a part of a protein constituting the layer containing a protein Can be formed.

(Measurement method using measurement probe)
Below, the measuring apparatus using the measuring probe of this invention is demonstrated in detail.

  FIG. 4 shows a schematic diagram of the measurement principle of the present invention. The beam splitter 2 is disposed on the optical path of the light beam emitted from the light source 1, the rod-shaped optical waveguide 3 is disposed along the optical path in the reflection direction of the beam splitter 2, and the beam splitter 2 on the optical path toward the optical waveguide 3 The detector 4 is provided on the opposite side. 4 schematically shows an example, and the relative arrangement and types of the light source 1, beam splitter 2, optical waveguide 3, and detector 4 are not limited to those shown in FIG. .

  The light beam emitted from the light source 1 is reflected by the beam splitter 2 and introduced into the optical waveguide 3 as total internal reflection light, and an evanescent wave is generated on the surface thereof. The fluorescence emitted from the fluorescently labeled antibody excited by the evanescent wave on the surface of the optical waveguide 3 returns through the optical waveguide 3, passes through the beam splitter 2, and is measured by the detector 4.

  In the optical system shown in FIG. 4, the optical waveguide 3 which is the sensor portion performs the guiding of the excitation light and the collection of the fluorescence together, so that the entire optical system can be miniaturized, and it is easy to ensure stability and maintain. Will also be easier.

  In addition, since the specimen is located outside the optical waveguide 3, various operations such as immersion and cleaning of the optical waveguide 3 are easily performed.

  In order to prevent the reflected light and the scattered light of the excitation light from being mixed and collected in the fluorescence that is the signal from the label, a black body is disposed on the non-observation side end face of the optical waveguide 3 or an optical filter is provided. May be introduced.

  When mixing of excitation light becomes a problem when dealing with a very small amount of fluorescence, the time-dependent change of antigen-antibody reaction is used as a method for separating unnecessary components other than fluorescence as a signal from the label. That is, the method utilizes the fact that only the amount of fluorescence from the fluorescently labeled antibody that increases based on the antigen-antibody reaction in the amount of light that is observed increases according to the contact time with the specimen. As a measurement using this method, the amount of fluorescence from the labeled antibody can be measured for each unit contact time with a predetermined contact time as a unit. The target substance can be detected from the amount of change in fluorescence obtained by repeating this specimen contact and fluorescence measurement a plurality of times (specified number of times) and comparing the fluorescence amount at each measurement. Obtained by subtracting the amount of fluorescence when the number of contacts is one less than the predetermined number of times (hereinafter referred to as prior fluorescence amount) from the amount of fluorescence of the labeled antibody when contacted with the specimen for a unit contact time a predetermined number of times. The obtained value is defined as the amount of change per unit contact time at the time of a predetermined number of contacts. You may use the value calculated | required as a ratio (henceforth a rate of change per unit contact time) with respect to the amount of prior fluorescence as an index which shows the change of the amount of fluorescence of a labeled antibody.

  FIG. 5 shows a flowchart of an example of the above-described measurement procedure, and FIG. 6 shows an operation flowchart in actual measurement, background measurement, and blank measurement. As shown in FIG. 5 as a basic measurement procedure, first, the optical waveguide 3 and the specimen are brought into contact with each other in step S1, the target substance in the specimen is brought into contact with the surface of the optical waveguide 3, and then the contact is made in step S2. Is released. In step S3, a labeled antibody having a fluorescent chromophore is brought into contact with and bound to the captured target substance, and then contact is released in step S4.

  Subsequently, in step S5, excitation light is introduced into the optical waveguide 3 to form total internal reflection light, an evanescent wave is generated on the surface of the optical waveguide 3, and the fluorescent chromophore is excited. Measure the light that propagates through 3 and is collected. Subsequently, after performing signal processing in step S7, it is stored in the memory in step S8.

  FIG. 6 shows how each measurement is combined, that is, the flow of the entire measurement. The “slope comparison calculation” in the figure is an operation for obtaining the rate of change in actual measurement and blank measurement and subtracting the rate of change in blank measurement from the rate of change in actual measurement. In actual measurement and blank measurement, the operation is the same only by the difference in specimen. In the background measurement, operations related to the specimen are omitted, but the other operations are the same. After contact between the optical waveguide 3 and each specimen or labeled antibody solution, washing with an appropriate liquid may be performed before proceeding to the next operation. As the rate of change, the rate of change per unit contact time described above can be used. The same applies to the following steps.

