阅读说明：本技术 一种光纤表面等离子体共振传感器、制备方法及检测系统 (Optical fiber surface plasma resonance sensor, preparation method and detection system ) 是由 陈郁芝 尹一敬 杨许峰 王鑫 肖磊 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种光纤SPR传感器,包括单模光纤,熔接于所述单模光纤两端的多模光纤,单模光纤的表面设置纳米传感层,纳米传感层表面设置纳米抗体层。采用纳米抗体作为光纤SPR传感器的特异性生物检测层,使传感器表面识别位点多且密,缩短传感器的光学感应距离,有效提高检测灵敏度和检测效率,纳米抗体与传感器表面的结合位点在尾部,特异性识别位点在头部,使得纳米抗体定向连接至传感器表面,纳米抗体上特异性的识别位点一致朝外,提高捕获待测生物分子的几率。本发明还提供一种利用上述光纤SPR传感器的检测系统,该检测系统响应快、检测过程耗时短。本发明还提供一种光纤SPR传感器的制备方法,具有操作简便,便于批量化生产的特点。(The invention discloses an optical fiber SPR sensor which comprises a single-mode optical fiber and a multimode optical fiber welded at two ends of the single-mode optical fiber, wherein a nano sensing layer is arranged on the surface of the single-mode optical fiber, and a nano antibody layer is arranged on the surface of the nano sensing layer. The nano antibody is used as a specific biological detection layer of the optical fiber SPR sensor, so that the number of recognition sites on the surface of the sensor is large and dense, the optical sensing distance of the sensor is shortened, the detection sensitivity and the detection efficiency are effectively improved, the binding site of the nano antibody and the surface of the sensor is arranged at the tail part, the specific recognition site is arranged at the head part, the nano antibody is directionally connected to the surface of the sensor, the specific recognition sites on the nano antibody are outward in a consistent manner, and the probability of capturing biomolecules to be detected is improved. The invention also provides a detection system using the optical fiber SPR sensor, which has the advantages of quick response and short detection process time. The invention also provides a preparation method of the optical fiber SPR sensor, which has the characteristics of simple and convenient operation and convenient batch production.)

1. The optical fiber surface plasma resonance sensor is characterized by comprising a single-mode optical fiber and a multi-mode optical fiber welded at two ends of the single-mode optical fiber, wherein a nano sensing layer is arranged on the surface of the single-mode optical fiber, and a nano antibody layer is arranged on the surface of the nano sensing layer.

2. The optical fiber surface plasmon resonance sensor of claim 1, wherein the nano antibody layer is composed of a nano antibody, the head end of the nano antibody is provided with a specific recognition site, and the tail end of the nano antibody is fixed on the surface of the nano sensing layer through a thiol group or a biological cross-linking agent.

3. The optical fiber surface plasmon resonance sensor of claim 2, wherein the specific recognition site of the nanobody head is a recognition site of virus S protein; the tail end of the nano antibody is connected with cysteine, and the nano antibody is directionally and self-assembled on the surface of the nano sensing layer through the sulfydryl of the cysteine.

4. The fiber surface plasmon resonance sensor of claim 3, wherein said nanosensor layer has a thickness of 30-90 nm; the nano sensing layer is made of one of gold, silver, copper, aluminum, composite metal and a semiconductor containing free electrons.

5. The fiber surface plasmon resonance sensor of claim 1 wherein the length of said single mode fiber is 1-20 mm.

6. A detection system, comprising the fiber surface plasmon resonance sensor of any of claims 1 to 5, a light source, a transmission fiber, a spectrum detector and an optical signal processing unit, wherein the light source enters the fiber surface plasmon resonance sensor through the transmission fiber, and the emergent light from the fiber surface plasmon resonance sensor passes through the transmission fiber to the spectrum detector and then to the optical signal processing unit after passing through the spectrum detector.

7. The detection system according to claim 6, further comprising an antibody modified by metal nanoparticles, wherein the antibody modified by metal nanoparticles is in a solution to be detected.

8. The detection system according to claim 7, wherein the metal nanoparticle-modified antibody is a nanobody modified with gold nanoparticles, and the nanobody has a site recognizing a virus S protein.

9. The detection system of claim 6, further comprising a fiber support to hold the transmission fiber.

10. The method for preparing an optical fiber surface plasmon resonance sensor according to claims 1 to 5, comprising the steps of:

(1) taking a section of single-mode fiber and welding the multimode fiber at two ends of the single-mode fiber to obtain an MSM (metal-semiconductor-metal) type heterochromatic fiber;

(2) depositing a nano sensing layer in the single-mode fiber area of the MSM type heterocored fiber in the step (1);

(3) and fixing the nano antibody on the surface of the nano sensing layer to obtain the product.

