CN115046986A - Biochemical sensor based on hollow anti-resonance optical fiber and surface enhanced Raman spectroscopy - Google Patents

Abstract

The invention relates to a biochemical sensor based on a hollow anti-resonance optical fiber and a surface enhanced Raman spectrum, which is used for solving the problems of low concentration sensitivity, single detection disease, strong invasiveness and poor specificity of the optical fiber biochemical sensor in a cancer detection method and belongs to the field of optical fiber sensing in biomedical photonics. The invention takes the hollow anti-resonance optical fiber modified with the specificity capture aptamer as a capture interface, and uses the Raman probe modified with the specificity recognition aptamer and the Raman reporter molecule to recognize the object to be detected, thereby constructing the novel multiplexing optical fiber biochemical sensor. The method utilizes the hollow anti-resonance optical fiber to enhance the interaction capacity of light and substances to enhance Raman signals, and realizes sensitive detection of low-concentration samples; the method utilizes the good linear relation between the Raman light intensity and the substance to be detected, and realizes the quantitative sensitive detection of the substance to be detected by detecting the change of the Raman light intensity; by utilizing the excellent reusability of the Raman spectrum, the simultaneous diagnosis of different objects to be detected is realized through the characteristic peaks corresponding to Raman reporter molecules on different Raman probes in the detection system. The invention can be used for detecting substances which can be identified by the aptamer, such as cancer exosomes, chemical molecules, glucose, cells, proteins and the like, has the characteristic of high sensitivity, and can realize the simultaneous detection of different noninvasive diseases.

Description

Biochemical sensor based on hollow anti-resonance optical fiber and surface enhanced Raman spectroscopy

Technical Field

The invention relates to a biochemical sensor applying surface enhanced Raman spectroscopy and based on a hollow anti-resonance optical fiber, can be used for simultaneously carrying out high-sensitivity specific detection on different types of objects to be detected, and belongs to the field of optical fiber sensing in biomedical photonics.

Background

Currently, the detection of some diseases still requires puncture and pathological examination to be diagnosed definitely, but the pathological examination has certain invasiveness and causes discomfort to patients. Some early diseases are difficult to be detected by conventional B-ultrasound, CT, nuclear magnetic resonance and gastrointestinal endoscopy because the onset is hidden or the focus is small.

Due to the defects of the detection means, the optical fiber biochemical sensor with the advantages of high sensitivity, non-invasiveness, low sample consumption, strong anti-electromagnetic interference capability and the like has inherent advantages in the aspects of high-precision biochemical analysis, environmental monitoring and the like. Optical fibers used in biochemical sensors can be classified into solid-core optical fibers, polished solid-core optical fibers, and hollow-core optical fibers. For the traditional solid core optical fiber, light is mainly transmitted in the fiber core, and the action of the light and a substance to be measured is weaker, so that the signal is weaker, and the sensitivity is lower. Polishing the fiber damages the structure of the fiber to increase the contact area of light and substance in the fiber core, making the fiber fragile. The hollow-core optical fiber is used for sensing and detecting, so that the acting distance between the light and substances filled in the hollow-core optical fiber is prolonged. In addition, the light guide principle of the hollow anti-resonance optical fiber is to block the transverse leakage of light in the fiber core through the anti-resonance effect formed by a special microstructure, so that the axial light transmission in the fiber core with low refractive index is realized. Light propagates in the air core, so that the optical fiber has the advantages of low absorption loss of materials to light, weak nonlinear effect, high mode purity, high damage threshold, small delay and the like, and more than 95% of light is limited in the fiber core. When the hollow-core optical fiber is used for sensing, the optical fiber can be used as a sample cell and an optical transmission channel, and only nL-muL-level substances to be detected are needed. Compared with the traditional method for detecting the interaction distance between the light and the substance in the light focus, the anti-resonance hollow-core resonant fiber greatly prolongs the interaction distance between the light and the substance.

