CN113406324B - S-shaped optical fiber cone immunosensor, preparation method and application thereof - Google Patents
S-shaped optical fiber cone immunosensor, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an S-shaped optical fiber cone immunosensor, a preparation method and application thereof, and belongs to the field of optical fiber biosensors. The method for preparing the optical immunosensor has the advantages of simple preparation process, low cost and short period, and the immunosensor improves sensitivity, specificity and stability, is quick in response, and can be used for label-free online detection.
Description
Technical Field
The invention belongs to the field of optical fiber biosensors, and particularly relates to an S-shaped optical fiber cone immunosensor, a preparation method and application thereof.
Background
Tumor marker detection is an indispensable operation in the process of diagnosing malignant tumor diseases, and can improve the accuracy of tumor disease diagnosis. Carcinoembryonic antigen is one of common tumor markers, is over-expressed in a plurality of human cancers, and has important clinical value in the aspects of differential diagnosis, curative effect evaluation, disease condition monitoring and the like of malignant tumors. The optical fiber biological sensing technology is a multidisciplinary crossed optical detection technology obtained by combining the optical fiber sensing technology and the biological specificity recognition technology, and can be used in the field of biochemical molecular detection. As a commonly used optical fiber structure, a sensing device based on the principle of optical fiber mode-to-mode interference has been widely researched and applied in recent years. The optical fiber biosensor has the characteristics of small volume, high sensitivity, no mark, online property, good biocompatibility, electromagnetic interference resistance and the like, and can be applied to the fields of biochemical detection such as immunodetection, gene diagnosis, cancer screening, drug research and development, environmental monitoring, food safety and the like. The working principle of the optical fiber biosensor is as follows: the surface of the optical fiber is functionally modified and processed, probe biomolecules are bound, and the optical fiber biosensor has the capability of specific recognition, so that the low-concentration specific detection of specific biomolecules can be realized.
Currently, the more popular optical fiber biosensors studied include long-period optical fiber grating biosensors, optical fiber bragg grating biosensors, inclined optical fiber grating biosensors, and D-type optical fiber biosensors. So far, most of the bio-sensitive materials used in the optical fiber immunosensor are simple graphene oxide or gold nanoparticles, etc., and most of the bio-probe molecules are traditional monoclonal antibodies. Although the optical fiber biosensors have simple structures and are easy to operate, the refractive index sensitivity, the specificity and the like are not high. In order to improve the sensitivity and specificity of the optical fiber biosensor and to miniaturize the device, many new microstructure optical fibers and sensitized materials are continuously proposed, and have prominent potential application value in the fields of biomedical diagnosis, food monitoring, drug research and development, environmental monitoring and the like. The S-shaped optical fiber cone structure is a Mach-Zehnder intermodal interference structure, is simple to manufacture, strong in robustness and high in sensitivity, and the optical fiber biosensor based on the structure has great research potential in the aspect of biomolecule detection. The graphene oxide and the gold nanoparticles have high stability, specific surface area, optical performance and biological affinity, and the combination of the graphene oxide and the gold nanoparticles can be used as a good sensitizing material. With the continuous development of antibody technology, nano antibodies with small volume, higher affinity and stronger specificity compared with the traditional antibodies have been researched. In addition, the nano antibody also has the characteristics of stability, simple preparation, short period, acid and alkali resistance and the like. Therefore, the nanobody is more suitable for use as a probe molecule in a biosensor to improve the characteristics of the biosensor, such as specificity, sensitivity and stability. Therefore, the development of the optical fiber immunosensor which is compact in structure, simple to manufacture, high in sensitivity and specificity and good in stability is of great significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to: provides a preparation method and application of an S-shaped optical fiber cone immunosensor functionalized by a graphene oxide/gold nanoparticle composite membrane. Firstly, an S-shaped optical fiber cone is prepared by a fusion splicer to serve as a sensing platform, so that the sensitivity of response to the