Method for detecting tumor marker PD-L1 by using composite structure optical fiber sensor based on gold nano sea urchin

Abstract

The invention discloses a method for detecting a tumor marker PD-L1 by using a composite structure optical fiber sensor based on gold nano sea urchin, which comprises the following steps: preparing a pSC4 solution, and soaking NS-LSPR-FOS in the pSC4 solution for 12 hours; washing to remove uncombined pSC4 molecules; immersing the end face of the sensor in a PBS solution containing 20 [mu] g / mL of PD-L1 antibody for 1 h to form a specific recognition layer; soaking the end face of the sensor in a BSA solution for 20 minutes; the end faces of the sensors are sequentially immersed into PD-L1 antigen solutions with different concentrations, each group is immersed for 45 minutes, and the concentrations are from small to large; and recording a reflection spectrum once every 15 seconds through a spectrograph, tracking resonance valley wavelength movement, and detecting the concentration of PD-L1. Compared with liquid biopsy methods such as ELISA, IFA and a micro-fluidic chip, the liquid biopsy method has the advantages of small size, no mark, real-time detection, in-vivo detection and the like, is expected to overcome two challenges of low concentration and spatial-temporal heterogeneity characterization of tumor marker PD-L1 detection, and plays an important role in cancer research and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for detecting a tumor marker PD-L1 using a composite structure optical fiber sensor based on gold nano-sea urchin. Background Art

[0002] As an important breakthrough in cancer treatment, immune checkpoint inhibitors (ICIs) have significantly prolonged the 5-year survival period to 15.5%-23.2%. However, their efficacy is limited by the complex tumor microenvironment and dynamic changes in biomarkers. Currently, there is an urgent need to design highly sensitive, real-time monitoring biosensors to track the dynamic changes of key targets such as PD-1 / PDL-1, optimize treatment strategies, and develop new inhibitors.

[0003] At present, the commonly used clinical detection methods for tumor markers in samples such as serum, cells, and urine, such as ELISA and flow cytometry, have three core defects: first, in situ monitoring in vivo cannot be achieved, which hinders the real-time evaluation of the dynamic response to immunotherapy; second, the sensitivity is limited by technology, especially when detecting protein markers, the existing surface enhanced Raman scattering (SERS) and other technologies often require fluorescent labeling and have weak anti-interference ability; third, the detection equipment is bulky and complicated to operate, which is difficult to meet the needs of clinical rapid detection or portable detection. At the same time, the efficacy of ICIs is closely related to the changes in the expression of circulating tumor antigens, cytokines, and extracellular vesicle proteins. Rapid, convenient, high-throughput and highly sensitive monitoring of the dynamic fluctuations of these biomarkers is of decisive significance for precision treatment.

[0004] Fiber optic sensors provide a revolutionary solution to break through the bottleneck of existing technologies. Their unique advantages are: 1) high sensitivity and precision, based on the nanostructured localized surface plasmon resonance (LSPR) effect, single-molecule level detection can be achieved; 2) strong anti-electromagnetic interference ability, suitable for complex body fluid environments; 3) strong real-time monitoring capability, miniaturized (<125μm) can be implanted in the body for continuous dynamic tracking; 4) multi-parameter detection potential, through wavelength / intensity multimodal analysis, PD-L1, CTLA-4 and other targets can be monitored simultaneously; 5) equipment integration advantages, the fiber optic sensor system has the characteristics of light weight, small size, flexibility, long transmission distance, etc., which is convenient for the development of bedside detection equipment. At present, the feasibility of fiber optic sensors based on nanostructured LSPR has been verified in prostate-specific antigen detection, but the sensitivity still needs to be improved.

