SPR (Surface Plasmon Resonance) optical fiber sensor for identifying bile duct cancer marker as well as preparation method and application of SPR optical fiber sensor

Abstract

The invention provides an SPR (Surface Plasmon Resonance) optical fiber sensor for identifying a bile duct cancer marker as well as a preparation method and application of the SPR optical fiber sensor. The CLU antibody is fixed on the surface of the optical fiber by utilizing a chemical cross-linking method, the SPR optical fiber sensor aiming at the bile duct cancer marker is prepared by combining the advantages of an optical fiber surface plasma resonance sensor, and a large number of CLU antibodies capable of specifically recognizing the bile duct cancer marker are fixed; when the optical fiber sensor is immersed in bile of a biliary duct cancer patient, antigen-antibody specific binding occurs, and the refractive index near the optical fiber probe is changed, so that the wavelength of the absorption peak of reflected light shifts. The optical fiber sensor disclosed by the invention has the capabilities of high-specificity recognition, rapid detection and accurate analysis on bile duct cancer, is simple to manufacture, convenient to use and low in production cost, only needs 200 microliters of detection quantity, and has huge clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of optical fiber chemical and biological sensing technology, and in particular to an SPR optical fiber sensor for identifying bile duct cancer markers, and a preparation method and application thereof. Background Art

[0002] Fiber optic chemical and biological sensors are sensors that convert the state of the measured chemical or biological object into a measurable optical signal. With the advantages of high sensitivity, strong specificity, and small size, they have been widely used in environmental food monitoring, biomedical diagnosis, and other fields. In recent years, with the rapid development of biomedicine, the detection of differences in protein components in body fluids and diseased tissues between symptomatic and non-symptomatic patients through comparative proteomics has become one of the important strategies for disease screening and diagnosis. However, the secretion of most disease markers (such as proteins, DNA, or RNA, etc.) in the human body is relatively low, and the changes in their content in the human body are even weaker. Therefore, the research and establishment of biosensors with high sensitivity, high specificity, and low detection limits are of great significance for the early diagnosis and treatment of diseases. The ultra-sensitive detection of disease markers using fiber optic biosensor technology plays a vital role in this.

[0003] Cholangiocarcinoma is a malignant tumor formed in the bile duct that transports digestive fluid bile. It is induced by uncontrolled cell proliferation and is the second most common primary hepatobiliary tumor after hepatocellular carcinoma. It has the characteristics of strong invasiveness and poor prognosis. However, the diagnostic accuracy and specificity of existing screening methods such as computed tomography (CT), magnetic resonance imaging (MRI) and endoscopy are not satisfactory. Therefore, some researchers expect to achieve accurate detection of it through cholangiocarcinoma markers. For example, the prior art discloses one or more of the artificial transport proteins SMAD4, SLC22A3 and GPRC5A as cholangiocarcinoma markers. Although the expression of the marker can be detected to determine whether the patient has cholangiocarcinoma, this method is not only time-consuming and costly, but also difficult to develop clinical applications. The prior art discloses a kit for cholangiocarcinoma prepared by using clusterin (CLU) and / or carbohydrate antigen 19-9 (CA19-9) separated from the bile of patients with bile duct stenosis. This invention gives a relatively accurate critical CLU concentration (726.2ng / mL) for bile duct cancer, but the detection process is not only very cumbersome and complicated, but also the ability to obtain the test results in a timely manner is also weak and still requires the assistance of other detection methods. Therefore, achieving rapid, real-time and accurate diagnosis of bile duct cancer is still a problem that needs to be solved in clinical medicine.