environment refractive index can be improved; then, graphene oxide and gold nanoparticles are respectively self-assembled on the surface of the S-shaped optical fiber cone to serve as a composite biological sensitive membrane, wherein the graphene oxide is of a sheet structure and can be fully connected with the surface of the optical fiber due to the fact that the graphene oxide has larger specific surface area, optical permeability and biological affinity, so that the graphene oxide has the functions of enlarging a biological sensing supporting surface and facilitating the connection of subsequent biochemical molecules, the gold nanoparticles are of a structure similar to an ellipsoid and can be combined with the graphene oxide and firmly combined with a nano antibody due to the fact that the gold nanoparticles have larger specific surface area and biological affinity which is superior to that of the graphene oxide, so that the two materials are combined to prepare the sensitive membrane which is superior to the sensitive membrane consisting of single components in characteristics; finally, the nano antibody with small volume, high affinity and strong specificity is connected to realize the specificity recognition and combination of the carcinoembryonic antigen, thereby playing the role of improving the specificity, stability and sensitivity of antigen detection. Therefore, the optical immunosensor prepared by the method has the advantages of simple preparation process, low cost and short period, the sensitivity, the specificity and the stability of the immunosensor are greatly improved, the response is fast, and label-free online detection can be realized.
The invention is realized by the following scheme:
a preparation method of an S-shaped optical fiber cone immunosensor comprises the following specific steps:
(1) And preparing the S-shaped optical fiber cone:
taking a section of 30-60cm long optical fiber, stripping a coating layer 2-4cm in the middle by using an optical fiber pliers, and finally wiping the optical fiber with the coating layer stripped off part cleanly by using alcohol cotton along the same axial direction of the optical fiber to finish optical fiber pretreatment; adjusting the optical fiber clamps at two ends of the fusion splicer to axially stagger the optical fiber clamps at 100-250 mu m, then flatly placing the pretreated optical fiber in a groove of the fusion splicer, adjusting the placement position of the optical fiber to enable a discharge electrode of the fusion splicer to be opposite to the middle part of the optical fiber with the coating layer stripped, and finally fixing the optical fiber clamps at two ends; the discharge time and the discharge current of the welding machine tapered bit are set, and the welding machine tapered bit is divided into two stages: the discharge time of the first stage is 7-15ms, and the discharge current is 8-16mA; the discharge time of the second stage is 6-12ms, and the discharge current is 6-10mA; finally, pressing a Fuse key on the welding machine, and starting discharging and tapering by the welding machine;
(2) The method comprises the following steps of (1) modifying the S-shaped optical fiber cone by using graphene oxide and gold nanoparticles:
performing biological functionalization on the S-shaped optical fiber cone prepared in the step (1), wherein the specific steps are as follows: immersing the S-shaped optical fiber cone into acetone, standing for 10-60min, cleaning, immersing into 0.5-2M NaOH aqueous solution, and standing for 1-4h; then 3-aminopropyltriethoxysilane-water solution with the volume concentration of 1-10% is used for soaking for 1-6h, after cleaning, the solution is heated for 5-20min at the temperature of 50-150 ℃, and then graphene oxide ethanol solution is used for soaking for 1-6h; soaking the mixture for 2 to 10 hours by using a 3-mercaptopropyltriethoxysilane-benzene solution with the volume concentration of 0.01 to 1 percent, and finally soaking the mixture for 5 to 30 hours by using a colloidal gold solution;
(3) And binding of the nano antibody:
further modifying the S-shaped optical fiber cone functionalized by the graphene oxide/gold nano particles prepared in the step (2) with a biological probe molecule to realize the specific recognition of the carcinoembryonic antigen, and the steps are as follows: immersing the S-shaped optical fiber cone functionalized by the graphene oxide/gold nanoparticles into a 10-40mM 11-mercaptoundecanoic acid ethanol solution, and standing for 10-60min; soaking in 2- (N-morpholinyl) ethanesulfonic acid buffer solution with concentration of 10-40mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for 19-50min, and then soaking in phosphate buffer solution with concentration of 30-90mM N-hydroxysuccinimide for 10-50min; soaking in phosphate buffered saline solution containing 0.1-3mg/mL of nano antibody for 10-60min, soaking in 0.2-2mg/mL of bovine serum albumin solution, incubating for 0.5-2h, and washing with phosphate buffered saline solution to remove unbound protein.
