WO2020007134A1 - 光纤免疫传感器、检测装置及光纤免疫传感器的制作方法 - Google Patents

光纤免疫传感器、检测装置及光纤免疫传感器的制作方法 Download PDF

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WO2020007134A1
WO2020007134A1 PCT/CN2019/088479 CN2019088479W WO2020007134A1 WO 2020007134 A1 WO2020007134 A1 WO 2020007134A1 CN 2019088479 W CN2019088479 W CN 2019088479W WO 2020007134 A1 WO2020007134 A1 WO 2020007134A1
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optical fiber
layer
metal film
immunosensor
monoclonal antibody
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PCT/CN2019/088479
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English (en)
French (fr)
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赵静
朱勇奇
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深圳信息职业技术学院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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  • the invention belongs to the technical field of immune disease detection, and more particularly, relates to a fiber-optic immunosensor, a detection device, and a method for manufacturing a fiber-optic immunosensor.
  • Tumor markers are chemical substances secreted by tumor microenvironmental stromal cells and tumor cells. They exist in cells, tissues, and body fluids and can be quantitatively detected by chemical or immune response methods. Generally, the more vigorous the tumor growth, the higher the level of tumor marker concentration; conversely, when the tumor growth is suppressed, its concentration expression decreases. Detection of tumor markers can detect tumor lesions earlier than imaging methods such as CT and MRI, which is an indispensable method for early diagnosis.
  • the detection of single markers for early tumor diagnosis still has the following weaknesses: first, the concentration of some tumor serum markers has not changed significantly in the early stage, and it has increased significantly in the middle and late stages; second, the concentration of markers has increased There is a limit to the inevitable correlation between high and tumor. Its increase does not necessarily represent the occurrence or recurrence of cancer. Third, an increase in a tumor marker can occur in a variety of tumor diseases, and further targeted an examination.
  • Mesothelin is a glycoprotein that exists on the surface of peritoneal, pleural, and pericardial mesothelial lining cells. It is a common biological indicator in the blood and urine of patients with early pancreatic cancer.
  • mesothelin is expressed in several tumors, for example, mesothelin is expressed in about 70% of all ovarian cancers, about 82% in papillary serous adenocarcinomas, about 83% in all pancreatic cancers, and all Pancreatic ductal adenocarcinoma accounts for approximately 86%.
  • Mesothelin has become a highly specific new marker for detecting malignant tumors such as pancreatic cancer.
  • Non-hazardous and label-free immune biosensor technology is a new detection technology gradually developed internationally, and Surface Plasmon Resonance (SPR: Surface Plasmon Resonance) biosensors are one of them.
  • SPR Surface Plasmon Resonance
  • the advantages of SPR sensors without labeling are: first, the detection method is simple, there is no need to mark the test object, saving time and effort, and real-time and dynamic monitoring of the immune response can be performed without damage; second, the detection signal comes directly from the immune response The real-time recording of the binding and analysis of the reaction helps to study the interaction between antibody and antigen molecules, and provides new possibilities for the dynamic study of complex reaction mechanisms.
  • SPR surface plasmon resonance
  • BIAcore series SPR detection equipment uses optical prisms (not optical fibers) as the optical coupling medium, which is bulky and expensive, and its promotion and application are limited.
  • Optical fiber sensing technology provides an effective way for miniaturization, high sensitivity, and real-time rapid detection applications.
  • Optical fiber sensing uses light as the detection signal and optical fiber as the transmission medium.
  • the parameters such as the amplitude, wavelength, and polarization state of the transmitted light are affected by external variables to achieve real-time detection of external factors. It has high sensitivity, fastness, and resistance to electromagnetic interference. Powerful, can be remote monitoring and other significant advantages.
  • the method is simple, sensitive, and specific, and it can provide rapid qualitative detection, but it cannot accurately and quantitatively measure the concentration of the marker.
  • the object of the present invention is to provide a fiber-optic immunosensor to solve the technical problem that the tumor markers cannot be detected in real time and accurately.
  • an optical fiber immunosensor including an optical fiber, an excitation metal film, a monoclonal antibody layer, and a molecular self-assembly for assembling the monoclonal antibody layer on the excitation metal film.
  • Floor
  • a groove is provided on the side of the optical fiber, and the bottom wall of the groove is adjacent to the core of the optical fiber;
  • the exciting metal film is deposited on the bottom wall of the groove
  • the molecular self-assembly layer is self-assembled on the excitation metal film
  • the monoclonal antibody layer is coated on the molecular self-assembly layer after the activation treatment.
  • the distance from the bottom wall of the groove to the core of the optical fiber is 1 to 2 ⁇ m.
  • the excitation metal film is a gold film or a silver film, and the thickness of the excitation metal film is 40-50 ⁇ m.
  • the molecular self-assembly layer is a graphene nanofilm layer or a graphene oxide nanofilm layer.
  • first connection layer is deposited on the bottom wall of the groove, and the excitation metal film is deposited on the first connection layer.
  • the first connection layer is a chromium film with a thickness of 2-3 nm.
  • connection layer is self-assembled on the excitation metal film
  • molecular self-assembly layer is self-assembled on the second connection layer
  • the monoclonal antibody layer is composed of a monoclonal antibody of a tumor marker.
  • the beneficial effect of the optical fiber immunosensor provided by the present invention is that compared with the prior art, an excitation metal film is deposited on the optical fiber to form an optical fiber SPR sensor, and a monoclonal antibody layer assembled on the excitation metal film through a molecular self-assembly layer is realized.
  • Optical fiber SPR sensor antibody modification the application of optical fiber to the SPR biosensor can quantitatively detect the immune disease marker-antigen through specific immune response, and integrates the real-time, accurate, lightweight, and strong anti-interference ability of optical fiber sensing technology and
  • the advantages such as network connectivity and the advantages of SPR sensing technology, such as no labeling and high sensitivity, enable high-sensitivity, real-time and convenient detection of immune disease markers; the bottom wall of the groove is close to the core of the optical fiber, which is beneficial for obtaining optical fiber. Qiang Qiang passed away.