[0004] Based on the problems in the above background technology, it is necessary to provide an SPR optical fiber sensor for identifying bile duct cancer markers and a preparation method and application preparation method thereof. Summary of the invention

[0005] The present invention aims to provide an SPR optical fiber sensor for identifying bile duct cancer markers and a preparation method and application thereof, so as to solve the problem that bile duct cancer marker detection methods in the prior art are difficult to apply clinically.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing an SPR optical fiber sensor for identifying bile duct cancer markers, comprising the following steps:

[0008] Preparing Au film on the surface of optical fiber;

[0009] The optical fiber with Au film on the surface is immersed in 11-mercaptoundecanoic acid ethanol solution to modify the surface of the optical fiber with carboxyl groups;

[0010] The optical fiber modified with carboxyl groups is immersed in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber;

[0011] The optical fiber activated with carboxyl group is immersed in CLU antibody solution to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

[0012] Preferably, the concentration of the 11-mercaptoundecanoic acid ethanol solution is 60-100 mM.

[0013] Preferably, the concentration of EDC in the mixed aqueous solution of EDC and NHS is 72-120 mM.

[0014] Preferably, the concentration of NHS in the mixed aqueous solution of EDC and NHS is 36-60 mM.

[0015] Preferably, the Au film is prepared on the surface of the optical fiber by ion sputtering;

[0016] The thickness of the Au film is 50-55 nm.

[0017] Preferably, the optical fiber having the Au film on the surface is immersed in the 11-mercaptoundecanoic acid ethanol solution for 24 to 30 hours;

[0018] And / or, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS for a immersion time of 0.5 to 1 hour.

[0019] Preferably, in the step of immersing the carboxyl-activated optical fiber in the CLU antibody solution, the immersion time is 12 to 24 hours.

[0020] Preferably, the concentration of the CLU antibody solution is 400-1000 ng / mL.

[0021] In a second aspect, the present invention further provides an SPR optical fiber sensor for identifying bile duct cancer markers, which is prepared by the preparation method.

[0022] In a third aspect, the present invention also provides an SPR optical fiber sensor for identifying bile duct cancer markers prepared by the preparation method or the use of the SPR optical fiber sensor for identifying bile duct cancer markers in the specific identification and detection of bile duct cancer markers.

[0023] The SPR optical fiber sensor for identifying bile duct cancer markers and its preparation method and application of the present invention have the following beneficial effects compared with the prior art:

[0024] 1. The preparation method of the SPR fiber optic sensor for identifying bile duct cancer markers of the present invention uses a chemical cross-linking method to fix the CLU antibody on the surface of the optical fiber, combines the advantages of the optical fiber surface plasmon resonance sensor, and prepares an SPR fiber optic sensor for bile duct cancer markers, fixing a large number of CLU antibodies that can specifically identify bile duct cancer markers; when the incident light enters the sensitive area of ​​the optical fiber sensor through the optical fiber, it will stimulate the SPR effect of the gold film, forming a strong absorption peak on the reflection spectrum; when the optical fiber sensor is immersed in the bile of a bile duct cancer patient, the antigen-antibody will undergo specific binding, causing the refractive index near the optical fiber probe to change, thereby causing the reflected light absorption peak wavelength to shift. By analyzing the corresponding relationship between the wavelength change and the concentration of the detected object, the linear relationship between the wavelength change and the risk of bile duct cancer can be obtained, thereby realizing rapid detection and analysis of unknown clinical samples;

[0025] 2. The SPR optical fiber sensor for identifying cholangiocarcinoma markers prepared by the present invention has the ability of highly specific recognition, rapid detection and accurate analysis of cholangiocarcinoma, and is simple to make, easy to use, and has low production cost. The detection volume only requires 200 μL, and it has great potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 This is a physical picture of an optical fiber after an Au film is sputtered on the surface of the optical fiber according to the method in Example 1;

[0028] Figure 2This is a physical picture of the SPR optical fiber sensor for identifying bile duct cancer markers finally prepared according to the method in Example 1;

[0029] Figure 3 This is a scanning electron microscope image of the surface state of the SPR optical fiber sensor for identifying bile duct cancer markers finally prepared according to the method in Example 1;

[0030] Figure 4 A spectral response curve of a bile sample of a bile duct cancer marker CLU solution detected by an SPR optical fiber sensor for identifying bile duct cancer markers prepared according to the method of Example 1;

[0031] Figure 5 This is a scanning electron microscope image of the surface state of the SPR optical fiber sensor for identifying bile duct cancer markers finally prepared according to the method in Example 2;