Further, the optical fiber used in step (1) is a single mode fiber (SMF-28 e).
Further, the single mode fiber used had a core diameter of 9 μm, a cladding diameter of 125 μm, and a coating diameter of 250 μm.
Further, the type of the optical fiber fusion splicer used in the step (1) is: ericsson FSU 995PM.
Further, the S-shaped optical fiber taper structure prepared in the step (1) has a taper waist diameter of 30-60 μm and a taper length of 600-900 μm.
Furthermore, the concentration of the used graphene oxide solution (NO: XF2241, nanjing XFNANNO) is 2mg/mL, and the diameter of the graphene oxide nanosheet is larger than 500nm.
Furthermore, the gold nanoparticles are prepared by a citric acid reduction method, and the diameter of the gold nanoparticles is 10-60nm.
Furthermore, the used nano-antibody is derived from alpaca, and the correspondingly detected antigen is carcinoembryonic antigen protein.
The invention also aims to provide an application of the S-shaped optical fiber cone immunosensor in detection.
Compared with the existing optical fiber immunosensor preparation method, the method has the following advantages:
(1) The S-shaped optical fiber cone structure is used as a sensing platform, and the preparation process is simple, small, low in cost and high in environmental refractive index sensitivity.
(2) The optical fiber immunosensor has the advantages of strong specificity, high sensitivity, good stability and good repeatability, and has wide application prospect in practice.
Drawings
FIG. 1: the invention discloses a schematic diagram of an experimental device of a preparation method of an S-shaped optical fiber cone immunosensor;
FIG. 2: an optical microscope image of the prepared S-shaped optical fiber cone;
FIG. 3: the surface of the S-shaped optical fiber cone modified by the biochemical molecules prepared by the invention is scanned by an electron microscope spectrogram;
FIG. 4: the prepared S-shaped optical fiber cone surface atomic force microscope spectrogram modified by biochemical molecules is prepared;
FIG. 5: the detection schematic diagram of the S-shaped optical fiber cone immunosensor prepared by the invention;
FIG. 6: the S-shaped optical fiber cone immunosensor prepared by the invention detects a transmission spectrogram of carcino-embryonic antigen;
FIG. 7 is a schematic view of: the specific detection analysis chart of the S-shaped optical fiber cone immunosensor prepared by the invention is shown;
FIG. 8 is a diagram showing the repetitive detection and analysis of the S-shaped optical fiber cone immunosensor prepared by the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1:
and preparing the oxidized graphene/gold nanoparticle functionalized S-shaped optical fiber cone immunosensor based on the single mode fiber (SMF-28 e) by combining a welding machine arc discharge technology and an in-situ self-assembly technology. The optical immunosensor has the advantages of simple preparation process, low cost, high specificity and sensitivity and wide application prospect.
The S-shaped optical fiber cone immunosensor comprises the following specific preparation steps:
(1) Preparation of S-shaped optical fiber cone
The preparation method comprises the following specific steps: (1) a50 cm length of single mode optical fiber was cut with the following parameters: the core diameter was 9 μm, the cladding diameter 125 μm, and the coating diameter 250 μm. Then, 3cm of the coating layer was removed from the middle portion of the optical fiber by fiber pliers, and the optical fiber from which the coating layer was removed was wiped clean with alcohol cotton in the same direction as the axial direction of the optical fiber. (2) Adjusting the optical fiber clamps at two ends of a welding machine (Ericsson FSU 995 PM) to axially stagger the optical fiber clamps at a distance of 120 μm, then flatly placing the optical fiber processed in the step (1) in a V-shaped groove of the welding machine, carefully adjusting the placing position of the optical fiber to enable a discharge electrode of the welding machine to be opposite to the middle part of the optical fiber with the coating layer removed, and then fixing the optical fiber clamps at two ends to enable the optical fiber to be in a natural flat state. (3) The discharge time and the discharge current of the tapering of the welding machine are set, the tapering process is divided into two stages in total, and the discharge time and the discharge current of each stage are set as follows: discharge time 1=9ms and discharge current 1=10ma; discharge time 2=7ms and discharge current 2=10ma. And finally, pressing a Fuse button on the welding machine, and starting discharging and tapering by the welding machine. During discharging, the temperature of the electrode discharging area can reach about 2000 ℃ instantly, so that the optical fiber near the electrode is in a molten state, and the optical fiber is tapered. The prepared S-shaped optical fiber cone has a cone waist diameter of 46.8 μm and a cone length of 859.3 μm.
