CN109060727B - Dual-channel optical fiber SPR biosensor - Google Patents
Dual-channel optical fiber SPR biosensor Download PDFInfo
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- 108091007433 antigens Proteins 0.000 claims description 35
- 108060003951 Immunoglobulin Proteins 0.000 claims description 32
- 102000018358 immunoglobulin Human genes 0.000 claims description 32
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 22
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The invention relates to a dual-channel optical fiber SPR sensor. The dual-channel SPR sensor comprises a reference channel and a sensing channel, wherein the reference channel is used as a blank reference channel, and the wavelength movement caused by nonspecific adsorption is detected; the sensing channel enhances the local electric field intensity by utilizing the coupling effect between the gold film and the gold nano particles to improve the detection sensitivity of the sensor, and enhances the fixing efficiency of the antibody by utilizing the graphene oxide film. Two channels in the dual-channel optical fiber SPR sensor respectively use different metal films, so that two resonance valleys are generated, dual-channel sensing is realized, and the dual-channel optical fiber SPR sensor has the unique advantages of eliminating measurement errors caused by nonspecific adsorption and being insensitive to temperature; meanwhile, the two SPR resonance valleys of the double channels are mutually separated, so that the two SPR resonance valleys cannot be adversely affected in the detection process, and the biological sensing is facilitated. The invention effectively solves the problems of low sensitivity and susceptibility to liquid refractive index change and temperature fluctuation of the sensor in the prior art.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to an SPR biosensor, and especially relates to a dual-channel optical fiber SPR biosensor.
Background
Surface Plasmon Resonance (SPR), chinese name surface plasmon resonance, is a common optical phenomenon, and refers to the resonance of an optical wave with a plasma wave generated on the surface of a metal (such as gold or silver) when incident light with a specific wavelength in an optical waveguide irradiates the metal film, which is called Surface Plasmon Resonance (SPR) effect. The SPR effect can make reflected light energy undergo the process of sharp reduction so as to form resonance trough, at the same time, SPR is very sensitive to external refractive index, when the measured solution concentration is changed, i.e. external solution refractive index is changed, the resonance trough of SPR can be moved along with it, so that said invention can implement biological sensing by means of detecting offset quantity of SPR resonance trough.
SPR biosensors have attracted considerable attention in biological and chemical detection due to their high sensitivity and biocompatibility. Detection of external biomolecules can be achieved by causing a shift in the resonance angle or resonance wavelength based on the interaction between the surface plasmon wave and the surrounding biomolecules. Compared with the traditional SPR biosensor platform based on a prism, the optical fiber SPR biosensor has the characteristics of simple manufacture, low cost, miniaturized structure, electromagnetic interference resistance and the like. However, conventional SPR biosensors lack sufficient sensitivity to detect biomolecules of lower molecular mass, such as DNA and lower concentration biological samples. In addition, most optical fiber biosensors have only one detection channel, and only one analyte can be detected, and in the detection process, the accuracy of the detection result is generally affected by nonspecific adsorption of a biological sample or external temperature fluctuation. Therefore, further improvement in detection sensitivity and accuracy of the sensor is required.