  • Another object of the present invention is to provide a detection device for detecting immunological diseases, so as to solve the technical problem that cannot detect tumor markers in real time and accurately.
  • a detection device for detecting immune diseases includes a broadband light source, a fiber optic spectrometer, a terminal, and a fiber optic immunosensor.
  • the broadband light source and the optical fiber spectrometer, and the terminal is communicatively connected with the optical fiber spectrometer.
  • the detection device provided by the present invention for the detection of immune diseases has the beneficial effect that, compared with the prior art, a broadband light source provides a covered input light for a fiber-optic immunosensor, and a light signal enters the fiber-optic immunosensor and is in the sensing area to be detected.
  • the liquid sample is measured for light-material interaction.
  • the output light signal is collected by a fiber optic spectrometer.
  • the terminal installs data processing software.
  • the data collected by the fiber spectrometer is calculated to obtain the resonance peak of the output light.
  • the wavelength of the resonance peak can quantitatively reflect the immune disease markers. Concentration value; rapid detection of tumor markers in liquid samples without labeling, non-destructive, real-time, and accurate, and can be further applied to the Internet of Things that require networked and remote detection.
  • the third object of the present invention is to provide a method for manufacturing an optical fiber immunosensor to solve the technical problem that the tumor marker cannot be detected in real time and accurately.
  • the technical solution adopted by the present invention is: a method for manufacturing an optical fiber immunosensor, including the following steps:
  • a groove is processed on the side of the optical fiber, and the bottom wall of the groove is adjacent to the core of the optical fiber;
  • a monoclonal antibody is added dropwise to the molecular self-assembly layer, and an amino group in the monoclonal antibody molecule undergoes an amide reaction with an activated carboxyl group on the surface of the molecular self-assembly layer, so that the monoclonal antibody is assembled and fixed on the molecular self-assembly layer.
  • the molecular self-assembly layer forms a monoclonal antibody layer.
  • the manufacturing method of the optical fiber immunosensor provided by the present invention has the beneficial effect that, compared with the prior art, the excitation metal film and the monoclonal antibody layer are integrated on the optical fiber through a simple process, and a highly selective and sensitive optical fiber immunity is prepared.
  • the sensor has high assembly efficiency.
  • the molecular self-assembly layer can protect the excitation metal film, and the excitation metal film and the monoclonal antibody layer are firmly attached to the optical fiber to avoid performance degradation.
  • FIG. 1 is a schematic diagram of a detection state of an optical fiber immunosensor provided by an embodiment of the present invention
  • FIG. 2 is a schematic structural diagram of a detection device for detecting an immune disease according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a manufacturing and detection process of an optical fiber immunosensor according to an embodiment of the present invention.
  • An optical fiber immunosensor 1 includes an optical fiber 11, an excitation metal film 12, a monoclonal antibody layer 13, and a molecular self-assembly layer 14 for assembling the monoclonal antibody layer 13 on the excitation metal film 12.
  • the side of the optical fiber 11 is provided with a groove 111, and a bottom wall of the groove 111 is adjacent to the core 113 of the optical fiber 11;
  • the excitation metal film 12 is deposited on the bottom wall of the groove 111;
  • the molecular self-assembly layer 14 self-assembles on the excitation metal film 12;
  • the monoclonal antibody layer 13 is coated on the molecular self-assembly layer 14 after the activation treatment.
  • the detection principle of the optical fiber immunosensor of the present invention is: the antibody 131 of the monoclonal antibody layer 13 specifically binds to the antigen 17 in the test sample, and the surface refractive index at the monoclonal antibody layer 13 of the optical fiber immunosensor 1 changes immediately, resulting in an optical fiber
  • the surface plasmon resonance resonance peak wavelength detected by the end test of the immunosensor 1 significantly shifts. Therefore, the amount of the wavelength shift of the resonance peak can be used to quantitatively reflect the change in the concentration of the antigen in the test sample.
  • the optical fiber immunosensor Compared with the prior art, the optical fiber immunosensor provided by the present invention deposits an excitation metal film 12 on the optical fiber 11 to form an optical fiber SPR sensor, and is implemented by a molecular self-assembly layer 14 assembled on the monoclonal antibody layer 13 on the excitation metal film 12
  • the antibody modification of optical fiber SPR sensor is applied; the application of optical fiber 11 to SPR biosensor can quantitatively detect the immune disease marker-antigen through specific immune response, and integrates the real-time, accurate, lightweight and anti-interference ability of optical fiber sensing technology
  • Strong and network-connected advantages, SPR sensing technology requires no labeling and high sensitivity, which enables high-sensitivity, real-time and convenient detection of immune disease markers; the bottom wall of the groove 111 is adjacent to the core 113 of the optical fiber 11, which is beneficial A strong evanescent field on the optical fiber 11 is obtained.
  • the distance from the bottom wall of the groove 111 to the core 113 of the optical fiber 11 is 1 to 2 ⁇ m, which is beneficial to obtain a strong evanescent field on the optical fiber 11. Improve the sensitivity of immune disease marker detection.
  • the excitation metal film 12 is a gold film or a silver film.
  • Both gold and silver films can excite SPR resonance peaks; gold films have stable chemical properties and can be directly modified biologically.
  • the disadvantage is that gold is expensive and the excited SPR resonance peaks are not as high resolution as silver films; silver films can be used to obtain Higher resolution SPR resonance peak, and the price is much cheaper than gold film;
  • the gold membrane is not efficient in assembling biomolecules directly.
  • the provision of a molecular self-assembly layer 14 can improve the assembly efficiency of antibodies;
  • the silver film is susceptible to chemical reactions such as oxidation and sulfuration in the air, which affects the stability of the optical fiber immunosensor 1.
  • the provision of a molecular self-assembly layer 14 can protect the silver film on the one hand, and ensure the assembly efficiency of the antibody on the other hand, thereby obtaining Fiber optic immunosensor 1 with high performance and relatively low cost.