Raman spectroscopy is a non-destructive analysis technique that results from the interaction of light with chemical bonds within a substance and is a unique "fingerprint" spectrum of a particular molecule. Raman scattered light is weak and attachment of a substance to a rough noble metal results in a large enhancement of the scattered light, i.e. surface enhanced raman effect (SERS). The SERS spectroscopy has the advantages of small damage to a sample, difficult quenching and strong signal. Because the existing detection technology can only detect a single sample, different substances can be detected simultaneously by utilizing the characteristics of narrow Raman spectrum width and strong reusability.

The Raman spectrum intensity is in direct proportion to the concentration of an object to be detected, and the SERS spectrum technology is combined with the optical fiber biochemical sensor, so that the high-sensitivity detection of different concentrations can be realized, and the composition analysis of a mixture can also be realized. Therefore, the biochemical sensor based on the hollow anti-resonance optical fiber and the SERS spectroscopy can reflect absolute concentration information through an absolute peak value with high sensitivity and can reflect different material compositions.

Disclosure of Invention

The purpose of the invention is as follows: the invention is used for solving the problems of low sensitivity, poor specificity and single detection of an optical fiber biochemical sensor in actual detection, and provides a biochemical sensor based on a hollow anti-resonance optical fiber and a surface enhanced Raman spectrum. The sensor takes a hollow anti-resonance optical fiber as a signal conversion system, takes the inner wall of the optical fiber modified with the aptamer as an exosome capture interface, and takes the silver nanoparticles modified with the aptamer specifically identified and the Raman reporter molecule as Raman probes, so that the novel hollow optical fiber biochemical sensor is formed.

The specific technical content is as follows:

a biochemical sensor based on hollow-core anti-resonant fiber and surface-enhanced raman spectroscopy, comprising: the laser micro-confocal Raman spectrometer comprises a laser (1) with adjustable output power, a 50X micro-focusing objective lens (2), a precise three-dimensional adjustable objective table (3) and a spectrometer (4), and an optical fiber sensing system which comprises a hollow anti-resonance optical fiber (5) and a Raman probe (6); the laser (1) is used for emitting laser, the 50X micro-focusing objective lens (2) is used for focusing light beams and collecting Raman scattering in optical fibers, the precise three-dimensional adjustable objective table (3) is used for fixing the hollow anti-resonance optical fibers (5), and the spectrometer (4) is used for collecting Raman scattering optical signals in the hollow anti-resonance optical fibers (5), and the laser is characterized in that: the inner wall of the hollow anti-resonance optical fiber (5) is modified with a nucleic acid aptamer 1, the Raman probe (6) is modified with a Raman reporter molecule and a specific nucleic acid aptamer 2, the nucleic acid aptamer 1 is used for capturing an object to be detected, the nucleic acid aptamer 2 is used for identifying the target object to be detected, and the Raman reporter molecule is used for generating a Raman signal;

the detection process of the whole sensor is that an object to be detected is filled into a hollow anti-resonance optical fiber (5), then the hollow anti-resonance optical fiber (5) is placed on a precise three-dimensional adjustable object stage (3), the object to be detected is captured on the inner wall of the optical fiber through a nucleic acid aptamer 1 on the inner wall of the optical fiber by the hollow anti-resonance optical fiber (5), and then a Raman probe (6) is introduced, when the object to be detected contains a target object to be detected, a specific nucleic acid aptamer 2 identifies the target object to be detected, a Raman reporter molecule is excited to generate a Raman scattering signal, the Raman scattering signal is collected by a spectrometer (4) after passing through a 50 x micro-focusing objective lens (2), a corresponding characteristic peak is presented in a spectrum, and the detection of the target object to be detected is realized.