Disclosure of Invention
In order to solve the problem of low sensitivity and accuracy of the existing optical fiber SPR biosensor, the invention provides a dual-channel optical fiber SPR sensor. The dual-channel SPR sensor comprises a reference channel and a sensing channel, wherein the reference channel is used as a blank reference channel, and the wavelength movement caused by nonspecific adsorption is detected; the sensing channel enhances the local electric field intensity by utilizing the coupling effect between the gold film and the gold nanoparticles to improve the detection sensitivity of the sensor, the graphene oxide film is fixed on the surface of the photonic crystal fiber sensor plated with the gold film by a covalent bond bonding method, the graphene oxide film is utilized to improve the fixing efficiency of the antibody, the specific bonding between the antibody and the antigen fixed by the gold nanoparticles causes resonance wavelength drift, and the measurement of antigen high sensitivity is realized according to the movement amount of the resonance wavelength displayed on a spectrometer; in the dual-channel optical fiber SPR sensor, two channels respectively use different metal films, so that two resonance valleys are generated, and dual-channel sensing is realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a dual channel optical fiber SPR biosensor comprising a reference channel and a sensing channel, the reference channel and the sensing channel being connected by a multimode optical fiber, wherein:
the reference channel comprises a first photonic crystal fiber, wherein two ends of the first photonic crystal fiber are respectively welded with a multimode fiber, and a silver film is plated on the surface of the first photonic crystal fiber; the reference channel is used as a blank control to detect the wavelength shift caused by nonspecific adsorption;
the sensing channel comprises a second photonic crystal fiber, multimode fibers are welded at two ends of the second photonic crystal fiber respectively, a gold plating film is plated on the surface of the second photonic crystal fiber, a graphene oxide film is fixed on the surface of the gold film, an antibody film layer which selectively absorbs biomolecules or chemical components is solidified on the graphene oxide film, antigens corresponding to the antibodies are fixed by the gold nanoparticles, and the antibodies are combined with the antigens in the detection process, so that the gold nanoparticles with the antigens fixed on the surface are fixed on the antibody film, and a coupling effect is generated between the gold film and the gold nanoparticles.
Further, the lengths of the first photonic crystal fiber of the reference channel and the second photonic crystal fiber of the sensing channel are 0.5-2 cm.
Further, the surface of the first photonic crystal of the reference channel is plated with a silver film, and the thickness of the silver film is 40-60 nm.
Further, the gold film is plated on the surface of the second photonic crystal fiber of the sensing channel, and the thickness of the gold film is 40-60 nm.
Further, the graphene oxide film is fixed on the surface of the gold film of the sensing channel, the graphene oxide film is fixed on the surface of the gold film through a covalent bond bonding method, and the fixing efficiency of the antibody is improved through the graphene oxide film.
Further, the graphene oxide film is fixed on the surface of the gold film of the sensing channel, and the thickness of the graphene oxide film is 0.2-0.6 nm.
Further, the diameter of the gold nano-particles of the sensing channel is 10-50 nm.
The principle of the invention is as follows:
the invention mainly utilizes a double channel to improve the sensitivity and accuracy of detection of an optical fiber SPR sensor, wherein one section of photonic crystal fiber is used as a sensing channel, a gold film and a graphene oxide film are plated on the surface of the photonic crystal fiber in sequence for fixing an antibody, the specific binding between an antigen and the antibody and the nonspecific adsorption of the antigen can cause the wavelength shift of the sensing channel, in order to eliminate the wavelength shift caused by the nonspecific adsorption of the antigen in the sensing channel, the photonic crystal fiber coated with a silver film is introduced as a reference channel for detecting the wavelength shift caused by the nonspecific adsorption of the antigen, and the wavelength shift caused by the specific binding between the antigen and the antibody is obtained by subtracting the wavelength shift of the reference channel from the wavelength shift of the sensing channel, thereby improving the detection accuracy.
The double-channel optical fiber SPR sensing system formed by the double-channel optical fiber SPR biosensor comprises the double-channel optical fiber SPR sensor taking the multimode optical fiber as the optical path, a broadband light source and a spectrometer, wherein the input end of the double-channel optical fiber SPR sensor transmits light emitted by the broadband light source to the double-channel optical fiber SPR sensor through the multimode optical fiber optical path, the output end of the double-channel optical fiber SPR sensor is connected with the spectrometer, the spectrometer is connected to a computer through a data interface, and the double-channel optical fiber SPR biosensor is arranged in a solution of biomolecules to be detected.
Further, the dual-channel optical fiber SPR sensing system is arranged in a closed tubular container to be detected, and a sample inlet and a sample outlet are arranged at two ends of the container to be detected.
Further, the tubular container to be detected is provided with a fixed bracket, and the fixed bracket supports the tubular container to be detected and is arranged at a fixed position.