  • the thickness of the excited metal film 12 is 40-50 ⁇ m, and a higher resolution SPR resonance peak is obtained, especially when the thickness of the silver film is 40-50 ⁇ m The resolution of the SPR resonance peak is significantly improved.
  • the molecular self-assembly layer 14 is a graphene nanofilm layer or a graphene oxide nanofilm layer, which has stable chemical properties, a large specific surface area, and adsorption of biomolecules. Stronger force, its six-membered carbon ring structure is more likely to combine with organic molecules to form a large ⁇ bond accumulation.
  • graphene nanofilm layers or graphene oxide nanofilm layers can achieve higher The biomolecule assembly efficiency improves the detection sensitivity and detection limit of the optical fiber immunosensor 1.
  • the molecular self-assembly layer 14 is a single-layer or oligo-layer graphene oxide film layer with a thickness of 1 nm to 3 nm, and the optical fiber immunosensor 1 has higher detection sensitivity.
  • the optical fiber immunosensor further includes a first connection layer, the first connection layer is deposited on the bottom wall of the groove 111, and the excitation metal film 12 is deposited on the first connection layer.
  • the first connection layer mainly can increase the adhesion between the excitation metal film 12 and the optical fiber 11, and avoid peeling and peeling of the excitation metal film 12.
  • the first connection layer is preferably a chromium film with a thickness of 2-3 nm.
  • the adhesion between the gold film (or silver film) and the optical fiber 11 can be greatly increased by the chromium transition.
  • the fiber-optic immunosensor as a specific embodiment of the fiber-optic immunosensor provided by the present invention, it further includes a second connection layer 16, the second connection layer 16 self-assembles on the excitation metal film 12, and the molecular self-assembly layer 14 Self-assembled on the second connection layer 16.
  • the second connection layer 16 provides protection for the excited metal film 12 (especially the silver film with poor chemical stability in the air), and on the other hand, the second connection layer 16 serves as a transition layer to connect the molecular self-assembly on the next layer.
  • the layer 14 improves the adhesion between the excited metal film 12 and the molecular self-assembled layer 14.
  • the excitation metal film 12 is a gold film or a silver film
  • the molecular self-assembly layer 14 is a graphene oxide layer
  • the second connection layer 16 adopts nano-octadecanethiol (ODT) with a thickness of nanometer order
  • the ODT containing a mercapto group is used as the connection layer between the gold film and the graphene oxide film layer to realize the alkylation of the gold film.
  • the gold film can also be alkylated by other methods; using the ODT containing a mercapto group as the gold film and The connection layer between the graphene oxide film layers, the ODT with a thickness of nanometers is self-assembled on the silver film through the Ag-S covalent bond, and a dense protective layer is formed on the silver film, which can effectively overcome the silver film
  • the shortcoming of the optical fiber immunosensor 1 is easy to degrade, and at the same time, the adhesion between the excited metal film 12 and the molecular self-assembly layer 14 is improved.
  • the monoclonal antibody layer 13 is composed of a monoclonal antibody of a tumor marker, such as mesothelin. Can achieve tumor diagnosis.
  • a detection device for detecting immune diseases includes a broadband light source 2, a fiber optic spectrometer, a terminal 4, and the fiber optic immunosensor 1 described in any of the foregoing embodiments.
  • the two ends of the fiber optic immunosensor 1 are respectively connected to the broadband light source 2 by connecting optical fibers 5.
  • optical fiber spectrometer, the terminal 4 is in communication connection with the optical fiber spectrometer.
  • the broadband light source 2 provides the input light (for example, a wavelength range of 400nm to 1100nm) for the optical fiber immunosensor 1.
  • the optical signal enters the optical fiber immunosensor 1 and interacts with the liquid sample to be measured in the sensing area.
  • the output optical signal is Fiber optic spectrometer acquisition, terminal 4 (such as a computer) installs data processing software, and calculates the resonance peak of the output light by calculating the data collected by the fiber optic spectrometer.
  • the wavelength of the resonance peak can quantitatively reflect the immune disease markers (such as tumor markers-mesothelin ) Concentration value; no marking, non-destructive, real-time, accurate detection of tumor markers in liquid samples (such as blood or urine), and it can be further applied to the Internet of Things that requires networked and remote detection.
  • immune disease markers such as tumor markers-mesothelin
  • the manufacturing method of the optical fiber immunosensor includes the following steps:
  • a groove 111 is processed on the side of the optical fiber 11, and the bottom wall of the groove 111 is adjacent to the core 113 of the optical fiber 11;
  • a layer of excitation metal film 12 is evaporated or sputtered on the bottom wall of the groove 111;
  • step S4 Activate the molecular self-assembly layer 14 so that the surface of the molecular self-assembly layer 14 forms an activated carboxyl group; (step S4 is not shown)
  • the monoclonal antibody is added dropwise to the molecular self-assembly layer 14.
  • the amino group in the monoclonal antibody molecule undergoes an amide reaction with the activated carboxyl group on the surface of the molecular self-assembly layer 14, so that the monoclonal antibody is assembled and fixed on the molecular self-assembly layer 14 on top of which a monoclonal antibody layer 13 is formed.
  • the manufacturing method of the optical fiber immunosensor can be used for manufacturing the optical fiber immunosensor 1 described in any one of the foregoing embodiments.
  • the excitation metal film 12 and the monoclonal antibody layer 13 are integrated on the optical fiber 11 through a simple process, and a highly selective and sensitive optical fiber immunosensor 1 is prepared.
  • the assembly efficiency is high, and the molecular self-assembly layer 14 can excite the metal film. 12 for protection, and the excitation metal film 12 and the monoclonal antibody layer 13 are firmly attached to the optical fiber 11 to avoid performance degradation.
  • step S1 includes: removing a portion of the cladding 112 of the optical fiber 11 by a processing method of polishing the optical fiber 11 on the side until the polishing surface is close to
  • the core 113 of the optical fiber 11 is formed with a groove 111 formed by removing a part of the cladding.