[0032] Figure 6 The linear relationship curve diagram of the response of the SPR optical fiber sensor for identifying bile duct cancer markers prepared according to the method in Example 1 to detect the bile sample of the bile duct cancer marker CLU solution;

[0033] Figure 7 This is a comparison chart of the detection results of the SPR optical fiber sensor for identifying bile duct cancer markers prepared according to the method in Example 1 and the existing medical detection method (ELISA kit). DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0036] The present invention provides a method for preparing an SPR optical fiber sensor for identifying a bile duct cancer marker, comprising the following steps:

[0037] S1, preparing Au film on the surface of optical fiber;

[0038] S2, immersing the optical fiber with Au film on the surface in 11-mercaptoundecanoic acid ethanol solution to modify the surface of the optical fiber with carboxyl groups;

[0039] S3, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber;

[0040] S4. Immerse the carboxyl-activated optical fiber in a CLU antibody solution to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

[0041] The preparation method of the SPR optical fiber sensor for identifying bile duct cancer markers of the present invention comprises the following steps: preparing an Au film on the surface of an optical fiber; immersing the optical fiber with the Au film on the surface in an 11-mercaptoundecanoic acid (11-MUA) ethanol solution, so that a large number of carboxyl groups are attached to the outer side of the gold film on the surface of the optical fiber; and immersing the optical fiber modified with the carboxyl groups in EDC (i.e., 1-ethyl-(3-dimethylaminopropyl) carbodiimide, whose chemical formula is C 8 H 17 N 3) and NHS (N-hydroxysulfosuccinimide) in a mixed aqueous solution to activate the hydroxyl groups on the optical fiber surface; EDC and ENHS are selected here for carboxyl activation mainly because: EDC is a hapten coupling agent, which can activate the carboxyl group without affecting the structure and activity of biological proteins or nucleic acids; when EDC and NHS exist at the same time, Sulfo-NHS ester with higher amino reactivity can be generated, that is, the mixed use of the two can produce a synergistic effect, enhance the activity of the carboxyl group, and thus improve the binding efficiency with subsequent biological antibody proteins; the optical fiber after carboxyl activation is immersed in the CLU antibody solution. In this process, the amino group (-NH 2 ) will combine with the highly activated hydroxyl group (-COOH) on the surface of the optical fiber. Since the -OH in the carboxyl group is negatively charged and can also gain an electron, when the CLU antibody in the solution contacts the optical fiber probe, -NH 2 H + Will react with OH in -COOH - Combined with dehydration, a peptide bond "-CO-NH-" (amide bond) is formed, thereby fixing the CLU antibody protein on the surface of the optical fiber probe, and an SPR optical fiber sensor with the function of recognizing bile duct cancer markers can be obtained.

[0042] In some embodiments, the concentration of the 11-mercaptoundecanoic acid ethanol solution is 60-100 mM. Specifically, 11-mercaptoundecanoic acid is added to ethanol to obtain the 11-mercaptoundecanoic acid ethanol solution.

[0043] In some embodiments, the concentration of EDC in the mixed aqueous solution of EDC and NHS is 72-120 mM, and the concentration of NHS is 36-60 mM. Specifically, the mixed aqueous solution of EDC and NHS is obtained by adding EDC and NHS into water.

[0044] In some embodiments, an Au film is prepared on the surface of the optical fiber by ion sputtering;

[0045] The thickness of the Au film is 50 to 55 nm.

[0046] In some embodiments, in the step of immersing the optical fiber having the Au film on the surface in the 11-mercaptoundecanoic acid ethanol solution, the immersion time is 24 to 30 hours.

[0047] In some embodiments, the optical fiber modified with carboxyl groups is immersed in a mixed aqueous solution of EDC and NHS for a period of 0.5 to 1 hour.

[0048] In some embodiments, in the step of immersing the carboxyl-activated optical fiber in the CLU antibody solution, the immersion time is 12 to 24 hours.