Technical Field

The invention relates to the technical field of biosensors, in particular to an optical fiber surface plasmon resonance sensor, a preparation method and a detection system.

Background

Surface Plasmon Resonance (SPR), an optical phenomenon that can be used to track the interaction between biomolecules in a natural state in real time, has been developed from the 90 s of the 20 th century. The method has no damage to the biological molecules and does not need any marker.

The optical fiber surface plasma resonance sensor is mainly based on the optical fiber surface plasma resonance sensing principle, and analyzes the characteristics of detected biomolecules by detecting the change of the refractive index in an evanescent field zone of the optical fiber surface. When the biological molecules in the evanescent field region are subjected to recognition reaction, the refractive index of the surface of the metal film can be changed along with the biological molecules, so that the resonance frequency of the surface plasma wave is changed. The amount of change in the resonant frequency depends on the average effective refractive index of the evanescent field, and by detecting this variable, the amount of analyte bound in this region can be determined. The sensor has the characteristics that the biological sample does not need to be marked and the dynamic reaction process can be monitored in real time, and is particularly suitable for detection of biological molecules and research of interaction between molecules.

However, the conventional fiber SPR sensor has various problems: poor detection repeatability, low sensitivity and the like, thereby affecting the detection efficiency. Because the traditional antibody modification method is adopted, the large-volume antibodies easily block the antigen recognition sites mutually, and the direction of the antibodies combined on the surface of the sensor is difficult to control, so that the antigen recognition sites cannot be accurately and consistently exposed, the individual difference of each sensor is large, and the judgment of the detection result is influenced.

In addition, in the detection process of the virus, the detection method mainly comprises the steps of extracting nucleic acid from a sample, adding an amplification detection reagent, expressing virus genes, and then judging the positivity or the negativity of the sample through fluorescence PCR detection so as to further judge whether a patient is infected. The fluorescent PCR detection equipment is generally high in cost, large in size, complex in detection steps and long in detection time, and is generally suitable for laboratory detection. Because special laboratory condition requirements are required, the system is usually equipped in large-scale medical institutions, and communities and remote areas often do not have detection conditions, so that the system has certain limitations. Meanwhile, the existing nucleic acid detection technology has a certain omission factor (generally two or three samples need to be collected to confirm diagnosis), and brings certain difficulty for effective investigation of communities.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the optical fiber surface plasmon resonance sensor which has the characteristics of high sensitivity and good consistency and can obviously improve the detection efficiency.

The invention also provides a detection system comprising the optical fiber surface plasmon resonance sensor.

The invention also aims to provide a preparation method of the optical fiber surface plasmon resonance sensor.

One of the purposes of the invention is realized by adopting the following technical scheme:

the optical fiber surface plasma resonance sensor comprises a single-mode optical fiber and a multi-mode optical fiber welded at two ends of the single-mode optical fiber, wherein a nano sensing layer is arranged on the surface of the single-mode optical fiber, and a nano antibody layer is arranged on the surface of the nano sensing layer.

Further, the nano antibody layer is composed of a nano antibody, the head end of the nano antibody is provided with a specific recognition site, and the tail end of the nano antibody is fixed on the surface of the nano sensing layer through a sulfydryl or biological cross-linking agent.

Further, the specific recognition site of the head end of the nano antibody is a recognition site of virus S protein; the tail end of the nano antibody is connected with cysteine, and the nano antibody is directionally and self-assembled on the surface of the nano sensing layer through the sulfydryl of the cysteine.

Further, the thickness of the nano sensing layer is 30-90nm, preferably 45 nm; the nano sensing layer is made of one of gold, silver, copper, aluminum, composite metal and a semiconductor containing free electrons.

Further, the length of the single mode fiber is 1-20mm, preferably 12 mm.

The second purpose of the invention is realized by adopting the following technical scheme:

a detection system comprises the optical fiber surface plasma resonance sensor, a light source, a transmission optical fiber, a spectrum detector and an optical signal processing unit, wherein the light source enters the optical fiber surface plasma resonance sensor through the transmission optical fiber, emergent light from the optical fiber surface plasma resonance sensor is transmitted to the spectrum detector through the transmission optical fiber, and then is transmitted to the optical signal processing unit through the spectrum detector.