The outer surface of the sensing channel is respectively provided with a gold-plated film and a graphene oxide film from inside and outside as a biological sensing channel, the graphene oxide film is utilized to improve the fixing efficiency of the antibody, the specific combination between the antibody and the antigen fixed by the gold nanoparticles causes the shift of resonance wavelength, and the measurement of high sensitivity of the antigen is realized according to the shift of the resonance wavelength displayed on the spectrometer. And silver film is plated on the outer surface of the reference channel, and the wavelength movement amount caused by nonspecific adsorption is detected as a blank control, so that the detection accuracy is improved.
The preparation method of the dual-channel optical fiber SPR sensor comprises the following steps:
(1) Preparation of double-channel optical fiber sensor
Respectively welding multimode fibers at two ends of the first photonic crystal fiber and the second photonic crystal fiber through a welding machine, namely, the two sections of photonic crystal fibers are also connected through the welded multimode fibers, wherein the lengths of the first photonic crystal fiber and the second photonic crystal fiber are 0.5-2 cm, the discharge intensity during welding is 50-100 mW, the discharge current is 3000-5000 mA, and the welding temperature is 2000-3000 ℃;
(2) Preparation of reference channel
Plating a silver film on the surface of the first photonic crystal fiber through silver mirror reaction, wherein the thickness of the silver film is 40-60 nm;
(3) Preparation of sensing channel
Gold-plating film
Putting the second photonic crystal fiber into a vacuum ion beam sputtering instrument, and plating a gold film on the surface of the second photonic crystal fiber, wherein the current of the vacuum ion beam sputtering instrument is 5-7 mA, the time is 2-4 minutes, and the thickness of the gold film is 40-60 nm;
ii) fixed graphene oxide film
Immersing the second photonic crystal fiber of the gold-plated film in 0.5-2 mmol/L of 4-aminophenylthiophenol ethanol solution for 6-24 hours to perform surface amination of the gold film, wherein 4-aminophenylthiophenol molecules can be connected with the gold film through Au-S covalent bonds, and amine groups (-NH 2) are left outside to be further combined with epoxy groups of graphene oxide; washing with distilled water, immersing a second photonic crystal fiber into a graphene oxide aqueous solution of 0.05-1 mg/mL, placing the graphene oxide aqueous solution into an incubator with the temperature of 30-60 ℃, evaporating the graphene oxide dispersion liquid after 20-60 minutes, and fixing a graphene oxide film on the surface of a gold film by a physical evaporation method, wherein the thickness of the graphene oxide film is 0.2-0.6 nm;
iii immobilized antibodies
Immersing the second photonic crystal fiber in a mixed solution of 0.5-1 mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 0.1-0.4 mol/L and 0.5-1 mL of N-hydroxysuccinimide with the concentration of 0.1-0.4 mol/L, and washing with distilled water after 15-60 minutes; then immersing the antibody into an antibody solution to be immobilized for antibody immobilization;
iv gold nanoparticle immobilized antigen
Adding an antigen to be detected into gold nanoparticle dispersion liquid with the concentration of 500-1000 ppm and the diameter of 10-50 nm, and fixing the antigen on the surface of gold nanoparticles through electrostatic self-assembly;
(4) Detection of antigens
At the temperature of 25-37 ℃, adding a human immunoglobulin solution fixed by gold nanoparticles into the dual-channel optical fiber SPR sensor, realizing detection of the antigen according to resonance wavelength drift caused by specific binding between the antibody and the antigen fixed by the gold nanoparticles, and simultaneously obtaining wavelength shift caused by specific adsorption between the actual antigen and the antibody according to the wavelength shift of a reference channel.