[0005] Therefore, in order to overcome the existing difficulties in real-time monitoring of tumor markers in vivo, a sensitive, micron-scale, and easy-to-operate detection strategy is urgently needed to address the challenges of low detection sensitivity and inability to real-time monitoring of fiber optic sensors based on nanostructured LSPR. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting the tumor marker PD-L1 using a composite structure optical fiber sensor based on gold nano-sea urchins, thereby solving the problems of low sensitivity, inability to monitor in real time, complex operation and high cost in the existing tumor marker PD-L1 detection technology.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] A method for detecting tumor marker PD-L1 based on a composite structure optical fiber sensor of gold nano-sea urchin comprises the following steps:

[0009] (1) Preparation of localized surface plasmon resonance fiber sensor (NS-LSPR-FOS) for specific detection of PD-L1: Prepare a 10 mM 4-sulfonylcaliphatic [4]arene hydrate (pSC4) solution, place the NS-LSPR-FOS in the pSC4 solution and soak for 12 hours; allow pSC4 to form a monolayer on the surface of the gold nano-sea urchin and connect via Au-S bonds;

[0010] (2) Connection of PD-L1 antibody to pSC4 monolayer: After pSC4 modification, the end face of the optical fiber was cleaned with deionized water to remove unbound pSC4 molecules on the surface; the end face of the optical fiber sensor was immersed in a PBS solution containing 20 μg / mL PD-L1 antibody for 1 hour to form a PD-L1-specific recognition layer on the surface of the gold nano-sea urchin; and the reflectance spectrum of the optical fiber sensor was recorded by a spectrometer.

[0011] (3) BSA blocking: Rinse the sensor end face with PBS and immerse the fiber optic sensor end face in a 10 mg / mL bovine serum albumin (BSA) solution for 20 minutes to block the active sites on the surface that are not bound to PD-L1 to avoid nonspecific binding;

[0012] (4) PD-L1 antigen detection:

[0013] (41) The end face of the optical fiber sensor was immersed in PD-L1 antigen solutions of different concentrations in sequence, with each group immersed for 45 minutes, from small to large concentrations, and then washed with PBS to remove the non-specifically bound PD-L1 antigen;

[0014] (42) The reflectance spectrum of the optical fiber sensor was recorded every 15 seconds by a spectrometer, and the wavelength shift of the resonance valley was tracked to achieve quantitative detection of different concentrations of PD-L1 protein;

[0015] (43) Based on the antigen detection calibration curve obtained by the optical fiber sensor in the early stage, the PD-L1 concentration was calculated according to the measured resonance valley wavelength shift value.

[0016] Preferably, in the above technical solution, in step (1), the thickness of the photon cavity of NS-LSPR-FOS is 340-460 nm, and the size and spacing of the gold nano-sea urchins are 50-80 nm.

[0017] Preferably, in the above technical solution, in step (2), during the connection process between the PD-L1 antibody and the pSC4 monolayer, the ambient temperature is maintained at 20±0.5°C.

[0018] Preferably, in the above technical solution, in step (41), the concentration of the PD-L1 antigen solution is 50 to 4000 ng / ml, and before and after immersion in antigen solutions of different concentrations, the samples are washed in a PBS solution to remove non-specifically bound PD-L1 antigens.

[0019] Preferably, in the above technical solution, step (42) is specifically as follows: first, a baseline is established using the environmental measurement value of the PBS solution after BSA blocking and before detection; after immersion in a solution containing PD-L1 antibodies, antigen-antibody binding occurs, and the wavelength corresponding to the resonance valley undergoes a red shift; thereafter, impurities not bound to the PD-L1 antibody are washed away with PBS buffer, the resonance wavelength reaches the binding point between the antibody and PD-L1 and remains stable, and the wavelength shift value of the resonance valley is measured.

[0020] Preferably, in the above technical solution, step (4) further comprises:

[0021] After preparing a PD-L1 antigen solution of known concentration, the optical fiber sensor was immersed in a SIBP-modified gold nanoparticle (AuNPs-SIBP) solution for 45 minutes to allow SIBP to bind to the PD-L1 antigen. The reflectance spectrum of the optical fiber sensor was recorded every 15 seconds to track the wavelength movement of the resonance valley to achieve amplification of the PD-L1 detection signal.