When the multi-target object to be detected is detected, the hollow anti-resonance optical fiber (5) captures the object to be detected on the inner wall of the optical fiber through the aptamer 1 on the inner wall of the optical fiber, then different types of Raman probe mixed liquid are filled, different Raman probes are combined with corresponding target objects to be detected, and whether the target objects to be detected of the type are contained or not is judged by observing the existence of corresponding Raman reporter molecule characteristic peaks on a Raman spectrum.

The intensity of the Raman light and the concentration of the target object to be detected form a linear relation, and the concentration of the object to be detected is quantified by detecting the intensity of the Raman light.

The output power adjustable laser (1) selects 785nm wavelength, thereby avoiding fluorescence interference.

The specific process of modifying the aptamer 1 on the inner wall of the optical fiber comprises the following steps: one end of the aptamer 1 modified on the optical fiber is modified with carboxyl, and the aptamer 1 is modified on the inner wall of the optical fiber by a chemical coupling method to form a specific capture interface.

The manufacturing method of the Raman probe comprises the following steps: the nucleic acid aptamer 2 and the Raman reporter molecule are modified on the Raman probe substrate, silver nanoparticles are selected as the substrate of the Raman probe, and the sulfydryl is modified at one end of the nucleic acid aptamer 2 modified on the Raman probe substrate, so that the nucleic acid aptamer can directly react with the silver nanoparticles; selecting a substance containing sulfydryl and benzene ring from the Raman reporter molecule, and directly reacting with the silver nanoparticles; firstly, mixing the aptamer 2 solution with the silver particle solution, adding the Raman reporter molecule, incubating and centrifuging to obtain the Raman probe.

The capture interface of the inner wall of the hollow anti-resonant fiber and the Raman probe are specifically described as follows:

1. the capture interface is based on a chemical coupling method, and the aptamer 1 is modified on the hollow-core anti-resonant optical fiber. The method is characterized in that a hollow anti-resonance optical fiber made of quartz material is used as a substrate, 3-aminopropyl triethoxysilane (APTES) is introduced by a precise injection instrument and incubated, and hydroxyl on the quartz optical fiber reacts with the APTES, so that amino groups are modified on the inner wall of the optical fiber. On the basis, the carboxylated aptamer 1 is introduced by a precise injection instrument and incubated, and the optical fiber interface with the capture function can be obtained. The sequence of the carboxylated aptamer 1 can recognize a corresponding target molecule, and when an object to be detected is added into the detection system, the target molecule can be combined with the aptamer 1. The higher the concentration of target molecules in a volume, the greater the number of target molecules that are captured on the inner wall of the fiber.

2. Raman probes are based on the surface enhanced raman effect. Due to electromagnetic enhancement and chemical enhancement, the Raman scattering spectrum intensity of a sample attached to the surface of the rough metal can be improved by 6-12 orders of magnitude, and silver nanoparticles with obvious Raman enhancement effect are selected as a substrate of the Raman probe. The material with larger scattering cross section has larger Raman light intensity, and the invention selects the material with benzene ring as Raman reporter molecule. In order to enable the Raman reporter molecule to be easily modified on the silver nanoparticles, the selected Raman reporter molecule is provided with sulfydryl. The combination of the silver particles and the Raman reporter molecules can be realized by mixing the Raman reporter molecule solution and the silver nanoparticles. When the Raman probe is manufactured, the silver nanoparticles are combined with a proper amount of thiolated specificity recognition aptamer 2, and then the selected Raman reporter molecule is added, so that the Raman probe with the specificity recognition function is formed. The thiolated aptamer 2 recognizes the corresponding target molecule. The higher the concentration of this type of target molecule in a volume, the more raman probes bind to this type of target molecule and the stronger the raman signal. When the object to be detected is filled into the hollow anti-resonance optical fiber by using the precision injection instrument, the optical fiber captures the object to be detected on the inner wall of the optical fiber, and then the Raman probe is filled into the precision injection instrument, so that the structure of the hollow anti-resonance optical fiber-aptamer 1-object to be detected-Raman probe can be formed. Different concentrations of target molecules within a volume will affect the amount of raman probe bound and thus the intensity of the raman signal. And the quantitative detection of the concentration of the target molecules is realized by detecting the change of the Raman intensity.