Compared with the prior art, the invention has the beneficial effects that:
1. the dual-channel optical fiber SPR sensor has the unique advantages of high sensitivity, measurement error elimination caused by nonspecific adsorption and insensitivity to temperature by arranging the reference channel and the sensing channel;
2. the two SPR resonance valleys of the double channels are mutually separated so as to ensure that the two SPR resonance valleys cannot have adverse effects on each other in the detection process, thereby being more beneficial to biological sensing;
in conclusion, the invention solves the problems of low sensitivity and easiness in being influenced by liquid refractive index change and temperature fluctuation of the sensor in the prior art, and is very suitable for wide popularization in the fields of biological sensing and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a dual channel fiber SPR-based biosensor in accordance with embodiment 1 of the present invention;
FIG. 2 is a schematic diagram of the structure of a dual-channel optical fiber SPR biosensing system in embodiment 1 of the present invention;
FIG. 3 is a graph showing the spectra of the dual channel fiber SPR sensor of example 1 of the present invention at different refractive indices;
FIG. 4 is a graph showing the refractive index fit of the dual-channel optical fiber SPR sensor of example 1 of the present invention at different refractive indices;
FIG. 5 is a graph showing the shift in wavelength corresponding to the detection of human immunoglobulins at different concentrations by the dual channel optical fiber SPR sensor of example 1 of the present invention;
in the figure: 1. the device comprises a first photonic crystal fiber, a second photonic crystal fiber, a multimode fiber, a silver film, a gold film, a graphene oxide film, an antibody-goat anti-human immunoglobulin, an antigen-human immunoglobulin, a gold nanoparticle, a reference channel, a 2', a sensing channel, an A high-sensitivity optical fiber SPR biosensor, a broadband light source, a C, a spectrometer, a D, a multimode fiber light path, an E, a sample inlet, an F, a sample outlet, a G, a computer, an H, a glass tube, an I and a bracket.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise. Meanwhile, it should be clear that the dimensions of the respective parts shown in the drawings are not drawn in actual scale for convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail, but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further discussion thereof is necessary in subsequent figures.
In the description of the present invention, it should be understood that the azimuth or positional relationships indicated by the azimuth terms such as "front, rear, upper, lower, left, right", "lateral, vertical, horizontal", and "top, bottom", etc., are generally based on the azimuth or positional relationships shown in the drawings, merely to facilitate description of the present invention and simplify the description, and these azimuth terms do not indicate and imply that the apparatus or elements referred to must have a specific azimuth or be constructed and operated in a specific azimuth, and thus should not be construed as limiting the scope of protection of the present invention: the orientation word "inner and outer" refers to inner and outer relative to the contour of the respective component itself.
Spatially relative terms, such as "above … …," "above … …," "upper surface at … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial location relative to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "above" or "over" other devices or structures would then be oriented "below" or "beneath" the other devices or structures. Thus, the exemplary term "above … …" may include both orientations of "above … …" and "below … …". The device may also be positioned in other different ways (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
In addition, the terms "first", "second", etc. are used to define the components, and are only for convenience of distinguishing the corresponding components, and the terms have no special meaning unless otherwise stated, and therefore should not be construed as limiting the scope of the present invention.
Example 1
In this embodiment, a dual-channel optical fiber SPR biosensor for detecting human immunoglobulin is prepared, i.e., the antigen to be detected is human immunoglobulin, and the detected antibody is goat anti-human immunoglobulin.
As shown in fig. 1, a dual-channel optical fiber SPR biosensor includes a reference channel 1 'and a sensing channel 2', the reference channel 1 'and a detection channel 2' being connected by a multimode optical fiber, wherein:
the reference channel 1' comprises a first photonic crystal fiber 1, the length of the first photonic crystal fiber 1 is 0.5-2 cm, and two ends of the first photonic crystal fiber 1 are respectively welded with a multimode fiber 2; plating a silver film 4 on the surface of the first photonic crystal fiber 1, wherein the thickness of the silver film 4 is 40-60 nm; the reference channel 1' is used as a blank control to detect the wavelength shift caused by nonspecific adsorption;
the sensing channel 2' comprises a second photonic crystal fiber 2, the length of the second photonic crystal fiber 2 is 0.5-2 cm, the two ends of the second photonic crystal fiber 2 are respectively welded with a multimode fiber 3, the surface of the second photonic crystal fiber 2 is plated with a gold film 5, and the thickness of the gold film 5 is 40-60 nm; the graphene oxide film 6 is fixed on the surface of the gold film 5 through a covalent bond bonding method, the thickness of the graphene oxide film is 0.2-0.6 nm, the antibody-goat anti-human immunoglobulin 7 film is solidified on the graphene oxide film 6, and the graphene oxide film 6 is utilized to improve the fixing efficiency of the antibody-goat anti-human immunoglobulin 7; the antigen to be detected, namely the human immunoglobulin 8, is fixed by gold nanoparticles 9, the diameter of the gold nanoparticles is 10-50 nm, and the antibody-goat anti-human immunoglobulin 7 and the antigen-human immunoglobulin 8 are combined in the detection process, so that the gold nanoparticles 9 with the antigen-human immunoglobulin 8 fixed on the surface are fixed on a film of the antibody-goat anti-human immunoglobulin 7, and a coupling effect is generated between the gold film 5 and the gold nanoparticles 9.