[0022] Preferably, in the above technical solution, AuNPs-SIBP is prepared by the following method:

[0023] Prepare 100 μg / mL SIBP solution and 0.25 mg / mL 10 nm gold nanoparticle solution;

[0024] The SIBP solution and the gold nanoparticle solution were mixed in a volume ratio of 1:1 and allowed to stand at 4°C for 12 h to form an Au-S bond between the thiol group (-SH) of cysteine ​​in the polypeptide and the gold nanoparticles to obtain AuNPs-SIBP.

[0025] Preferably, the above technical solution further comprises: cleaning the end face of the optical fiber sensor with a PBS solution to remove unbound AuNPs-SIBP.

[0026] The above technical solution of the present invention has the following beneficial effects:

[0027] (1) High-sensitivity detection: This application achieves high-sensitivity detection of PD-L1 by designing a nanostructured localized surface plasmon resonance fiber sensor (NS-LSPR-FOS). Its sensitivity is significantly better than traditional detection methods, such as traditional fiber sensors based on gold nanoparticles (70 nm / RIU). The sensitivity of this application can reach 386.3 nm / RIU.

[0028] (2) Real-time dynamic monitoring: The fiber optic sensor of the present application can monitor the changes in PD-L1 concentration in real time and dynamically. By recording the reflection spectrum every 15 seconds through a spectrometer, the antigen-antibody binding process can be tracked in real time, helping doctors to adjust the treatment plan in a timely manner.

[0029] (3) Label-free detection: The detection method of the present application does not require the use of fluorescent or radioactive labels, thus avoiding the errors and background interference that may be introduced by the labeling process. This simplifies the detection process and improves the accuracy and reliability of the detection.

[0030] (4) Strong anti-interference ability: Fiber optic sensors are based on optical signal transmission and are not affected by electromagnetic interference. They can work stably in complex biological samples and medical environments. In addition, by optimizing the nanostructure design, the sensor can effectively eliminate the interference of other components in the complex body fluid background and focus on the target detection object.

[0031] (5) Miniaturization and implantability: Fiber optic sensors are small, light, and flexible, making them easy to detect in the body. Through miniaturized design, they can be easily inserted into specific parts of the human body for body fluid testing, and can even be implanted for testing, providing strong support for precision medicine.

[0032] (6) Multi-parameter detection capability: The optical fiber sensor of the present application is capable of simultaneously detecting multiple biomarkers or physicochemical parameters. For example, multiple indicators such as PD-L1 concentration, pH value, and glucose concentration can be monitored simultaneously in one test, providing rich data support for a comprehensive assessment of the patient's physiological state.

[0033] (7) Easy operation and low cost: The detection method of the present application is easy to operate and does not require complicated sample pretreatment. At the same time, the preparation and use costs of the optical fiber sensor are low, which is suitable for large-scale production and clinical application.

[0034] (8) Signal amplification: This application introduces SIBP-modified gold nanoparticles to form a sandwich structure, which significantly improves the detection signal, making the optical fiber sensor feasible for trace, real-time, label-free quantitative detection of tumor markers and heterogeneity characterization.

[0035] In summary, compared with liquid biopsy methods such as ELISA, IFA, and microfluidic chips, the fiber optic sensor proposed in this application has many advantages such as small size, label-free, real-time detection, and in vivo detection. It is expected to overcome the two major challenges of low concentration and spatiotemporal heterogeneity characterization of tumor marker PD-L1 detection, and play an important role in cancer research and immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0037] Figure 1 Schematic diagram of the functionalization and detection steps of NS-LSPR-FOS for PD-L1 detection.

[0038] Figure 2 Real-time detection of PD-L1 by PPR fiber optic sensor based on gold nanourchin, including: (a) real-time reflectance spectrum response of PD-L1 detection; (b) relationship between resonance valley wavelength shift and different concentrations of PD-L1.