  • the groove 111 can also be processed by other methods.
  • the single-mode optical fiber is coarsely ground, finely ground, and finely polished by using 3,000-mesh, 7000-mesh, and 12000-mesh sandpaper in order to produce a side-polished optical fiber.
  • the polishing surface of the optical fiber 11 is 1 to 2 ⁇ m away from the core region (that is, the distance from the bottom wall of the groove 111 to the core 113 of the optical fiber 11 is 1 to 2 ⁇ m). It is beneficial to obtain a strong evanescent field on the optical fiber 11.
  • step S2 includes: polishing the optical fiber 11 on the side, and applying the method of vacuum thermal evaporation or magnetron sputtering to the optical fiber 11 A layer of excitation metal film 12 is uniformly deposited on the polishing surface to obtain an optical fiber SPR sensor.
  • the excitation metal film 12 is a gold film or a silver film
  • a chromium film is deposited on the surface of the optical fiber 11, and then a gold film or a silver film is deposited.
  • the thickness of the chromium film may be specifically 2 to 3 nm.
  • step S3 includes:
  • step S31 when the excitation metal film 12 is a silver film, n-octadecanethiol (ODT) containing a mercapto group is used as silver.
  • ODT n-octadecanethiol
  • the second connection layer 16 between the film and the graphene oxide film layer self-assembles the ODT having a thickness on the order of nanometers on the silver film through an Ag-S covalent bond.
  • an ODT solution with a concentration of 10 mM is used to self-assemble the surface in a closed environment at normal temperature for 36 hours.
  • a thiol-containing ODT is used as the second connection layer 16 for intermediate transition, and a dense protective layer is formed on the silver film, which can effectively avoid the shortcoming of the silver-based optical fiber SPR sensor that is easily degraded.
  • step S32 the optical fiber SPR sensor with the ODT self-assembly completed in step S31 is placed in a graphene oxide sol and placed in a sealed room temperature Surface self-assembly is performed in the environment. After the assembly is completed, the ultrapure water is taken out for cleaning, and then dried at room temperature to obtain a sensitized optical fiber SPR sensor.
  • the self-assembly time of the graphene oxide film layer is preferably more than 40 minutes, and an oligo-layer graphene oxide nano film layer can be assembled on the second connection layer 16.
  • the biomolecules monoclonal antibodies—are assembled on the silver film by oligo graphene oxide.
  • the graphene oxide film layer provides further protection for the silver film on the one hand, and the graphene oxide has stable chemical properties and specific surface area on the other. Huge, strong adsorption of biomolecules, its six-membered carbocyclic structure is more likely to combine with organic molecules to form a large ⁇ bond accumulation.
  • the graphene oxide film layer can achieve higher biomolecule assembly efficiency, thereby improving the detection sensitivity and detection limit of the sensor.
  • the graphene oxide sol can be prepared by the following method:
  • N, N-dimethylformamide (DMF) as a solvent, dissolve the purchased commercial graphene oxide powder in it and stir well, place it in a five-sided vibration ultrasonic machine, and perform high-power ultrasound on the graphene oxide solution. dispersion.
  • the temperature in the water area is constant at about 25-30 ° C., and the ultrasonic wave is applied for 4 hours to obtain a better graphene oxide sheet peeling effect, and a stable graphene oxide sol system is obtained.
  • step S4 includes:
  • sensitized optical fiber SPR sensor prepared in step S3 into 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxypropionimide (NHS )
  • EDC 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxypropionimide
  • step S5 includes: drip-coating a monoclonal antibody specific to a tumor marker on the sensitized fiber-optic SPR sensor processed in step S4.
  • the sensing area is placed in a refrigerator at 4 ° C to dry and air dry, and a fiber-optic immunosensor 1 for detecting corresponding markers is prepared, which expands the new application of the fiber-optic 11 sensor in the field of biochemical detection.
  • mesotheliin is used as a detection target for markers of tumors such as pancreatic cancer and ovarian cancer, and a mesothelin monoclonal antibody is added dropwise to the sensing area.
  • the activated carboxyl group (-COOH) on the surface graphene oxide film layer reacts with the amino group (-NH2) in the monoclonal antibody molecule, so that the antibody is assembled and fixed on the surface of the sensor, realizing real-time, accurate and high specificity Biomolecule recognition.