As shown in FIG. 1, it is a schematic diagram of an experimental apparatus for preparing an S-shaped optical fiber taper, and the specific steps are as described above. The prepared S-shaped fiber taper was then connected to a broadband light source (NKTPhotonics, denmark, super Compact) at one end and a spectrum analyzer (Yokogawa, japan, AQ 6370D) at the other end. The broadband light source outputs supercontinuum light, the supercontinuum light enters the spectrum analyzer through the S-shaped optical fiber cone structure, and the spectrum analyzer monitors the transmission spectrum in real time. FIG. 2 is an optical microscope photograph of an S-shaped fiber taper prepared on a single mode fiber, having a waist diameter of 46.8 μm and a taper length of 859.3 μm.
(2) The S-shaped optical fiber cone is modified by graphene oxide and gold nanoparticles;
surface functionalization is carried out on the S-shaped optical fiber cone prepared in the step (1), and the specific steps are as follows: firstly, immersing the S-shaped optical fiber cone into acetone and standing for 30min to remove organic impurities; after cleaning, immersing the mixture into 1.0M NaOH and standing for 2 hours; then soaking the optical fiber cone in a 5% volume concentration 3-aminopropyltriethoxysilane-water solution for 3h, heating at 95 ℃ for 10min after cleaning, and soaking in a graphene oxide solution for 4h; then 3-mercaptopropyltriethoxysilane-benzene solution with volume concentration of 0.1% is used for soaking for 6 hours, and finally the gold colloid solution is used for soaking for 24 hours. Namely, the functionalization of the S-shaped optical fiber taper surface is completed.
(3) Binding the nano antibody;
and (3) further binding the surface functionalized S-shaped optical fiber cone in the step (2) with a nano antibody probe molecule to realize specific binding to the carcinoembryonic antigen, and specifically comprising the following steps: (1) immersing in 20mM 11-mercaptoundecanoic acid-ethanol solution, and standing for 30min; (2) soaking in 25mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 2- (N-morpholino) ethanesulfonic acid buffer (0.1M pH =5.6, 0.9% NaCl) for 20min, followed by transferring to 60mM N-hydroxysuccinimide phosphate buffered saline (0.1M phosphate buffered saline, pH = 7.4) for 20min; (3) soaking in phosphate buffer solution containing 1mg/mL nano antibody for 20min, soaking in 1mg/mL bovine serum albumin solution, incubating for 1h to block excessive binding sites, and washing with phosphate buffer solution to remove unbound protein.
FIG. 3 is the surface scanning electron microscope spectrogram of the prepared S-shaped optical fiber cone immunosensor. The uneven appearance of the modified graphene oxide, gold nanoparticles and nanobody can be seen. FIG. 4 is the surface atomic force microscope spectrum of the prepared S-shaped optical fiber cone immunosensor. The surface morphology structure based on the elliptic gold nanoparticles can be more clearly seen. Therefore, the nano material is successfully assembled on the surface of the S-shaped optical fiber cone.