[0049] In some embodiments, the concentration of the CLU antibody solution is 0.4-1 μg / μL; the CLU antibody is a clusterin (CLU) antibody, which can specifically recognize and bind to the CLU antigen; specifically, a commercially purchased CLU antibody solution with a concentration of 200-300 μg / mL is diluted 300-500 times with deionized water to obtain a CLU antibody solution with a concentration of 400-1000 ng / mL.

[0050] In some embodiments, preparing the Au film on the surface of the optical fiber specifically includes:

[0051] S11. Cut the 600μm diameter optical fiber into small sections of 8 to 10cm, and polish both ends of the optical fiber to a mirror finish with sandpaper. The thickness of the optical fiber can also be adjusted according to actual needs.

[0052] S12, then spraying gold on one end for 200 seconds using an ion sputtering apparatus to form a 50-55 nm thick gold mirror on its surface;

[0053] S13, then peel off the outer cladding, the stripping length is 1.0 to 1.5 cm;

[0054] S14, then soaking in acetone solution for 3 hours to completely remove the residual cladding on the surface of the fiber core, so that the fiber core is completely exposed;

[0055] S15. Control the required power to 14W, and use an ion sputtering device to spray the circumferential surface of the fiber core at one end with the gold mirror for more than 200s until the thickness of the gold film sprayed on the circumferential surface of the fiber core reaches 50-55nm, that is, the Au film is prepared on the surface of the optical fiber.

[0056] The preparation method of the SPR optical fiber sensor for identifying bile duct cancer markers of the present invention uses a chemical crosslinking method to fix CLU antibodies on the surface of optical fiber, combines the advantages of optical fiber surface plasma resonance sensors, and prepares a SPR optical fiber sensor for bile duct cancer markers, fixing a large number of CLU antibodies that can specifically identify bile duct cancer markers; when the incident light enters the sensitive area of ​​the optical fiber sensor through the optical fiber, it will stimulate the SPR effect of the gold film, forming a strong absorption peak on the reflection spectrum; when the optical fiber sensor is immersed in the bile of a bile duct cancer patient, the antigen-antibody will undergo specific binding, causing the refractive index near the optical fiber probe to change, thereby causing the reflected light absorption peak wavelength to shift. By analyzing the corresponding relationship between the wavelength change amount and the concentration of the detected object, the linear relationship between the wavelength change amount and the risk of bile duct cancer can be obtained, thereby realizing the rapid detection and analysis of unknown clinical samples; the optical fiber sensor of the present invention has high specific recognition, rapid detection and accurate analysis capabilities for bile duct cancer, and is simple to make, easy to use, and has low production cost. The detection amount only requires 200 μL, and has huge clinical application potential.

[0057] Based on the same inventive concept, the present invention also provides an SPR optical fiber sensor for identifying bile duct cancer markers, which is prepared by the above-mentioned preparation method.

[0058] Based on the same inventive concept, the present invention also provides an SPR optical fiber sensor for identifying bile duct cancer markers prepared by the above preparation method or the use of the above SPR optical fiber sensor for identifying bile duct cancer markers in the specific identification and detection of bile duct cancer markers.

[0059] Specifically, the SPR fiber optic sensor used to identify bile duct cancer markers was connected to the Y-shaped fiber optic port between the light source and the spectrometer. After the incident light was introduced, 0 ng / mL, 400 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL and 1000 ng / mL of bile duct cancer marker CLU solutions were detected in turn. When the CLU antibody on the surface of the fiber optic sensor specifically binds to CLU, the refractive index near the fiber optic sensor will change, causing the absorption peak wavelength of the reflection spectrum to shift. In this process, the fiber optic responds quickly, and a stable spectral curve can be obtained in only 5 to 10 seconds.

[0060] The following further describes the SPR fiber optic sensor for identifying bile duct cancer markers of the present application and its preparation method and application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] In the following examples, the CLU antibody solution used was purchased from MacLean's Reagent.