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Abstract

一种光纤免疫传感器、包含该传感器的检测装置及制备方法，该传感器包括光纤(11)、激发金属膜(12)、分子自组装层(14)和单克隆抗体层(13)；激发金属膜(12)沉积于光纤(11)侧边的凹槽的底壁上；分子自组装层(14)自组装于激发金属膜(12)上；单克隆抗体层(13)涂于分子自组装层(14)上；该检测装置包括依次连接的宽带光源(2)、光纤免疫传感器、光纤光谱仪(3)和终端(4)；制作方法为在光纤的侧边加工出的凹槽上蒸镀或溅射激发金属膜；在激发金属膜上表面自组装分子自组装层并活化分子自组装层的表面的羧基；向分子自组装层上滴加单克隆抗体。上述传感器及其制备方法具有选择性高、反应灵敏，组装效率高，能够对激发金属膜进行保护，激发金属膜和单克隆抗体层牢固的附着在光纤上，避免性能发生退化的优点。

Description

光纤免疫传感器、检测装置及光纤免疫传感器的制作方法 技术领域
本发明属于免疫疾病检测技术领域，更具体地说，是涉及光纤免疫传感器、检测装置及光纤免疫传感器的制作方法。
背景技术
肿瘤的早期诊断是挽救患者生命的关键。
现有的肿瘤诊断技术中，除临床征象及影像学手段外，肿瘤标志物检测法日益被人们重视。肿瘤标志物是由肿瘤微环境基质细胞及肿瘤细胞分泌的化学物质，存在于细胞、组织、体液中，可通过化学或免疫反应的方法进行定量检测。通常，肿瘤生长越旺盛，肿瘤标志物浓度水平越高；反之，当肿瘤生长被压制时，其浓度表达减少。检测肿瘤标志物能比CT、MRI等影像学手段更早地发现肿瘤病灶，是早期诊断不可或缺的方法。
然而，通过检测单项标志物进行早期肿瘤诊断还存在以下弱点：其一，某些肿瘤血清标志物在早期的浓度并无大变化，到中晚期才有明显升高；其二，标志物浓度升高与肿瘤的必然相关性存在局限，其升高并不一定代表癌症的发生或复发；其三，一项肿瘤标志物的升高会发生在多种肿瘤疾病中，还需要进一步进行针对性的检查。肿瘤早期诊断为标志物检测提出了新的要求：其一，选择灵敏度、特异性更高的肿瘤标志物，即该标志物在某种肿瘤早期就有明显的上调表达；其二，不断完善检测手段，提高传感器灵敏度、准确性、检测极限，并能够灵活地对血清标志物进行实时、动态的监测。
间皮素(mesothelin)是存在于腹膜、胸膜和心包体腔间皮衬里细胞表面上的糖蛋白，是早期胰腺癌患者血液和尿液中常有的一种生物指标。此外，间皮 素在若干种肿瘤中有表达，例如，间皮素表达于所有卵巢癌的约占70％、乳头状浆液性腺癌的约占82％、所有胰腺癌的约占83％和所有胰腺导管腺癌的约占86％。间皮素成为胰腺癌等恶性肿瘤检测的高特异性新型标志物。
无危害、无需标记的免疫生物传感技术是国际上逐渐发展起来的新检测技术，表面等离子体共振(SPR：Surface Plasmon Resonance)生物传感器就是其中之一。无需标记的SPR传感器的优越性在于：其一，检测方法简单，无需对待测物进行标记，省时省力，可无损地对免疫反应进行实时、动态的监测；其二，检测信号直接来自免疫反应，实时记录反应的结合与解析过程，有助于研究抗体-抗原分子间的相互作用，为动态研究复杂反应机理提供新的可能。但现有的表面等离子体共振(SPR)检测设备，如通用电气公司BIAcore系列SPR检测设备，以光学棱镜(而非光纤)为光耦合介质，体积庞大、造价昂贵，其推广应用受到限制。
光纤传感技术为小型化、高灵敏度、实时的快速检测应用提供了一种有效途径。光纤传感以光为检测信号，以光纤为传输介质，利用传输光的振幅、波长、偏振态等参量受外界变量的影响，实现对外界因素的实时探测，具有高灵敏度、快速、抗电磁干扰力强、可进行远程监测等显著优点。结合光纤传感技术与表面等离子体共振检测技术的优势，研制出轻巧、柔韧、高灵敏度、响应迅速的光纤SPR传感器，是实现无标记、无损害、实时动态检测的优选方案，并可进一步适用于远程监测。
2014年，美国人Jack Andraka发明了一种检测间皮素的“癌症试纸”(WO2013172866A2)用于胰腺癌的早期筛查，其灵敏度、响应速度相对于现有的常规肿瘤检测技术显著提高。该“癌症试纸”将间皮素抗体通过碳纳米管固定在滤纸上，当间皮素抗原与其抗体结合发生特异性免疫反应，碳纳米管的导电率将发生改变，通过检测电信号即可间接反应间皮素浓度的变化；但该测癌试纸的导电性可能受其它非检测对象的因素影响，从而影响真实间皮素测试的准确性。
申请号为CN201610309318的发明专利申请，将CA19-9抗体与间皮素抗体通过胶体金等颗粒进行显色标记并固定在试纸上，制作出检测CA19-9与间皮素的双联检测试剂盒，用于胰腺癌的快速筛查。该方法操作简便、敏感度高、特异性强，能提供快速的定性检测，但无法准确定量测量标志物浓度。
然而，利用光纤传感技术与表面等离子体共振传感技术结合，用来实现间皮素等肿瘤标志物的免疫检测技术尚未见报道。现有检测肿瘤标志物的免疫生物传感技术无法实时检测、无法联网、无法进行远程监测，无法面向物联网应用中万物互连、实时监测的需求。
发明内容
本发明的目的在于提供一种光纤免疫传感器，以解决现有技术中存在的不能实时、准确检测肿瘤标志物的技术问题。
为实现上述目的，本发明采用的技术方案是：光纤免疫传感器，包括光纤、激发金属膜、单克隆抗体层和用于将所述单克隆抗体层组装于所述激发金属膜上的分子自组装层；
所述光纤的侧边设有凹槽，所述凹槽的底壁邻近所述光纤的纤芯；
所述激发金属膜沉积于所述凹槽的底壁上；
所述分子自组装层自组装于所述激发金属膜上；
所述单克隆抗体层涂于经活化处理后的所述分子自组装层上。
进一步地，所述凹槽的底壁到所述光纤的纤芯的距离为1～2μm。
进一步地，所述激发金属膜为金膜或银膜，所述激发金属膜的厚度为40～50μm。
进一步地，所述分子自组装层为石墨烯纳米膜层或氧化石墨烯纳米膜层。
进一步地，还包括第一连接层，所述第一连接层沉积于所述凹槽的底壁上，所述激发金属膜沉积于所述第一连接层上。