Example 2: carcinoembryonic antigen detection
Step (1) → (3) same as in example 1
And (4) connecting one end of the S-shaped optical fiber cone structure with a broadband light source (Superk Compact, NKTPHOTONics, denmark) and the other end with an optical spectrum analyzer (AQ 6370D, yokogawa, japan), and setting the resolution of the optical spectrum analyzer to be 0.02nm and the wavelength scanning range to be 1000nm-1700nm. The broadband light source outputs supercontinuum, and the spectrum analyzer monitors the transmission spectrum in real time during antigen detection. The carcinoembryonic antigen concentrations are respectively set to be 0nM, 0.2nM, 0.4nM, 0.6nM, 0.8nM, 1.0nM, 1.2nM and 1.4nM, and the concentration is detected from low to high during detection.
FIG. 5 is a schematic diagram of detection of an S-shaped optical fiber taper immunosensor.
FIG. 6 is a transmission spectrum of the prepared S-shaped optical fiber taper immunosensor. In the wavelength range of 1000nm-1700nm, a plurality of interference peaks exist in the transmission spectrum, and in a carcinoembryonic antigen detection experiment, one peak is selected to monitor the change of the spectrum. From the figure, it can be found that when the antigen concentration is increased from 0.0nM to 1.4nM, the transmission spectrum moves to the long wave direction, the red shift occurs, the total shift is 33.6nM, and the sensitivity of carcinoembryonic antigen detection is 24nM/nM and the lowest detection line is 0.015nM through linear fitting. It can be shown that the S-shaped fiber cone immunosensor prepared by the method has linear correlation between antigen concentration and spectrum drift distance in a detection experiment of carcinoembryonic antigen, and has higher sensitivity and lower detection limit.
Example 3: specificity detection and reproducibility detection
Firstly, the specificity detection of the S-shaped optical fiber cone immunosensor is verified, and the specific experimental steps are as follows:
step (1) → (3) same as in example 2.
And (4) connecting one end of the S-shaped optical fiber cone structure with a broadband light source (Superk Compact, denmark NKTPHOTONics), connecting the other end with a spectrum analyzer (AQ 6370D, yokogawa, japan), and setting the resolution of the spectrum analyzer to be 0.02nm and the wavelength scanning range to be 1000nm-1700nm. The broadband light source outputs super-continuous light, and the spectrum analyzer monitors the transmission spectrum in real time during antigen detection. The analytes to be detected were sequentially changed to phosphate buffered saline, bovine serum albumin, and goat serum albumin to observe the spectral changes, to verify that the spectral shift in example 2 was caused by the specific binding of carcinoembryonic antigen.
FIG. 7 shows the results of a specific detection verification experiment for the S-shaped fiber-cone immunosensor, and it can be seen that several unrelated analytes do not cause significant drift in the output spectrum. It is known that the spectral shift in example 2 is caused by the specific binding of carcinoembryonic antigen to nanobody.
Then, the repeatability of the S-shaped optical fiber cone immunosensor is verified, and the specific experimental steps are as follows:
an S-shaped fiber-cone immunosensor was prepared again according to the above-described method to verify the reproducibility of the sensors of the present invention. The repetition detection step (1) → (4) is the same as in example 2.
FIG. 8 is a line-fit plot of a reconstituted S-shaped fiber-cone immunosensor to carcinoembryonic antigen detection. As can be seen from the figure, the antigen concentration and the spectral drift distance of the re-prepared S-shaped fiber cone immunosensor in the detection experiment of carcinoembryonic antigen are linearly related, and the effect is the same as that of the S-shaped fiber cone immunosensor prepared for the first time. Therefore, the S-shaped optical fiber cone immunosensor prepared by the method has good repeatability.