[0062] Example 1

[0063] The present invention provides a method for preparing an SPR optical fiber sensor for identifying a bile duct cancer marker, comprising the following steps:

[0064] S1. Preparing an Au film on the surface of an optical fiber, specifically comprising the following steps:

[0065] S11, cut the optical fiber with a diameter of 600 μm into small sections of 8 cm, and polish both ends of the optical fiber to a mirror surface with sandpaper;

[0066] S12, then one end is sprayed with gold for 200 seconds using an ion sputtering apparatus to form a 50 nm thick gold mirror on its surface;

[0067] S13, then peel off the outer cladding, the stripping length is 1.0 cm;

[0068] S14, then soaking in acetone solution for 3 hours to completely remove the residual cladding on the surface of the fiber core, so that the fiber core is completely exposed;

[0069] S15, controlling the required power to 14W, using an ion sputtering apparatus to spray the circumferential surface of the fiber core at one end with the gold mirror for more than 200s, until the thickness of the gold film sprayed on the circumferential surface of the fiber core reaches 50nm, that is, an Au film is prepared on the surface of the optical fiber;

[0070] S2, immersing the optical fiber with Au film on the surface in 11-mercaptoundecanoic acid ethanol solution, so that the surface of the optical fiber is modified with carboxyl groups; the concentration of 11-mercaptoundecanoic acid ethanol solution is 60mM, and the immersion time is 24h;

[0071] S3, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber; the concentration of EDC in the mixed aqueous solution of EDC and NHS is 72mM, the concentration of NHS in the mixed aqueous solution of EDC and NHS is 36mM, and the optical fiber modified with carboxyl groups is immersed in the mixed solution of EDC and NHS for 0.5h;

[0072] S4. Immerse the carboxyl-activated optical fiber in a 300-fold diluted CLU antibody solution (i.e., a commercially available 200 μg / mL CLU antibody solution is diluted 300 times with deionized water to obtain a CLU antibody solution with a concentration of 667 ng / mL) for 12 hours to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

[0073] Example 2

[0074] The present invention provides a method for preparing an SPR optical fiber sensor for identifying a bile duct cancer marker, comprising the following steps:

[0075] S1. Preparing an Au film on the surface of an optical fiber, specifically comprising the following steps:

[0076] S11, cut the optical fiber with a diameter of 600 μm into small sections of 10 cm, and polish both ends of the optical fiber to a mirror surface with sandpaper;

[0077] S12, then one end is sprayed with gold for 200 seconds using an ion sputtering apparatus to form a 50 nm thick gold mirror on its surface;

[0078] S13, then peel off the outer cladding, the stripping length is 1.5 cm;

[0079] S14, then soaking in acetone solution for 3 hours to completely remove the residual cladding on the surface of the fiber core, so that the fiber core is completely exposed;

[0080] S15, controlling the required power to 14W, using an ion sputtering apparatus to spray the circumferential surface of the fiber core at one end with the gold mirror for more than 200s, until the thickness of the gold film sprayed on the circumferential surface of the fiber core reaches 50nm, that is, an Au film is prepared on the surface of the optical fiber;

[0081] S2, immersing the optical fiber with Au film on the surface in 11-mercaptoundecanoic acid ethanol solution, so that the surface of the optical fiber is modified with carboxyl groups; the concentration of 11-mercaptoundecanoic acid ethanol solution is 100mM, and the immersion time is 24h;

[0082] S3, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber; the concentration of EDC in the mixed aqueous solution of EDC and NHS is 120mM, the concentration of NHS in the mixed aqueous solution of EDC and NHS is 60mM, and the optical fiber modified with carboxyl groups is immersed in the mixed solution of EDC and NHS for 0.5h;

[0083] S4. Immerse the carboxyl-activated optical fiber in a 200-fold diluted CLU antibody solution (i.e., a commercially available 200 μg / mL CLU antibody solution is diluted 200 times with deionized water to obtain a 1000 ng / mL CLU antibody solution) for 12 hours to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

[0084] Example 3

[0085] The present invention provides a method for preparing an SPR optical fiber sensor for identifying a bile duct cancer marker, comprising the following steps:

[0086] S1. Preparing an Au film on the surface of an optical fiber, specifically comprising the following steps:

[0087] S11, cut the optical fiber with a diameter of 600 μm into small sections of 9 cm, and polish both ends of the optical fiber to a mirror surface with sandpaper;