3. Different Raman reporter molecules have different characteristic peaks on a Raman spectrum, and the identification of different types of target molecules can be realized by changing the nucleic acid aptamer sequence on the silver particles and the Raman reporter molecules. When the object to be detected contains different types of target objects to be detected simultaneously and the target objects to be detected can be identified by the aptamer 1 on the inner wall of the optical fiber, different types of Raman probe mixed liquid are filled in the precise injection instrument, and different Raman probes are combined with the corresponding target objects to be detected. Whether the type of the substance to be detected is contained is judged by observing whether the Raman spectrum has the corresponding Raman reporter molecule characteristic peak or not.

The hollow anti-resonance optical fiber is used as a sensing platform, and has the advantages of strong interaction between light and substances, wide spectrum transmission bandwidth, low transmission loss, low bending loss, high laser damage threshold, high mode purity and the like. Due to the special structural design of the hollow anti-resonance optical fiber, liquid to be detected can be directly filled into the fiber core to form the liquid core optical fiber, when the refractive index of the liquid to be detected is smaller than that of quartz, light can still be limited to be transmitted in the liquid core, the interaction between the light and Raman reporter molecules is enhanced, and the Raman effect is enhanced. The silver particles can further greatly enhance the Raman light, so that the Raman signal in the optical fiber can be more easily measured by a laser micro-confocal Raman spectrometer.

For sensor performance, the detection limit of the instrument, the volume of the analyte, the sensitivity of the instrument and the multiplexing capability are several important parameters for evaluating the performance of the sensor.

Advantageous effects

Compared with the prior art, the invention has the following advantages:

1. the detection limit is low. The hollow anti-resonance optical fiber can limit more than 95% of light in the fiber core, and greatly enhances the interaction between the light and Raman reporter molecules; instead of focusing the laser directly on the sample, the optical fiber of the present invention can lengthen the interaction distance between the light and the raman reporter molecule, thereby further increasing the raman signal. Based on SERS effect, the silver particles are used for preparing the Raman probe, so that the Raman signal is further increased. The hollow anti-resonance optical fiber has strong Raman signals when the concentration of the object to be measured is low due to the light intensity limiting effect and the SERS enhancing effect.

2. The required object to be measured is small in volume. The diameter of the fiber core of the hollow anti-resonance optical fiber is dozens of microns, and the fiber core can be filled with the object to be detected with the volume of nL-muL magnitude, which is significant in the application of the biochemical field and is beneficial to realizing the trace detection of the sample.

3. The detection sensitivity is high. The aptamer 1 modified on the inner wall of the optical fiber and the aptamer on the Raman probe can be selected according to a substance to be detected, the aptamer 1 modified on the inner wall of the optical fiber can capture the substance to be detected in a complex detection environment, the aptamer modified on the Raman probe can accurately identify the target substance to be detected in the complex detection environment, and the detection sensitivity is greatly improved. And because the strong limiting effect of the optical fiber on light enhances the effect of light and substances and the enhancing effect of SERS, the Raman signal is improved, and the sensitivity is further improved.

4. High efficiency. The Raman spectrum has stronger multiplexing capability due to narrower wave crests. The aptamer on the Raman probe used by the invention can be selected according to the type of the object to be detected, and the Raman reporter molecule on the Raman probe also has multiple choices. When a plurality of target substances exist in a sample to be detected, the aptamers corresponding to different target substances can be selected to be modified on the Raman probe, the mixture of the Raman probes with different types is introduced into the optical fiber, so that different target substances in the sample to be detected can be identified, and whether the different target substances are identified can be characterized by whether characteristic peaks of different Raman reporter molecules exist on the spectrum.