  After this series of steps, the change rate of the observation light quantity with respect to the contact time between the optical waveguide 3 and the specimen is taken as the measurement value. In the above repeating process, when the observation light quantity does not increase monotonously and starts to decrease from a certain point, for example, this is abnormal and a warning is issued to the operator. This step is preferably repeated at least twice in order to obtain a more stable signal.

  Further, in the above measurement procedure, when non-specific adsorption that does not depend on the antigen-antibody reaction of the labeled antibody is strong, an increase in fluorescence due to this may be observed. If there is such a fear, it is desirable to contact only with the labeled antibody prior to the actual measurement to saturate the nonspecific adsorption in advance, and the background measurement in step S11 is performed. Subsequently, in step S12, an increase in fluorescence due to non-specific adsorption is determined. If it is significantly large, the measurement is interrupted and a warning is issued to the operator.

  When there is a possibility that a fluorescent impurity exists in the specimen, the measurement is performed as follows. Prior to the actual measurement in step S13, the measurement of the blank sample which is a reference sample not containing the target substance, that is, the blank measurement is performed through the same measurement procedure as the actual measurement, and then the actual measurement is performed in step S14. This operation is also effective when the non-specific adsorption component once bound is eluted for some reason.

  Further, in step S15, the change rate with respect to the contact time of the fluorescence amount obtained in the blank measurement is subtracted from the change rate with respect to the contact time obtained in the actual measurement to obtain a net fluorescence change rate by performing a slope comparison calculation. Displayed in S16. The blank measurement described above performs the same contact time and the number of times of contact, that is, repetition, from the statistical point of view.

  The method for capturing the target substance on the surface of the optical waveguide 3 can use direct physical adsorption or capture with an adsorbent prepared on the surface in advance, but in particular, capture with an antibody immobilized on the surface in advance is more effective. It is desirable as a highly selective method.

  In applying the above measurement procedure, the sample and the blank sample are contacted by rotating the open-type container containing the sample and sticking it in a portion parallel to the rotation axis except on the rotation axis in the container. An apparatus configuration in which the optical waveguide 3 is placed is preferable.

  Next, more specific embodiments of the present invention will be described. FIG. 2 shows a side view of a measurement probe which is a fiber-type optical waveguide with a lens in which polystyrene resin is injection-molded in the same shape as that described in US Pat. No. 6,136,611, and FIG. 3 shows a front view thereof. ing. FIG. 7 is an explanatory diagram of the measurement method, and FIG. 8 is a block diagram of the detection unit.

  In the measurement probe 10 having the optical waveguide 3, a flange 11 is provided at one end of the optical waveguide 3, a convex lens 12 is disposed at the center of the flange 11, and the optical axis of the convex lens 12 coincides with the central axis of the optical waveguide 3. . Further, a light absorbing portion 13 for suppressing end surface reflection is provided at the other end of the optical waveguide 3.

  The measurement probe 10 manufactured by the above-described manufacturing method uses a flange 11 between the standby unit 21, the sample action unit 22, the labeled antibody container 23, the sample container 24, and the measurement container 25 to insert an air suction type pad. It is moved by the robot arm 26 included in In the sample action unit 22, the measurement probe 10 is suspended in the sample injected into the sample container 24 to capture the target substance. At this time, it is preferable that the specimen container 24 is rotated by a motor to facilitate capture.

  The light quantity measurement is performed by fitting the optical system of the detection unit 27 to the upper part of the measurement container 25 in which the measurement probe 10 is set. In the measurement container 25, the measurement probe 10 is washed, laser light of 635 nm is introduced by a semiconductor laser light source, and fluorescence is condensed. A water supply pipe 30 to which a buffer reservoir tank 28 and a liquid feed pump 29 are connected and a drain pipe 31 are connected above and below the side of the measurement container 25.

  The detection unit 27 includes an optical fiber 32, a dye filter 33, a lens 34, a dichroic filter 35, a lens 36, and a photodiode in which light from a semiconductor laser light source (not shown) is guided from the measurement probe 10 side and the tip is cut by 45 degrees. 37 are arranged.

  The fluorescence generated by the target substance excited by the semiconductor laser light guided from the optical fiber 32 and captured by the measurement probe 10 enters the detection unit 27 from the convex lens 12 of the measurement probe 10 and passes through the optical system. It is detected by the photodiode 37. The obtained electric signal is amplified and digitized in the signal processing unit 38 and stored and calculated in the personal computer 50. Each drive command to the robot arm 26, the semiconductor laser light source, and the liquid feed pump 29 is also performed by the personal computer 50.

(Another specific configuration example of the detection device)
Hereinafter, a specific configuration example using the measurement probe of the present invention will be described.