As shown in fig. 2, the sensing system formed by the dual-channel optical fiber SPR biosensor comprises a dual-channel optical fiber SPR biosensor a, the system uses multimode optical fibers as light paths, namely multimode optical fibers are light paths D, the input end of the dual-channel optical fiber SPR biosensor a is connected with a broadband light source B with a spectrum of visible light band, the output end of the dual-channel optical fiber SPR biosensor a is connected with a spectrometer C, the spectrometer C is connected to a computer G through a data interface, the dual-channel optical fiber SPR biosensor a is arranged in a sealed tubular container to be detected, in this embodiment, the container to be detected is a glass tube H, two ends of the glass tube H are provided with a sample inlet E and a sample outlet F, and a solution to be detected enters through the sample inlet E of the glass tube H and flows out from the sample outlet F, so that the dual-channel optical fiber SPR biosensor a is arranged in the solution to be detected. The glass tube H is provided with a fixing bracket I which supports the glass tube H and is arranged at a fixed position.
The preparation method of the dual-channel optical fiber SPR biosensor comprises the following steps:
(1) Preparation of double-channel optical fiber sensor
The outer diameters of the first photonic crystal fiber 1 and the second photonic crystal fiber 2 of the sensing channel and the reference channel are 125 mu m, the sensing channel and the reference channel comprise 5 layers of air holes which are arranged in a regular hexagon, the diameters of the air holes are 4.8 mu m, the distance between the air holes is 7.7 mu m, the lengths of the first photonic crystal fiber 1 and the second photonic crystal fiber 2 are 1cm, the two ends of the first photonic crystal fiber 1 and the second photonic crystal fiber 2 are respectively welded with a multimode fiber 3 through a welding machine, the outer diameter of the multimode fiber 3 is 125 mu m, the core diameter is 62.5 mu m, the first photonic crystal fiber 1, the second photonic crystal fiber 2 and the multimode fiber 3 are made of quartz glass materials, the discharge intensity of the welding machine is 130mW, the discharge time is 3000ms, and the welding temperature is 2000 ℃;
(2) Preparation of reference channel
Plating a silver film 4 on the surface of the first photonic crystal fiber 1 through silver mirror reaction, wherein the thickness of the silver film 4 is 50nm;
(3) Preparation of sensing channel
Gold-plating film
Putting the second photonic crystal fiber 2 into a vacuum ion beam sputtering instrument, and plating a gold film 5 on the surface of the second photonic crystal fiber 2, wherein the current of the vacuum ion beam sputtering instrument is 7mA, the time is 2 minutes, and the thickness of the gold film 5 is 50nm;
ii) fixed graphene oxide film
Immersing the second photonic crystal fiber 2 of the gold-plated film in a petri dish containing 20mL of an ethanol solution of 4-aminophenylthiophenol having a concentration of 0.5mmol/L, washing with distilled water after 24 hours, allowing the 4-aminophenylthiophenol molecule to be connected to the gold film through an Au-S covalent bond, and forming an amine group (-NH) on the outer surface of the gold film 2 ) The method comprises the steps of carrying out a first treatment on the surface of the Fixing the second photonic crystal fiber 2 on a glass slide, taking 0.5mL of graphene oxide dispersion liquid with the concentration of 0.5mg/mL, immersing the second photonic crystal fiber 2, placing the second photonic crystal fiber 2 in a constant temperature box at 40 ℃, evaporating the graphene oxide dispersion liquid after 40 minutes, and fixing a graphene oxide film 6 on the surface of a gold film 5 by a physical evaporation method, wherein the thickness of the graphene oxide film 6 is 0.5nm;
iii immobilized antibody-goat anti-human immunoglobulin