Claims (8)
1. A preparation method of an S-shaped optical fiber cone immunosensor is characterized by comprising the following specific steps:
(1) Preparing an S-shaped optical fiber cone:
taking a section of 30-60cm long optical fiber, stripping a coating layer 2-4cm in the middle by using an optical fiber pliers, and finally wiping the optical fiber with the coating layer stripped off part cleanly by using alcohol cotton along the same axial direction of the optical fiber to finish optical fiber pretreatment; adjusting optical fiber clamps at two ends of the fusion splicer to axially stagger the optical fiber clamps at the two ends by a distance of 100-250 mu m, then flatly placing the pretreated optical fiber in a groove of the fusion splicer, adjusting the placing position of the optical fiber to ensure that a discharge electrode of the fusion splicer faces the middle part of the optical fiber with a coating layer removed, and finally fixing the optical fiber clamps at the two ends; the discharge time and the discharge current of the cone of the welding machine are set, and the welding machine is divided into two stages: the discharge time of the first stage is 7-15ms, and the discharge current is 8-16mA; the discharge time of the second stage is 6-12ms, and the discharge current is 6-10mA; finally, pressing a Fuse key on the welding machine, and starting discharging and tapering by the welding machine;
(2) The method comprises the following steps of (1) modifying the S-shaped optical fiber cone by using graphene oxide and gold nanoparticles:
performing biological functionalization on the S-shaped optical fiber taper prepared in the step (1), and specifically comprising the following steps: immersing the S-shaped optical fiber cone into acetone, standing for 10-60min, cleaning, and then immersing into NaOH aqueous solution with the concentration of 0.5-2M, standing for 1-4h; then soaking the substrate for 1-6h by using a 3-aminopropyltriethoxysilane-water solution with the volume concentration of 1-10%, heating the substrate for 5-20min at the temperature of 50-150 ℃ after cleaning, and then soaking the substrate for 1-6h by using a graphene oxide ethanol solution; soaking the mixture for 2 to 10 hours by using a 3-mercaptopropyltriethoxysilane-benzene solution with the volume concentration of 0.01 to 1 percent, and finally soaking the mixture for 5 to 30 hours by using a colloidal gold solution;
(3) And binding of the nano antibody:
further modifying the S-shaped optical fiber cone functionalized by the graphene oxide/gold nano particles prepared in the step (2) with a biological probe molecule to realize the specific recognition of the carcinoembryonic antigen, and the steps are as follows: immersing the S-shaped optical fiber cone functionalized by the graphene oxide/gold nanoparticles into a 10-40mM 11-mercaptoundecanoic acid ethanol solution, and standing for 10-60min; soaking in 2- (N-morpholinyl) ethanesulfonic acid buffer solution with concentration of 10-40mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for 19-50min, and then soaking in phosphate buffer solution with concentration of 30-90mM N-hydroxysuccinimide for 10-50min; soaking in phosphate buffered saline solution containing 0.1-3mg/mL of nano antibody for 10-60min, then soaking in 0.2-2mg/mL of bovine serum albumin solution, incubating for 0.5-2h, and washing with phosphate buffered saline solution to remove unbound protein.
2. The method according to claim 1, wherein the optical fiber used in step (1) is a single mode optical fiber.
3. The method according to claim 1, wherein the single-mode optical fiber has a core diameter of 9 μm, a cladding diameter of 125 μm, and a coating diameter of 250 μm.
4. The method according to claim 1, wherein the S-shaped optical fiber taper immunosensor has a taper waist diameter of 30-60 μm and a taper length of 600-900 μm in the S-shaped optical fiber taper structure prepared in the step (1).
5. The method for preparing an S-shaped optical fiber taper immunosensor according to claim 1, wherein a concentration of the used graphene oxide solution is 2mg/mL, and a diameter of the graphene oxide nanosheet is greater than 500nm.
6. The method according to claim 1, wherein the gold nanoparticles are prepared by citric acid reduction, and the diameter of the gold nanoparticles is 10-60nm.
7. The method according to claim 1, wherein the nanobody is derived from alpaca, and the antigen to be detected is carcinoembryonic antigen protein.
8. An S-shaped fiber taper immunosensor prepared by the method of any one of claims 1-7.
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| CN208705230U (en) * | 2018-08-22 | 2019-04-05 | 东北大学 | A kind of double channel optical fiber surface plasmon resonance biosensor |
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