5. The repeatability is high. Raman light is generated by changing the frequency phase of light due to the vibrational rotation of molecular bonds in a substance, and has almost no bleaching property; the required exciting light power is lower than 0.5mW, the damage to the sample is small, and the repeatability is high.

6. The detection range is wide. The present invention can be used to detect substances specifically recognized by nucleic acid aptamers, and thus the scope of detection includes, but is not limited to, cancer, Parkinson's disease, kidney disease, nucleic acids, and the like. Depending on the choice of the corresponding aptamer, different substances can be detected.

Therefore, the hollow anti-resonance optical fiber provides a good platform for the interaction of an object to be measured and light, the combination of the hollow anti-resonance optical fiber and the SERS technology further enhances Raman signals on the basis of the surface Raman enhancement effect, and the optical fiber has lower optical loss and higher laser damage threshold value on a longitudinal light path, thereby being an ideal sensing platform.

Description of the drawings:

FIG. 1 is a schematic view of an overall inspection system;

FIG. 2(a) is a cross-sectional view of a hollow-core antiresonant optical fiber for use as an optical fiber biochemical sensor

FIG. 2(b) is a cross-sectional view of a hollow core antiresonant fiber core for fiber optic biochemical sensing

FIG. 3 is a schematic view of an optical fiber structure

FIG. 4 is a schematic diagram showing a flow of manufacturing an optical fiber having an inner wall modified with an aptamer;

FIG. 5 is a schematic diagram of a Raman probe preparation process;

FIG. 6 is a schematic diagram of the detection step in a hollow core antiresonant fiber;

FIG. 7(a) is a Raman spectrum intensity chart obtained by detecting different concentrations of analytes;

FIG. 7(b) is a graph showing the linear relationship between the concentration of the analyte and the Raman intensity obtained in FIG. 7 (a).

Detailed Description

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.

The sensor comprises a laser micro-confocal Raman spectrometer and an optical fiber sensing system, wherein the laser micro-confocal Raman spectrometer comprises a laser with adjustable output power, a 50 Xmicro-focusing objective lens, a precise three-dimensional adjustable objective table and a spectrometer, and the optical fiber sensing system comprises a hollow anti-resonance optical fiber and a Raman probe; the laser is used for emitting laser, the 50X micro-focusing objective lens is used for focusing light beams and collecting Raman scattering in the optical fiber, the precise three-dimensional adjustable objective table is used for fixing the hollow anti-resonance optical fiber, and the spectrometer is used for collecting Raman scattering optical signals in the hollow anti-resonance optical fiber.

When sensing detection is carried out, a substance to be detected is injected into the hollow anti-resonance optical fiber of which the inner wall is modified with the aptamer 1 through a precision injection instrument, and the substance capable of being specifically combined with the aptamer 1 in the substance to be detected is captured by the hollow anti-resonance optical fiber; injecting a Raman probe modified with Raman reporter molecules and specific aptamer 2 into the anti-resonance hollow-core resonance optical fiber through a precise injection instrument, wherein the Raman probe is specifically combined with a substance to be detected; a light source emitted by a laser with 785nm of output power is coupled into a hollow anti-resonance optical fiber through a 50 multiplied micro focusing objective; the laser scatters with the Raman reporter molecules on the Raman probe in the process of transmitting in the hollow anti-resonance optical fiber, the silver nanoparticles can enhance Raman scattering signals, the enhanced scattering signals are collected by a 50 Xmicro-focusing objective lens and a spectrometer, the intensity of the Raman light and the concentration of the object to be detected form a good linear relation, and the concentration of the object to be detected is quantified by detecting the intensity of the Raman light.