进一步地，所述第一连接层为铬膜，厚度为2-3nm。
进一步地，还包括第二连接层，所述第二连接层自组装于所述激发金属膜上，所述分子自组装层自组装于所述第二连接层上。
进一步地，所述单克隆抗体层由肿瘤标志物的单克隆抗体构成。
本发明提供的光纤免疫传感器的有益效果在于：与现有技术相比，在光纤上沉积激发金属膜，形成光纤SPR传感器，通过分子自组装层组装于激发金属膜上的单克隆抗体层实现了光纤SPR传感器的抗体修饰；将光纤应用到SPR生物传感器，能够通过特异性免疫反应对免疫疾病标志物—抗原进行定量检测，集成了光纤传感技术的实时、准确、轻巧、抗干扰能力强和可联网等优势及SPR传感技术的无需标记和灵敏度高等优势，能高灵敏度、实时及便捷地进行免疫疾病标志物检测；凹槽的底壁邻近所述光纤的纤芯，有利于获得光纤上的强倏逝场。
本发明的目的之二在于提供一种用于免疫性疾病检测的检测装置，以解决现有技术中存在的不能实时、准确检测肿瘤标志物的技术问题。
为实现上述目的，本发明采用的技术方案是：用于免疫性疾病检测的检测装置，包括宽带光源、光纤光谱仪、终端和光纤免疫传感器，所述光纤免疫传感器的两端分别通过连接光纤连接所述宽带光源和所述光纤光谱仪，所述终端与所述光纤光谱仪通信连接。
本发明提供的用于免疫性疾病检测的检测装置的有益效果在于：与现有技术相比，宽带光源为光纤免疫传感器提供覆盖的输入光，光信号进入光纤免疫传感器并在传感区域与待测液体样本发生光-物质作用，输出光信号由光纤光谱仪采集，终端安装数据处理软件，对光纤光谱仪采集的数据进行计算得到输出光的谐振峰，谐振峰的波长可定量反映免疫疾病标志物的浓度值；无需标记、无损、实时、准确地对液体样本中的肿瘤标志物进行快速检测，并可进一步应用于需要联网、远程检测的物联网中。
本发明的目的之三在于提供一种光纤免疫传感器的制作方法，以解决现有技术中存在的不能实时、准确检测肿瘤标志物的技术问题。
为实现上述目的，本发明采用的技术方案是：光纤免疫传感器的制作方法，包括如下步骤：
在光纤的侧边加工出凹槽，且所述凹槽的底壁邻近所述光纤的纤芯；
在所述凹槽的底壁上蒸镀或溅射一层激发金属膜；
在所述激发金属膜上表面自组装一层分子自组装层；
活化所述分子自组装层，使所述分子自组装层的表面形成活化羧基；
将单克隆抗体滴加于所述分子自组装层，所述单克隆抗体分子中的氨基与所述分子自组装层的表面的活化羧基发生酰胺反应，使得所述单克隆抗体被组装固定于所述分子自组装层上而形成单克隆抗体层。
本发明提供的光纤免疫传感器的制作方法的有益效果在于：与现有技术相比，通过简单工艺在光纤上集成了激发金属膜和单克隆抗体层，制得选择性高、反应灵敏的光纤免疫传感器，组装效率高，分子自组装层能够对激发金属膜进行保护，且激发金属膜和单克隆抗体层牢固的附着在光纤上，避免性能发生退化。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的一种光纤免疫传感器的检测状态示意图；
图2为本发明实施例提供的用于免疫性疾病检测的检测装置的结构示意图；
图3为本发明实施例提供的光纤免疫传感器的制作及检测流程的示意图。
其中，图中各附图标记：
1、光纤免疫传感器；
11、光纤；111、凹槽；12、激发金属膜；13、单克隆抗体层；131、抗体；14、分子自组装层；16、第二连接层；17、抗原；
2、宽带光源；3、光纤光谱仪；4、终端；5、连接光纤。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
需要说明的是，当元件被称为“固定于”或“设置于”另一个元件及类似用语，它可以直接在另一个元件上或者间接在该另一个元件上。当一个元件被称为是“连接于”另一个元件，它可以是直接连接到另一个元件或间接连接至该另一个元件上。
此外，术语“第一”、“第二”及类似用语仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。
请参阅图1，现对本发明提供的光纤免疫传感器进行说明。光纤免疫传感器1，包括光纤11、激发金属膜12、单克隆抗体层13和用于将单克隆抗体层13组装于激发金属膜12上的分子自组装层14；
光纤11的侧边设有凹槽111，凹槽111的底壁邻近光纤11的纤芯113；
激发金属膜12沉积于凹槽111的底壁上；
分子自组装层14自组装于激发金属膜12上；
单克隆抗体层13涂于经活化处理后的分子自组装层14上。
本发明光纤免疫传感器的检测原理为：单克隆抗体层13的抗体131与测试样本中的抗原17发生特异性结合，光纤免疫传感器1的单克隆抗体层13处的表面折射率立即改变，导致光纤免疫传感器1末端测试检测得到的表面等离子体共振谐振峰波长发生明显漂移，因此,可通过谐振峰的波长漂移量定量反应测试样本中抗原的浓度变化。
本发明提供的光纤免疫传感器，与现有技术相比，在光纤11上沉积激发金属膜12，形成光纤SPR传感器，通过分子自组装层14组装于激发金属膜12上的单克隆抗体层13实现了光纤SPR传感器的抗体修饰；将光纤11应用到SPR生物传感器，能够通过特异性免疫反应对免疫疾病标志物—抗原进行定量检测，集成了光纤传感技术的实时、准确、轻巧、抗干扰能力强和可联网等优势及SPR传感技术的无需标记和灵敏度高等优势，能高灵敏度、实时及便捷地进行免疫疾病标志物检测；凹槽111的底壁邻近光纤11的纤芯113，有利于获得光纤11上的强倏逝场。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，凹槽111的底壁到光纤11的纤芯113的距离为1～2μm，有利于获得光纤11上的强倏逝场，提高免疫疾病标志物检测的灵敏度。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，激发金属膜12为金膜或银膜。
金膜或银膜均能够激发SPR谐振峰；金膜化学性能稳定并且可以直接进行生物修饰，缺点是金价格贵，激发的SPR谐振峰并不如银膜的分辨率高；采用银膜，能获得更高分辨率的SPR谐振峰，而且价格比金膜廉价许多；