Further, the kit also comprises an antibody modified by the metal nanoparticles, wherein the antibody modified by the metal nanoparticles is positioned in a solution to be detected.

Further, the antibody modified by the metal nanoparticles is a nanobody modified by gold nanoparticles, and the nanobody has a site for recognizing virus S protein.

Further, the optical fiber bracket is used for fixing the transmission optical fiber.

The third purpose of the invention is realized by adopting the following technical scheme:

the preparation method of the optical fiber surface plasma resonance sensor comprises the following steps:

(1) taking a section of single-mode fiber and welding the multimode fiber at two ends of the single-mode fiber to obtain an MSM (metal-semiconductor-metal) type heterochromatic fiber;

(2) depositing a nano sensing layer in the single-mode fiber area of the MSM type heterocored fiber in the step (1);

(3) and fixing the nano antibody on the surface of the nano sensing layer to obtain the product.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts the nano antibody as the specific biological detection layer of the optical fiber SPR sensor, compared with the traditional antibody, the invention ensures that the surface recognition sites of the sensor are more and dense, shortens the optical sensing distance of the sensor and effectively improves the detection sensitivity and the detection efficiency.

The binding site of the nano antibody and the sensor surface is at the tail, and the specific recognition site is at the head, so that the nano antibody is directionally connected to the sensor surface, the specific recognition site on the nano antibody faces outwards, and the probability of capturing the biomolecule to be detected is improved.

2. The optical fiber SPR sensor can be used at one time, the safety of operators is ensured, and the cross contamination caused by the repeated use of the traditional sensor can be effectively avoided. The device has the advantages of high sensitivity, good consistency, simple structure, small volume and convenience in use or carrying, and the reliability of the detection result is improved.

3. When the detection system is used, the detection of the sample can be realized only by placing the optical fiber SPR sensor into the sample, various complicated biological operation steps are omitted, the sample is subjected to real-time activity detection, the false detection rate is reduced, and the detection efficiency is improved. The detection system disclosed by the invention is quick in response, only takes the time for capturing the to-be-detected biomolecule by the nano antibody during the whole detection, and is short in time consumption.

4. Under the condition that a sample to be detected only contains a very small amount of biomolecules to be detected, the detection can be realized by adding the metal nanoparticle modified antibody into the sample to be detected. The optical fiber SPR sensor actually detects the mass of biomolecules adsorbed on the surface, the metal nanoparticle modified antibody can be specifically connected with the biomolecules to be detected, when detection is carried out, the optical fiber SPR sensor captures the biomolecules to be detected, then the mass of the biomolecules adsorbed on the surface of the optical fiber SPR sensor is increased by adding the metal nanoparticle modified antibody, so that amplification of optical detection signals is realized, the sensitivity and the detection limit of the optical fiber SPR sensor are improved, and the omission factor is reduced.

5. The invention also provides a preparation method of the optical fiber SPR sensor, which is simple and convenient to operate and convenient for batch production.

Drawings

FIG. 1 is a schematic structural diagram of an unmodified nanoantibody layer of an optical fiber SPR sensor of the present invention;

FIG. 2 is a schematic structural diagram of the connection between the nano sensing layer and the nano antibody on the single-mode fiber surface of the fiber SPR sensor of the present invention through thiol groups;

FIG. 3 is a schematic structural diagram of a nanobody of the present invention and a conventional antibody;

FIG. 4 is a schematic diagram of the structure of the nano-antibody capture virus when the fiber SPR sensor of the present invention is used for sample detection;

FIG. 5 is a schematic structural diagram of the detection system of the present invention during sample detection;

FIG. 6 is a schematic structural diagram of a nano antibody capturing virus when an antibody modified by gold nanoparticles is added to the optical fiber SPR sensor of the present invention;

FIG. 7 shows the detection results of the sensitivity of the optical fiber SPR sensor of the present invention before being modified by the nanobody;

FIG. 8 shows the results of the detection before and after the addition of the gold nanoparticle-modified antibody in the detection of porcine epidemic diarrhea virus by the detection system of the present invention.

In the figure: 1. an optical fiber SPR sensor; 11. a single mode optical fiber; 111. a nano-sensing layer; 112. a nanobody; 113. a viral S protein recognition site; 114. (ii) cysteine; 115. gold nanoparticles; 116. s protein of the virus surface; 12. a multimode optical fiber; 2. a light source; 3. a transmission optical fiber; 4. a spectral detector; 5. an optical signal processing unit; 6. an optical fiber holder; 7. and (5) testing the sample to be tested.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.