When a single type of object to be detected is detected, an exosome is injected into the hollow anti-resonance optical fiber modified with the aptamer 1 by using a precision injection instrument, and the exosome is fixed on the inner wall of the optical fiber by the aptamer 1 on the inner wall; then injecting the Raman probe into the optical fiber, grasping the Raman probe capable of sending signals by the exosomes, and completing the grasping process by the specific recognition of the aptamer 2 on the probe and the exosomes; the laser is focused into the fiber by a focusing objective lens, and the generated raman signal is collected by a spectrometer. The presence or absence of a raman signal indicates the presence or absence of exosomes that can bind to the raman probe, thereby indicating whether the disease is diseased or not.

Method for the simultaneous detection of different types of exosomes: injecting the mixed solution of the exosomes of different types into the hollow anti-resonance optical fiber modified with the aptamer 1 by using a precise injection instrument, and capturing all types of exosomes by using the hollow anti-resonance optical fiber; injecting mixed liquid of different types of Raman probes into the hollow anti-resonance optical fiber by using a precise injection instrument, and combining the Raman probes and corresponding exosomes into a stable hybrid; different Raman reporter molecules on the Raman probe can be excited to form different characteristic peaks, and Raman scattering signals are collected by a laser micro-confocal Raman spectrometer, so that different characteristic peaks are presented at different positions of a spectrum. Whether the corresponding exosomes exist or not is represented by the existence of the corresponding characteristic peaks in the Raman spectrum, so that the exosomes of different types can be detected simultaneously.

Example 1

The experiment is based on the detection system shown in fig. 1, and the optical fiber biochemical sensor is used for detecting SKBR3 breast cancer cell exosomes.

The optical fiber used by the invention is a hollow anti-resonance optical fiber; the aptamer 1 modified on the inner wall of the optical fiber can be combined with all types of exosomes; the aptamer 2 modified on the silver particle can be combined with a specific type of exosome; the constructed sensor can sensitively detect cancer cell exosomes in blood and other body fluids, such as breast cancer exosomes, pancreatic cancer exosomes and the like.

Because exosomes in the cancer patients contain abnormally-increased HER2 protein compared with healthy individuals, whether the patients are ill or not is judged by the fact that the HER2 content is negative. Firstly, introducing APTES into an optical fiber by using a precision injection instrument to perform silanization treatment on the inner wall of the optical fiber, so that the inner wall of the optical fiber is modified with amino functional groups; next, the carboxyl group-modified aptamer 1, which uses a DNA strand recognizing the CD63 protein contained on all the exosome surfaces, was introduced and incubated for 12 hours. The amino group on the inner wall of the optical fiber is covalently coupled to the carboxyl group on the aptamer 1, thereby immobilizing the aptamer 1 on the inner wall of the optical fiber. After the aptamer capture substrate is manufactured, exosomes of SKBR3 breast cancer cells can be introduced and incubated for 2h, and the combination of aptamer 1 and the exosomes is realized.

The preparation of the Raman probe selects 80nm silver nanoparticles with better SERS characteristics as a substrate. And incubating the silver nanoparticles and the aptamer 2 for 2h, and adding a Raman reporter molecule MMBN to incubate for 2.5h, so that the preparation of the Raman probe can be completed. The aptamer 2 is a DNA chain which is modified with sulfydryl and can be specifically combined with HER2 protein, and the sulfydryl directly reacts with the silver nanoparticles to realize the connection of the silver particles and the aptamer 2; the MMBN is tetra-mercapto benzonitrile and can also be directly reacted with the silver nanoparticles to realize connection. The prepared probe is introduced into the optical fiber in which the exosome is captured, and if the surface of the captured exosome contains HER2 protein, the probe is specifically bound to the exosome and immobilized on the inner wall of the optical fiber. Finally, the unidentified probe is washed by introducing water and the optical fiber is placed in the detection system of fig. 1 to detect the raman signal in the optical fiber, wherein the detected raman signal is the signal of the raman probe bound to exosomes. The more high-concentration exosomes are combined with the more Raman probes, so that the Raman light intensity is increased, and a correlation curve of the exosome concentration and the Raman light intensity can be drawn. Fig. 7(a) is a raman spectrum intensity diagram obtained by detecting analytes with different concentrations, and a linear relationship diagram of the analyte concentration and the raman intensity is obtained, as shown in fig. 7 (b).