  FIG. 9 shows the shape of a measurement probe that is one embodiment of the present invention. The measurement probe 1 has a fiber-like part 38, a flange 39, and a convex lens-like part 40.

  FIG. 10 is a schematic diagram showing a specific configuration of a measurement apparatus using the measurement probe.

  The flow cell 41 has an inlet 41a and an outlet 41b, and is configured to be set in a state where the fiber-like portion of the measurement probe 10 is arranged in the cell.

  As a measurement optical system, a plurality of lenses 42, a half mirror 43, a semiconductor laser 44, an optical filter 45, and a photodiode 46 are provided.

Laser light emitted from the semiconductor laser 44 is condensed using a plurality of lenses 42 and irradiated to a convex lens-shaped portion of the measurement probe. The signal light from the convex lens-shaped part is guided to the photodiode 46 by using the half mirror 43 and photometrically measured as the signal intensity.
The measurement probe according to the present invention will be described in more detail below using examples.

  An example in which the fluorescence immunization method using the measurement probe of the present invention is applied to a fiber type probe is shown. In addition, the following example is one of the more suitable forms of this invention, and does not limit the application range of this invention.

  The anti-PSA (prostate specific antigen) monoclonal antibody was diluted to 20 μg / mL with a phosphate buffer (0.01 mol / L, pH 7.4, the same applies hereinafter), and the fiber-like portion of the polystyrene transparent probe shown in FIG. Was immersed at 4 ° C. for 24 hours to immobilize the antibody by physical adsorption. The antibody was selected from a lot that had been previously known to be easily peeled off from the support. A probe on which the antibody was immobilized only by this physical adsorption was used as a control sample (hereinafter also referred to as a probe without crosslinking treatment).

The surface antibody concentration by physical adsorption was calculated from the decrease in antibody concentration of the immersed antibody solution. In a solution in which a plurality of probes were immersed, the antibody concentration before and after immersion was measured by a protein assay (manufactured by Bio-Rad), and the mass of the adsorbed antibody was determined by dividing the decrease by the number of probes (0.052 μg ). In addition, the area of the adsorption layer is set from the immersion length (35 mm) and the probe diameter (0.7 mm), and the thickness of the adsorption layer is set from the size of the antibody (single-layer adsorption, assuming 10 nm). It was determined (7.7 × 10 ⁻¹⁰ L). From the above values, the surface antibody concentration (average value) in the adsorption layer was estimated to be 4.5 × 10 ⁻⁴ mol / L.

  Plural probes were selected from the adsorption-immobilized probes, and immersed in a phosphate buffer solution containing 0.01% by volume (1 mmol / L, about 2.2 times molar concentration) of glutaraldehyde at 25 ° C. for 30 minutes. Subsequently, it was immersed in a tris (hydroxymethyl) aminomethane hydrochloride aqueous solution (1 mol / L, pH 7.4) at 25 ° C. for 1 hour. This was air-dried to obtain a crosslinked probe.

Measurements using these probes were performed using the apparatus shown in FIG. The apparatus shown in FIG. 10 includes an optical system for introducing semiconductor laser light into a probe and simultaneously observing light emitted from the probe, and a liquid feeding system for supplying a liquid to the probe surface. The following operation was performed on a 1 ng / mL PSA sample (phosphate buffer solution). Here, what is called a cleaning solution uses a phosphate buffer containing 0.1% by mass of polyoxyethylene (20) sorbitan monolaurate. The labeled antibody was prepared by allowing Cy5 bisfunctional reactive dye (Amersham Biosciences) to act on the same antibody as the capture antibody, and diluted with a phosphate buffer containing 1% by weight of bovine serum albumin to give a 2 μg / mL solution. .
(1) The sample is injected and discharged after 5 minutes.
(2) The cleaning liquid is injected, and the signal light is measured in the liquid immersion state (signal A).
(3) Drain the washing solution, inject the labeled antibody solution, and drain after 5 minutes.
(4) The cleaning liquid is injected, and the signal light is measured in the liquid immersion state (signal B).
(5) Subtract signal A from signal B to obtain a net measurement.

  A measurement value (average) of 29.8 picoamperes was obtained with the probe without crosslinking treatment and 173 picoamperes with the probe after crosslinking treatment, and an increase in sensitivity due to the crosslinking treatment of the antibody adsorption layer was confirmed.