Immersing the second photonic crystal fiber 2 in a mixed solution (1:1 mixture) of 1mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 0.4mol/L and 1mL of N-hydroxysuccinimide with the concentration of 0.1mol/L, and washing with distilled water after 20 minutes; then immersing the second photonic crystal fiber 2 into 500 mu L of antibody-goat anti-human immunoglobulin 7 solution with the concentration of 200 mu g/mL for 1 hour, so as to realize the fixation of the antibody-goat anti-human immunoglobulin 7 on the surface of the graphene oxide film 6;
iv gold nanoparticle immobilized antigen-human immunoglobulin
Adding antigen-human immunoglobulin 8 solutions with different concentrations into gold nanoparticle 9 dispersion liquid with the volume of 10mL, the concentration of 1000ppm and the diameter of 10nm, and fixing the antigen-human immunoglobulin 8 on the surface of the gold nanoparticle 9 through electrostatic self-assembly;
(4) Detection of antigens
At the temperature of 25 ℃, an antigen-human immunoglobulin 8 solution immobilized by gold nanoparticles 9 is added into a dual-channel optical fiber SPR sensor, an antibody-goat anti-human immunoglobulin 7 is specifically combined with the antigen-human immunoglobulin 8 immobilized by the gold nanoparticles 9 to cause the movement of resonance wavelength, the detection of human immunoglobulins with different concentrations is realized according to the movement amount of wavelength, the movement amount of wavelength corresponding to the human immunoglobulins with different concentrations is shown as a graph in fig. 5, a sensing channel 2 'is used for biological sensing, a reference channel 1' is used as a blank control, the nonspecific adsorption is detected, and the shift amount of resonance wavelength caused by the specific combination between an actual antibody (goat anti-human immunoglobulin) and an antigen (human immunoglobulin) is obtained by subtracting the movement amount of wavelength of the reference channel 1 'from the movement amount of wavelength of the sensing channel 2'.
The refractive index and the biosensing characteristic of the prepared dual-channel optical fiber SPR biosensor are tested:
in order to study the refractive index sensing performance of the dual-channel optical fiber SPR sensor provided by the invention, the sensor is connected into a sensing system taking a multimode optical fiber as a light path, and a deuterium-halogen lamp with the wavelength range of 215nm to 2500nm is used at the input endAs a light source, a marine optical spectrometer was used to detect resonance spectra, and then the dual-channel optical fiber SPR sensor was immersed in sodium chloride salt solutions of different refractive indexes, the resonance spectra being shown in fig. 3, and the resonance wavelength of the dual-channel optical fiber SPR sensor shifted rightward as the refractive index increased. The sensitivity of the sensor can be expressed as the shift Δλ of the resonance peak p Change delta n of refractive index from sample to be measured a Ratio of (2), i.e
FIG. 4 is a refractive index sensitivity fitting curve of the dual-channel optical fiber SPR sensor provided by the invention, according to the slope of the fitting curve shown in FIG. 4, the refractive index sensitivity of the dual-channel optical fiber SPR sensor provided by the invention can reach 13592nm/RIU, the linearity is 0.98896, the refractive index sensitivity of a reference channel 1 'is 2524nm/RIU, the linearity is 0.9659, the refractive index sensitivity of a sensing channel 2' is 5.4 times that of the reference channel 1 'only coated with a silver film, and the sensing channel 2' is coated with a gold film and a graphene oxide film fixed with the graphene oxide film, so that the graphene oxide has high-efficiency electron migration rate, the interaction between the electromagnetic field intensity of the sensor surface and an external medium is enhanced, the refractive index sensitivity of the sensor is improved, and the important effect of the dual-channel optical fiber SPR sensor provided by the invention on improving the detection sensitivity is illustrated.