金膜直接组装生物分子的效率不高，设置分子自组装层14，能够提高抗体的组装效率；
银膜在空气中容易发生氧化、硫化等化学反应，从而影响光纤免疫传感器1的稳定性，设置分子自组装层14，一方面能够保护银膜，另一方面能够保证抗体的组装效率，从而得到性能高且成本相对较低的光纤免疫传感器1。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，激发金属膜12的厚度为40～50μm，获得更高分辨率的SPR谐振峰，尤其是银膜的厚度为40～50μm时，SPR谐振峰的分辨率显著提高。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，分子自组装层14为石墨烯纳米膜层或氧化石墨烯纳米膜层，化学性能稳定、比表面 积巨大，对生物分子的吸附力较强，其六元碳环结构更易与有机分子结合形成大π键的堆积，相对于常规的采用金膜组装生物分子，石墨烯纳米膜层或氧化石墨烯纳米膜层能获得更高的生物分子组装效率，从而提高光纤免疫传感器1的检测灵敏度及检测极限。
优选地，分子自组装层14为单层或寡层的氧化石墨烯膜层，厚度为1nm～3nm，光纤免疫传感器1检测灵敏度更高。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，还包括第一连接层，第一连接层沉积于凹槽111的底壁上，激发金属膜12沉积于第一连接层上。第一连接层主要能增加激发金属膜12与光纤11的粘附力，避免激发金属膜12起皮、脱膜。
当激发金属膜12为金膜或银膜，优选第一连接层为铬膜，厚度为2-3nm。金膜(或银膜)时，通过铬过渡，能够大幅增加金膜(或银膜)与光纤11的粘附力。
进一步地，请参阅图1，作为本发明提供的光纤免疫传感器的一种具体实施方式，还包括第二连接层16，第二连接层16自组装于激发金属膜12上，分子自组装层14自组装于第二连接层16上。一方面第二连接层16为激发金属膜12(尤其是在空气中化学稳定性差的银膜)提供了保护，另一方面第二连接层16作为过渡层，连接下一层设置的分子自组装层14，提高激发金属膜12与分子自组装层14的粘附力。
当激发金属膜12为金膜或银膜时，且分子自组装层14为氧化石墨烯层时，优选第二连接层16采用厚度为纳米量级的正十八硫醇(ODT)；
采用含有巯基的ODT作为金膜与氧化石墨烯膜层之间的连接层，实现金膜的烷基化，当然金膜也可通过其它方式进行烷基化；用含有巯基的ODT作为金膜与氧化石墨烯膜层之间的连接层，通过Ag-S共价键将厚度为纳米量级的ODT自组装在银膜上，在银膜上会形成致密的保护层，能有效克服包含银膜的光纤免疫传感器1容易退化的缺点，同时还提高了激发金属膜12与分子自组装层 14的粘附力。
进一步地，作为本发明提供的光纤免疫传感器的一种具体实施方式，单克隆抗体层13由肿瘤标志物的单克隆抗体构成，如间皮素。能够实现肿瘤诊断。
请一并参阅图1和图2，现对本发明提供的用于免疫性疾病检测的检测装置进行说明。用于免疫性疾病检测的检测装置，包括宽带光源2、光纤光谱仪、终端4和前述任一实施例述及的光纤免疫传感器1，光纤免疫传感器1的两端分别通过连接光纤5连接宽带光源2和光纤光谱仪，终端4与光纤光谱仪通信连接。
宽带光源2为光纤免疫传感器1提供覆盖的输入光(如波长范围为400nm～1100nm)，光信号进入光纤免疫传感器1并在传感区域与待测液体样本发生光-物质作用，输出光信号由光纤光谱仪采集，终端4(如电脑)安装数据处理软件，对光纤光谱仪采集的数据进行计算得到输出光的谐振峰，谐振峰的波长可定量反映免疫疾病标志物(如肿瘤标志物—间皮素)的浓度值；无需标记、无损、实时、准确地对液体样本(如血液或尿液)中的肿瘤标志物进行快速检测，并可进一步应用于需要联网、远程检测的物联网中。
请一并参阅图1和图3，现对本发明提供的光纤免疫传感器的制作方法进行说明。光纤免疫传感器的制作方法，包括如下步骤：
S1、在光纤11的侧边加工出凹槽111，且凹槽111的底壁邻近光纤11的纤芯113；
S2、在凹槽111的底壁上蒸镀或溅射一层激发金属膜12；
S3、在激发金属膜12上表面自组装一层分子自组装层14；
S4、活化分子自组装层14，使分子自组装层14的表面形成活化羧基；(步骤S4未图示)
S5、将单克隆抗体滴加于分子自组装层14，单克隆抗体分子中的氨基与述分子自组装层14的表面的活化羧基发生酰胺反应，使得单克隆抗体被组装固定于分子自组装层14上而形成单克隆抗体层13。
该光纤免疫传感器的制作方法能够用于制作前述任一实施例述及的光纤免疫传感器1。该方法通过简单工艺在光纤11上集成了激发金属膜12和单克隆抗体层13，制得选择性高、反应灵敏的光纤免疫传感器1，组装效率高，分子自组装层14能够对激发金属膜12进行保护，且激发金属膜12和单克隆抗体层13牢固的附着在光纤11上，避免性能发生退化。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，步骤S1包括：通过侧边抛磨光纤11的加工方法，将光纤11的部分包层112去除直至抛磨面接近光纤11纤芯113，去除部分包层处形成凹槽111。当然凹槽111也可通过其它方式加工。
优选地，依次采用3000目、7000目、12000目砂纸，对单模光纤进行粗磨、细磨、精抛光加工制作侧边抛磨光纤。通过监控光纤11抛磨过程中光功率损耗，发现光纤11抛磨面距离纤芯区域1～2μm(即凹槽111的底壁到光纤11的纤芯113距离为1～2μm)为最佳，有利于获得光纤11上的强倏逝场。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，步骤S2包括：在侧边抛磨光纤11上，采用真空热蒸镀或磁控溅射的方法，于光纤11抛磨面上均匀沉积一层激发金属膜12，制得光纤SPR传感器。