  In addition, a phosphate buffer containing 0.5% by mass of polyoxyethylene (20) sorbitan monolaurate is continuously fed to the probe after signal detection at a flow rate of 10 mL / min for 5 minutes instead of the washing solution. The attenuation of signal intensity was monitored under conditions where peeling was more likely to occur. In the probe without crosslinking treatment, 82% signal attenuation was observed, but the signal attenuation with the crosslinking treatment probe was 50%, and it was confirmed that peeling was suppressed.

  When the cross-linking agent was replaced with bis (sulfosuccinimidyl) suberate, similar results were obtained by setting the reaction time to 1 hour.

It is the schematic which shows the example of the suitable shape in the measurement probe of this invention. (1) Fiber shape. (2) Sword mountain shape. (3) Mesh shape. (4) Non-linear. (5) Missing ring (C shape). It is a flowchart figure of a measurement procedure. It is an operation flowchart figure of an actual measurement, a background measurement, and a blank measurement. It is a side view of a measurement probe. It is a front view of a measurement probe. It is explanatory drawing of a measuring method. It is explanatory drawing of a measuring method. It is a block diagram of the detection part of a measuring method. It is the schematic which shows the shape of the measurement probe in an Example. It is the schematic which shows the structure of the measuring apparatus in an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Beam splitter 3 Optical waveguide 4 Detector 5 Convex lens 10 Measurement probe 11 Flange 12 Convex lens 13 Light absorption part 21 Waiting part 22 Sample action part 40
23 Labeled antibody container 24 Sample container 25 Measurement container 26 Robot arm 27 Detector 32 Optical fiber 33 Dye filter 34 Lens 35 Dichroic filter 36 Lens 37 Photodiode 50 Personal computer 38 Fiber-like part 39 Flange 40 Convex-lens-like part 41 Flow cell 41a Inlet 41b Discharge port 42 Lens 43 Half mirror 44 Semiconductor laser 45 Optical filter 46 Photo diode

Claims (14)

A measurement probe for detecting the presence or concentration of a target substance in a sample by contacting the sample,
A support having an optical waveguide comprising a fiber-shaped part, a flange part, and a convex lens-like part;
A layer formed by cross-linking between proteins, formed in an endless ring shape on the surface of the fiber-shaped part,
Measurement probe proteins that constitute the layer made by cross-linking of the protein to each other, characterized in that it is crosslinked by treatment with a crosslinking agent solution molarity of 1 to 5 times with respect to the protein. The measurement probe according to claim 1, comprising an antibody, an antigen protein, an enzyme, or a receptor, which is a component for detecting the target substance, as at least a part of a protein constituting a layer formed by cross-linking of the proteins. The measurement probe according to claim 1, wherein a component formed by cross-linking of the proteins is used as a base, and the target substance detection component is held in the layer.   The measurement probe according to claim 3, wherein the detection component includes at least one selected from an antibody, an antigen, an enzyme, and a receptor.   The measurement probe according to any one of claims 2 to 4, wherein the presence or absence or concentration of the target substance is detected by detecting a signal based on the binding between the detection component and the target substance.   The measurement probe according to claim 5, wherein the signal is a color developed from a chromophore held by the target substance.   The measurement probe according to claim 6, wherein the chromophore is a fluorescent chromophore, and the fluorescent chromophore is excited by excitation light introduced into the optical waveguide and led to the surface of the optical waveguide.   The measurement probe according to claim 1, wherein the cross-linking agent is a divalent cross-linking agent, and the functional group for cross-linking is an aldehyde, a carboxylic acid, or a carboxylic acid ester.   The measurement probe according to claim 8, wherein the cross-linking agent is glutaraldehyde. A method for producing a measurement probe for contacting a specimen to detect a target substance in the specimen,
A layer formed by cross-linking proteins with each other via a cross-linking agent on the fiber-shaped portion of the support composed of a fiber-shaped portion, a flange portion, and a convex lens-shaped portion, encircles one axis of the support. Forming a portion having
Production of a measurement probe characterized in that cross-linking of proteins constituting a layer formed by cross-linking of the proteins is performed by treatment with a cross-linking agent solution having a molar concentration of 1 to 5 times the protein. Method. As at least a part of a protein constituting a layer formed by cross-linking the proteins, at least one selected from the group consisting of an antibody, an antigen protein, an enzyme and a receptor, which is a component for detecting the target substance, is included The manufacturing method according to claim 10. The manufacturing method according to claim 10, further comprising a step of holding a component for detecting the target substance in the layer using a layer formed by crosslinking the proteins as a base.   The production method according to claim 12, comprising at least one selected from an antibody, an antigen, an enzyme, and a receptor as the detection component.   The manufacturing method according to claim 12, wherein only the fiber-shaped portion of the support is immersed in the crosslinking agent solution.

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