The dual-channel optical fiber SPR biosensor utilizes a dual-channel structure, a reference channel is used as a blank control, wavelength movement caused by non-specific adsorption is detected, a sensing channel is used as a biosensing channel, the coupling effect between a gold film and gold nanoparticles is utilized to enhance the local electric field intensity to improve the detection sensitivity of the sensor, a graphene oxide film is fixed on the surface of a photonic crystal fiber plated with the gold film through a covalent bond bonding method, the fixation efficiency of an antibody (goat anti-human immunoglobulin) is improved by utilizing the graphene oxide film, resonance wavelength drift is caused by the specific combination between the antibody (goat anti-human immunoglobulin) and an antigen (human immunoglobulin) fixed by the gold nanoparticles, the antigen (human immunoglobulin) high-sensitivity measurement is realized according to the movement of the resonance wavelength, and the detection accuracy is also improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (8)
1. The utility model provides a binary channels optic fibre SPR biosensor which characterized in that includes reference channel and sensing channel, reference channel and detection channel pass through multimode fiber connection, wherein:
the reference channel comprises a first photonic crystal fiber, wherein two ends of the first photonic crystal fiber are respectively welded with a multimode fiber, and a silver film is plated on the surface of the first photonic crystal fiber; the reference channel is used as a blank control to detect the wavelength shift caused by nonspecific adsorption;
the sensing channel comprises a second photonic crystal fiber, two ends of the second photonic crystal fiber are respectively welded with multimode fibers, a gold plating film is coated on the surface of the second photonic crystal fiber, a graphene oxide film is fixed on the surface of the gold film, an antibody film layer which selectively absorbs biomolecules or chemical components is solidified on the graphene oxide film, an antigen corresponding to the antibody is fixed by the gold nanoparticles, and the antibody is combined with the antigen in the detection process, so that the gold nanoparticles with the antigen fixed on the surface are fixed on the antibody film, and a coupling effect is generated between the gold film and the gold nanoparticles;
the lengths of the first photonic crystal fiber of the reference channel and the second photonic crystal fiber of the sensing channel are 0.5-2 cm;
and a silver film is plated on the surface of the first photonic crystal of the reference channel, and the thickness of the silver film is 40-60 nm.
2. The dual-channel optical fiber SPR biosensor according to claim 1, wherein the gold film is coated on the surface of the second photonic crystal fiber of the sensing channel, and the thickness of the gold film is 40-60 nm.
3. The dual-channel optical fiber SPR biosensor according to claim 1, wherein the graphene oxide film is fixed on the surface of the gold film of the sensing channel, the graphene oxide film is fixed on the surface of the gold film by a covalent bonding method, and the fixing efficiency of the antibody is improved by using the graphene oxide film.
4. The dual-channel optical fiber SPR biosensor according to claim 1, wherein the graphene oxide film is fixed on the surface of the gold film of the sensing channel, and the thickness of the graphene oxide film is 0.2-0.6 nm.
5. The dual-channel optical fiber SPR biosensor according to claim 1, wherein the diameter of the gold nanoparticles of the sensing channel is 10-50 nm.
6. A dual-channel optical fiber SPR sensing system formed by the dual-channel optical fiber SPR biosensor as claimed in any one of claims 1 to 5, comprising a dual-channel optical fiber SPR sensor taking multimode optical fiber as an optical path, a broadband light source and a spectrometer, wherein the input end of the dual-channel optical fiber SPR sensor transmits light emitted by the broadband light source to the dual-channel optical fiber SPR sensor through the multimode optical fiber optical path, the output end of the dual-channel optical fiber SPR sensor is connected with the spectrometer, the spectrometer is connected to a computer through a data interface, and the dual-channel optical fiber SPR biosensor is placed in a solution of biomolecules to be detected.
7. The dual-channel optical fiber SPR sensing system according to claim 6, wherein the dual-channel optical fiber SPR sensing system is arranged in a closed tubular container to be detected, and a sample inlet and a sample outlet are arranged at two ends of the container to be detected; the tubular container to be detected is provided with a fixed bracket, and the fixed bracket supports the tubular container to be detected and is arranged at a fixed position.