[0088] S12, then one end is sprayed with gold for 200 seconds using an ion sputtering apparatus to form a 50 nm thick gold mirror on its surface;

[0089] S13, then peel off the outer cladding, the stripping length is 1.5 cm;

[0090] S14, then soaking in acetone solution for 3 hours to completely remove the residual cladding on the surface of the fiber core, so that the fiber core is completely exposed;

[0091] S15, controlling the required power to 14W, using an ion sputtering apparatus to spray the circumferential surface of the fiber core at one end with the gold mirror for more than 200s, until the thickness of the gold film sprayed on the circumferential surface of the fiber core reaches 50nm, that is, an Au film is prepared on the surface of the optical fiber;

[0092] S2, immersing the optical fiber with Au film on the surface in 11-mercaptoundecanoic acid ethanol solution, so that the surface of the optical fiber is modified with carboxyl groups; the concentration of 11-mercaptoundecanoic acid ethanol solution is 80mM, and the immersion time is 24h;

[0093] S3, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber; the concentration of EDC in the mixed aqueous solution of EDC and NHS is 96mM, the concentration of NHS in the mixed aqueous solution of EDC and NHS is 48mM, and the optical fiber modified with carboxyl groups is immersed in the mixed aqueous solution of EDC and NHS for 0.5h;

[0094] S4. Immerse the carboxyl-activated optical fiber in a 400-fold diluted CLU antibody solution (i.e., a commercially available 200 μg / mL CLU antibody solution is diluted 400 times with deionized water to obtain a 500 ng / mL CLU antibody solution) for 12 hours to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

[0095] Performance Characterization

[0096] Figure 1 This is a real picture of the optical fiber after the Au film is sputtered on the surface of the optical fiber according to the method in Example 1.

[0097] Figure 2 This is a physical picture of the SPR optical fiber sensor for identifying bile duct cancer markers that is finally prepared according to the method in Example 1.

[0098] Figure 3 This is a scanning electron microscope image of the surface state of the SPR optical fiber sensor for identifying bile duct cancer markers finally prepared according to the method in Example 1.

[0099] from Figure 3 It can be seen that Figure 3 The raised part in the figure is the CLU antibody fixed on the surface of the optical fiber sensor, and its lateral dimension is about 50 to 150 nm.

[0100] The SPR optical fiber sensor for identifying bile duct cancer markers prepared in Example 1 is used to detect bile duct cancer markers, and the specific steps are as follows:

[0101] The SPR fiber optic sensor used to identify the bile duct cancer marker was connected to the Y-type fiber optic port between the light source and the spectrometer. After the incident light was introduced, the bile duct cancer marker CLU aqueous solutions of 0 ng / mL, 400 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL and 1000 ng / mL were detected in sequence, and the spectral response curve of the bile sample was obtained, as shown in FIG. Figure 4 shown.

[0102] from Figure 4 It can be seen that with the continuous increase of CLU protein concentration, the absorption peak position of the spectral response curve is also shifting regularly, and the shift increases with the increase of CLU protein concentration. This shows that the prepared cholangiocarcinoma optical fiber sensor can accurately identify CLU protein in the sample.

[0103] Figure 5 This is a scanning electron microscope image of the surface state of the SPR optical fiber sensor for identifying bile duct cancer markers finally prepared according to the method in Example 2.

[0104] from Figure 5 It can be seen that a higher concentration of the modification reagent and a lower antibody dilution factor can link more CLU antibodies on the surface of the fiber optic detector, making it suitable for detecting high-concentration clinical samples.

[0105] The SPR optical fiber sensor for identifying bile duct cancer markers prepared in Example 2 is used to detect bile duct cancer markers, and the specific steps are as follows:

[0106] The SPR fiber optic sensor used to identify the bile duct cancer marker was connected to the Y-type fiber optic port between the light source and the spectrometer. After the incident light was introduced, the bile duct cancer marker CLU aqueous solutions of 0 ng / mL, 400 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL and 1000 ng / mL were detected in sequence, and the linear relationship curve of the bile sample response was obtained, as shown in FIG. Figure 6 shown.