Example 2

The experiment is based on the detection system shown in fig. 1, and the optical fiber biochemical sensor is used for realizing the simultaneous detection of breast cancer SKBR3 and pancreatic cancer Panc01 cell exosomes. The surface of the exosome contains CD63 protein, and if the two exosomes are simultaneously filled into the optical fiber modified with the CD63 aptamer, the two exosomes can be captured by the inner wall of the optical fiber. Firstly, introducing APTES into an optical fiber by using a precision injection instrument to perform silanization treatment on the inner wall of the optical fiber, so that the inner wall of the optical fiber is modified with amino functional groups; subsequently, the carboxyl group-modified aptamer 1 was introduced and incubated for 12 hours, and the aptamer 1 selected from a DNA strand recognizing the CD63 protein contained on the surface of all exosomes, thereby immobilizing all types of exosomes on the inner wall of the optical fiber. The amino group on the inner wall of the optical fiber is covalently coupled to the carboxyl group on the aptamer 1, thereby immobilizing the aptamer 1 on the inner wall of the optical fiber. After the production of the aptamer capture substrate is completed, exosomes of SKBR3 breast cancer cells and exosomes of pancreatic cancer Panc01 cells can be introduced and incubated for 2h, so that the combination of the aptamer 1 and all exosomes is realized.

SKBR3 breast cancer cell exosome contains HER2 protein and does not contain GPC-1 protein, and pancreatic cancer Panc01 cell contains GPC-1 and does not contain HER2 protein. The thiolated aptamer 2 specifically bound with HER2 protein and the thiolated aptamer 3 specifically bound with GPC-1 are respectively used for preparing two Raman probes. Preparation of raman probe for identifying HER2 protein: after the silver nanoparticles are incubated with the aptamer 2 for 2h, the silver nanoparticles are incubated with the Raman reporter MMBN for 2.5 h. Preparation of Raman probes for identifying GPC-1 protein: after incubating the silver nanoparticles with aptamer 3 for 2h, incubating the silver nanoparticles with Raman reporter MGITC for 2.5 h.

Introducing a mixture of two cancer cell exosomes into an optical fiber modified with a capture aptamer for incubation, fixing the exosomes on the inner wall of the optical fiber, introducing a mixture of two probes for incubation overnight, finally washing the unbound Raman probe by filling water with a precise injection instrument, putting the Raman probe into the detection system shown in the figure 1, and fixing the optical fiber on a precise three-dimensional stage to measure Raman spectrum. If the surface of the exosome contains HER2 and GPC-1, a characteristic peak of MMBN and a characteristic peak of MGITC simultaneously appear in a Raman spectrum, so that the simultaneous detection of two diseases is realized.

Claims (7)

1. A biochemical sensor based on hollow-core anti-resonant fiber and surface-enhanced raman spectroscopy, comprising: the laser micro-confocal Raman spectrometer comprises a laser (1) with adjustable output power, a 50X micro-focusing objective lens (2), a precise three-dimensional adjustable objective table (3) and a spectrometer (4), and an optical fiber sensing system which comprises a hollow anti-resonance optical fiber (5) and a Raman probe (6); the laser (1) is used for emitting laser, the 50X micro-focusing objective lens (2) is used for focusing light beams and collecting Raman scattering in optical fibers, the precise three-dimensional adjustable objective table (3) is used for fixing the hollow anti-resonance optical fibers (5), and the spectrometer (4) is used for collecting Raman scattering optical signals in the hollow anti-resonance optical fibers (5), and the laser is characterized in that: the inner wall of the hollow anti-resonance optical fiber (5) is modified with a nucleic acid aptamer 1, the Raman probe (6) is modified with a Raman reporter molecule and a specific nucleic acid aptamer 2, the nucleic acid aptamer 1 is used for capturing an object to be detected, the nucleic acid aptamer 2 is used for identifying the target object to be detected, and the Raman reporter molecule is used for generating a Raman signal;