8. The method for preparing the dual-channel optical fiber SPR biosensor according to any one of claims 1 to 5, which is characterized by comprising the following steps:
(1) Preparation of double-channel optical fiber sensor
Respectively welding multimode fibers at two ends of the first photonic crystal fiber and the second photonic crystal fiber through a welding machine, namely, the two sections of photonic crystal fibers are also connected through the welded multimode fibers, wherein the lengths of the first photonic crystal fiber and the second photonic crystal fiber are 0.5-2 cm, the discharge intensity during welding is 50-100 mW, the discharge current is 3000-5000 mA, and the welding temperature is 2000-3000 ℃;
(2) Preparation of reference channel
Plating a silver film on the surface of the first photonic crystal fiber through silver mirror reaction, wherein the thickness of the silver film is 40-60 nm;
(3) Preparation of sensing channel
Gold-plating film
Putting the second photonic crystal fiber into a vacuum ion beam sputtering instrument, and plating a gold film on the surface of the second photonic crystal fiber, wherein the current of the vacuum ion beam sputtering instrument is 5-7 mA, the time is 2-4 minutes, and the thickness of the gold film is 40-60 nm;
ii) fixed graphene oxide film
Immersing the second photonic crystal fiber of the gold-plated film in 0.5-2 mmol/L of 4-aminophenylthiophenol ethanol solution for 6-24 hours to perform surface amination of the gold film, connecting 4-aminophenylthiophenol molecules with the gold film through Au-S covalent bonds, and leaving amine groups (-NH 2) outside to be further combined with epoxy groups of graphene oxide; washing with distilled water, immersing a second photonic crystal fiber into a graphene oxide aqueous solution of 0.05-1 mg/mL, placing the graphene oxide aqueous solution into an incubator with the temperature of 30-60 ℃, evaporating the graphene oxide dispersion liquid after 20-60 minutes, and fixing a graphene oxide film on the surface of a gold film by a physical evaporation method, wherein the thickness of the graphene oxide film is 0.2-0.6 nm;
iii immobilized antibodies
Immersing the second photonic crystal fiber in a mixed solution of 0.5-1 mL of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 0.1-0.4 mol/L and 0.5-1 mL of N-hydroxysuccinimide with the concentration of 0.1-0.4 mol/L, and washing with distilled water after 15-60 minutes; then immersing the antibody into an antibody solution to be immobilized for antibody immobilization;
iv gold nanoparticle immobilized antigen
Adding an antigen to be detected into gold nanoparticle dispersion liquid with the concentration of 500-1000 ppm and the diameter of 10-50 nm, and fixing the antigen on the surface of gold nanoparticles through electrostatic self-assembly;
(4) Detection of antigens
At the temperature of 25-37 ℃, adding a human immunoglobulin solution fixed by gold nanoparticles into the dual-channel optical fiber SPR sensor, realizing detection of the antigen according to resonance wavelength drift caused by specific binding between the antibody and the antigen fixed by the gold nanoparticles, and simultaneously obtaining wavelength shift caused by specific adsorption between the actual antigen and the antibody according to the wavelength shift of a reference channel.
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CN109959635A (en) * | 2019-02-22 | 2019-07-02 | 东北大学 | A kind of optical fiber SPR sensor based on field coupling |
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CN111272703B (en) * | 2020-03-19 | 2022-10-14 | 南京信息工程大学 | Array type multi-channel optical fiber sensor and preparation method thereof |
CN112304906B (en) * | 2020-10-23 | 2024-01-23 | 重庆理工大学 | Dual-channel probe type 81-degree inclined fiber bragg grating sensor system and preparation method and application thereof |
CN113533259A (en) * | 2021-06-04 | 2021-10-22 | 天津大学 | Optical fiber SPR sensor based on dual-channel structure and detection method thereof |
CN113406324B (en) * | 2021-06-30 | 2023-01-24 | 吉林大学 | S-shaped optical fiber cone immunosensor, preparation method and application thereof |
WO2023123142A1 (en) * | 2021-12-29 | 2023-07-06 | 北京建工环境修复股份有限公司 | Surface plasma resonance sensing chip, and preparation method therefor and use thereof |
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