具体地，为增强激发金属膜12与光纤11的粘附力，优选先在光纤11上沉积第一连接层，再沉积激发金属膜12。当激发金属膜12为金膜或银膜时，先在光纤11表面沉积铬膜，再沉积金膜或银膜，铬膜的厚度具体可为2～3nm。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，步骤S3包括：
S31、在激发金属膜12的表面自组装第二连接层16；
S32、在第二连接层16的表面自组装分子自组装层14，如氧化石墨烯膜层。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，在步骤S31中：当激发金属膜12采用银膜时，采用含有巯基的正十八硫醇(ODT)作为银膜与氧化石墨烯膜层之间的第二连接层16，通过Ag-S共价键 将厚度为纳米量级的ODT自组装在银膜上。
优选地，采用浓度为10mM的ODT溶液于常温封闭环境下表面自组装36小时。
优选地，采用含巯基的ODT作为起中间过渡作用的第二连接层16，在银膜上会形成致密的保护层，能有效避免基于银的光纤SPR传感器容易退化的缺点。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，在步骤S32中：将步骤S31完成ODT自组装的光纤SPR传感器放入氧化石墨烯溶胶中，置于密封的室温环境中进行表面自组装，组装完成后取出超纯水清洗，室温下晾干制得增敏型光纤SPR传感器。
具体地，氧化石墨烯膜层自组装时间为40分钟以上为宜,能够在第二连接层16上组装出寡层氧化石墨烯纳米膜层。在银膜上通过寡层氧化石墨烯来组装生物分子——单克隆抗体，氧化石墨烯膜层一方面为银膜提供了进一步的保护，另一方面由于氧化石墨烯的化学性能稳定、比表面积巨大，对生物分子的吸附力较强，其六元碳环结构更易与有机分子结合形成大π键的堆积。相对于常规的采用金膜组装生物分子，氧化石墨烯膜层能获得更高的生物分子组装效率，从而提高传感器的检测灵敏度及检测极限。
具体地，氧化石墨烯溶胶可通过下述方法制得：
采用水或N，N-二甲基甲酰胺(DMF)为溶剂，将购买的商用氧化石墨烯粉末溶于其中搅拌均匀，置于五面震动超声波机中，对氧化石墨烯溶液进行大功率超声分散。优选地，水域温度恒定在25～30℃左右，超声4小时可获得较好的氧化石墨烯片层剥离效果，制得稳定的氧化石墨烯溶胶体系。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，步骤S4包括：
将步骤S3制得的增敏型光纤SPR传感器放入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基珀酰亚胺(NHS)溶液中，低温(4℃)活化30min，取出室温下晾干。从而对增敏型光纤SPR传感器表面的氧化石墨烯的羧 基(—COOH)进行活化。
进一步地，作为本发明提供的光纤免疫传感器的制作方法的一种具体实施方式，步骤S5包括：将肿瘤标志物的特异性单克隆抗体滴涂于步骤S4处理过的增敏型光纤SPR传感器的传感区域，置于4℃冰箱中干燥晾干，制得一种用于检测相应标志物的光纤免疫传感器1，拓展了光纤11传感器在生化检测领域中的新应用。
作为实施案列，针对胰腺癌、卵巢癌等肿瘤的标志物，以间皮素作为检测对象，将间皮素单克隆抗体滴加在传感区域。表层氧化石墨烯膜层上活化后的羧基(—COOH)与单克隆抗体分子中的氨基(—NH2)发生酰胺反应，从而抗体被组装固定于传感器表面，实现肿瘤的实时、准确、特异性高的生物分子识别。
以上仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 光纤免疫传感器，其特征在于，包括光纤、激发金属膜、单克隆抗体层和用于将所述单克隆抗体层组装于所述激发金属膜上的分子自组装层；
    所述光纤的侧边设有凹槽，所述凹槽的底壁邻近所述光纤的纤芯；
    所述激发金属膜沉积于所述凹槽的底壁上；
    所述分子自组装层自组装于所述激发金属膜上；
    所述单克隆抗体层涂于经活化处理后的所述分子自组装层上。
  2. 如权利要求1所述的光纤免疫传感器，其特征在于，所述凹槽的底壁到所述光纤的纤芯的距离为1～2μm。
  3. 如权利要求1所述的光纤免疫传感器，其特征在于，所述激发金属膜为金膜或银膜，所述激发金属膜的厚度为40～50μm。
  4. 如权利要求3所述的光纤免疫传感器，其特征在于，所述分子自组装层为石墨烯纳米膜层或氧化石墨烯纳米膜层。
  5. 如权利要求3所述的光纤免疫传感器，其特征在于，还包括第一连接层，所述第一连接层沉积于所述凹槽的底壁上，所述激发金属膜沉积于所述第一连接层上。
  6. 如权利要求5所述的光纤免疫传感器，其特征在于，所述第一连接层为铬膜，厚度为2-3nm。
  7. 如权利要求3所述的光纤免疫传感器，其特征在于，还包括第二连接层，所述第二连接层自组装于所述激发金属膜上，所述分子自组装层自组装于所述第二连接层上。
  8. 如权利要求1所述的光纤免疫传感器，其特征在于，所述单克隆抗体层由肿瘤标志物的单克隆抗体构成。
  9. 用于免疫性疾病检测的检测装置，包括宽带光源、光纤光谱仪和终端，其特征在于，还包括如权利要求1-8任一项所述的光纤免疫传感器，所述光纤免疫传感器的两端分别通过连接光纤连接所述宽带光源和所述光纤光谱仪，所 述终端与所述光纤光谱仪通信连接。
  10. 光纤免疫传感器的制作方法，其特征在于，包括如下步骤：
    在光纤的侧边加工出凹槽，且所述凹槽的底壁邻近所述光纤的纤芯；
    在所述凹槽的底壁上蒸镀或溅射一层激发金属膜；
    在所述激发金属膜上表面自组装一层分子自组装层；
    活化所述分子自组装层，使所述分子自组装层的表面形成活化羧基；
    将单克隆抗体滴加于所述分子自组装层，所述单克隆抗体分子中的氨基与所述分子自组装层的表面的活化羧基发生酰胺反应，使得所述单克隆抗体被组装固定于所述分子自组装层上而形成单克隆抗体层。
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