[0107] from Figure 6 It can be seen that there is a good linear relationship between the CLU protein content in the sample and the offset of the spectral absorption peak in the range of 200-1000 ng / mL. When detecting bile solutions of unknown concentration, the specific concentration can be calculated based on the offset of the absorption peak of the absorption spectrum.

[0108] Furthermore, the SPR optical fiber sensor for identifying bile duct cancer markers prepared in Example 1 and the existing medical detection method (ELISA reagent, purchased from McLean C772760 human clusterin (CLU) ELISA research kit) were used to detect the actual bile duct cancer marker CLU solution. The detection results are as follows: Figure 7 shown.

[0109] Specifically, Figure 7Fiber optical sensor represents the test result of the SPR optical fiber sensor in Example 1, Elisa represents the test result using the existing Elisa kit, and Standard represents the actual cholangiocarcinoma marker CLU solution concentration (the concentrations are 400 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, and 1000 ng / mL, respectively)

[0110] from Figure 7 It can be seen that the CLU protein contents detected by the SPR optical fiber sensor for identifying bile duct cancer markers of the present invention are: 475.51ng / mL, 641.99ng / mL, 717.72ng / mL, 741.09ng / mL, 956.85ng / mL, respectively, and the results obtained by using the Elisa kit are: 334.53ng / mL, 418.13ng / mL, 515.70ng / mL, 567.63ng / mL, 830.71ng / mL. By comparison, it can be seen that the SPR optical fiber sensor for identifying bile duct cancer markers of the present invention has higher accuracy and can better reflect the true value of the CLU protein content in the bile sample.

[0111] In summary, the present invention uses chemical cross-linking method to fix CLU antibody on the surface of optical fiber, combines the advantages of optical fiber surface plasmon resonance sensor, and prepares an optical fiber sensor with high specific recognition, rapid detection and accurate analysis capabilities for bile duct cancer markers. The optical fiber sensor is simple to make, easy to use, and has low production cost, which is easy to realize and easy to promote and apply.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an SPR optical fiber sensor for identifying bile duct cancer markers, characterized in that: The following steps are involved: Preparing Au film on the surface of optical fiber; The optical fiber with Au film on the surface is immersed in 11-mercaptoundecanoic acid ethanol solution to modify the surface of the optical fiber with carboxyl groups; The optical fiber modified with carboxyl groups is immersed in a mixed aqueous solution of EDC and NHS to activate the carboxyl groups on the surface of the optical fiber; The optical fiber activated with carboxyl group is immersed in CLU antibody solution to obtain an SPR optical fiber sensor for identifying bile duct cancer markers.

2. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The concentration of the 11-mercaptoundecanoic acid ethanol solution is 60-100 mM.

3. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The concentration of EDC in the mixed aqueous solution of EDC and NHS is 72-120 mM.

4. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The concentration of NHS in the mixed aqueous solution of EDC and NHS is 36-60 mM.

5. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The Au film was prepared on the optical fiber surface by ion sputtering; The thickness of the Au film is 50-55 nm.

6. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The step of immersing the optical fiber having the Au film on the surface in an 11-mercaptoundecanoic acid ethanol solution for 24 to 30 hours; And / or, immersing the optical fiber modified with carboxyl groups in a mixed aqueous solution of EDC and NHS for a immersion time of 0.5 to 1 hour.

7. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The optical fiber activated by the carboxyl group is immersed in the CLU antibody solution for 12 to 24 hours.

8. The method for preparing the SPR optical fiber sensor for identifying bile duct cancer markers according to claim 1, characterized in that: The concentration of the CLU antibody solution is 400-1000 ng / mL.

9. An SPR optical fiber sensor for identifying bile duct cancer markers, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of an SPR optical fiber sensor for identifying a cholangiocarcinoma marker prepared by the preparation method according to any one of claims 1 to 8 or an SPR optical fiber sensor for identifying a cholangiocarcinoma marker according to claim 9 in specific identification and detection of a cholangiocarcinoma marker.

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