the detection process of the whole sensor is that an object to be detected is filled into a hollow anti-resonance optical fiber (5), then the hollow anti-resonance optical fiber (5) is placed on a precise three-dimensional adjustable object stage (3), the object to be detected is captured on the inner wall of the optical fiber through a nucleic acid aptamer 1 on the inner wall of the optical fiber by the hollow anti-resonance optical fiber (5), and then a Raman probe (6) is introduced, when the object to be detected contains a target object to be detected, a specific nucleic acid aptamer 2 identifies the target object to be detected, a Raman reporter molecule is excited to generate a Raman scattering signal, the Raman scattering signal is collected by a spectrometer (4) after passing through a 50 x micro-focusing objective lens (2), a corresponding characteristic peak is presented in a spectrum, and the detection of the target object to be detected is realized.

2. The biochemical sensor according to claim 1, wherein the biochemical sensor is based on a hollow-core antiresonant fiber and surface-enhanced Raman spectroscopy, and comprises:

when the multi-target object to be detected is detected, the hollow anti-resonance optical fiber (5) captures the object to be detected on the inner wall of the optical fiber through the aptamer 1 on the inner wall of the optical fiber, then different types of Raman probe mixed liquid are filled, different Raman probes are combined with corresponding target objects to be detected, and whether the target objects to be detected of the type are contained or not is judged by observing the existence of corresponding Raman reporter molecule characteristic peaks on a Raman spectrum.

3. The biochemical sensor according to claim 1 or 2, wherein the optical fiber is a hollow-core antiresonant optical fiber and the surface-enhanced Raman spectroscopy is based on the following characteristics: the hollow-core anti-resonant optical fiber can limit more than 95% of light to be transmitted in the core.

4. The biochemical sensor according to claim 1 or 2, wherein the optical fiber is a hollow-core antiresonant optical fiber and the surface-enhanced Raman spectroscopy is based on the following characteristics: the intensity of the Raman light and the concentration of the target object to be detected form a linear relation, and the concentration of the object to be detected is quantified by detecting the intensity of the Raman light.

5. The biochemical sensor according to claim 1 or 2, wherein the optical fiber is a hollow-core antiresonant optical fiber and the surface-enhanced Raman spectroscopy is based on the following characteristics: the output power adjustable laser (1) selects 785nm wavelength, thereby avoiding fluorescence interference.

6. The biochemical sensor according to claim 1 or 2, wherein the optical fiber is a hollow-core antiresonant optical fiber and the surface-enhanced Raman spectroscopy is based on the following characteristics: the specific process of modifying the aptamer 1 on the inner wall of the optical fiber comprises the following steps: one end of the aptamer 1 modified on the optical fiber is modified with carboxyl, and the aptamer 1 is modified on the inner wall of the optical fiber by a chemical coupling method to form a specific capture interface.

7. The biochemical sensor according to claim 1 or 2, wherein the optical fiber is a hollow-core antiresonant optical fiber and the surface-enhanced Raman spectroscopy is based on the following characteristics: the manufacturing method of the Raman probe comprises the following steps: the nucleic acid aptamer 2 and the Raman reporter molecule are modified on the Raman probe substrate, silver nanoparticles are selected as the substrate of the Raman probe, and the sulfydryl is modified at one end of the nucleic acid aptamer 2 modified on the Raman probe substrate, so that the nucleic acid aptamer can directly react with the silver nanoparticles; selecting a substance containing sulfydryl and benzene ring from the Raman reporter molecule, and directly reacting with the silver nanoparticles; firstly, mixing the aptamer 2 solution with the silver particle solution, adding the Raman reporter molecule, incubating and centrifuging to obtain the Raman probe.

Images