[0039] Figure 3 The sensitivity of the optical fiber sensor, including: (a) the reflectance spectrum of the sensor in aqueous glucose solutions with different concentrations, (b) the real-time response of the sensor in glucose solutions with different concentrations. Inset: the relationship between the resonance valley wavelength and the corresponding refractive index.

[0040] Figure 4 Signal detection through the sandwich structure method, including: a) signal amplification principle diagram; b) wavelength shift of PD-L1 detection and signal amplification. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0042] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified. The equipment used in the experiments are all well known to those skilled in the art unless otherwise specified.

[0043] Nouns and abbreviations

[0044] ICIs: immune checkpoint inhibitors, ICIs, immune checkpoint inhibitors

[0045] PD-L1: Programmed Death-Ligand 1

[0046] LSPR: Localized Surface Plasmon Resonance

[0047] LSPR-FOS: Fiber Optic Sensor based on Localized Surface Plasmon Resonance, localized surface plasmon resonance fiber sensor

[0048] NS-LSPR-FOS: Fiber Optic Sensors Based on Nanostructure - LocalizedSurface Plasmon Resonance, Fiber Optic Sensors Based on Nanostructure - LocalizedSurface Plasmon Resonance

[0049] PPR: Plasomonic-Photonic Resonance

[0050] APTES:3-Aminopropyltriethoxysilane

[0051] PS- b -P4VP:Poly(styrene)-b-poly(4-vinyl pyridine)

[0052] THF:Tetrahydrofuran

[0053] pSC4: 4-Sulfonylcalixa[4]arene hydrate

[0054] SIBP: Specific Intracellular Binding Peptide

[0055] AuNP:Gold Nanoparticles

[0056] AuNU: Gold Nanourchins

[0057] SERS: Surface - Enhanced Raman Scatterin, Surface Enhanced Raman Scatterin

[0058] ELISA: enzyme-linked immunosorbent assay

[0059] To further verify the fiber optic sensing performance and its potential application in tumor marker detection, a series of real-time detections of PD-L1, a representative predictive biomarker for cancer immunotherapy, were performed.

[0060] Example 1 Real-time detection of PD-L1 based on nanostructured localized surface plasmon resonance fiber sensor (NS-LSPR-FOS)

[0061] Experimental Materials:

[0062] Experimental procedures (see Figure 1 , where (a) is the start):

[0063] 1. Preparation of NS-LSPR-FOS for PD-L1-specific detection: Prepare 10 mM 4-sulfonylcalixa[4]arene hydrate (pSC4) solution, place the prepared NS-LSPR-FOS (localized surface plasmon resonance fiber sensor, photon cavity thickness: 340-460nm, gold nanourchin size and spacing 50-80nm) in the above solution for 12 hours, and form a pSC4 monolayer on the surface of the gold nanourchin, connected by a strong Au-S bond. The calixarene in pSC4 is a commonly used protein linker, which can maintain the natural activity of the antibody through several soft non-covalent bonds.

[0064] Among them, the preparation process of NS-LSPR-FOS (localized surface plasmon resonance fiber sensor) is as follows:

[0065] (1) Cut a 10 cm long section of optical fiber and use a stripping pliers to remove a 2 cm long coating from the tip of the optical fiber;

[0066] (2) To obtain a smooth and clean fiber end face, use a fiber cleaver to cut the fiber tip to leave only the cladding and core for effective sensing;

[0067] (3) Hydroxylate the optical fiber in a plasma cleaner for 1 minute;

[0068] (4) Prepare a solution with a volume ratio of water: isopropanol: APTES of 100:100:1 and let it stand for 2 hours;

[0069] (5) Soak the tip of the optical fiber in the APTES solution for 15 minutes. After silanization, wash it with deionized water for one minute to remove the residual liquid left on the end face of the optical fiber.

[0070] (6) Chemical vapor deposition of Parylene-C on the surface of the optical fiber tip forms a photon cavity with a thickness of 340-460nm;

[0071] (7) Hydroxylate the optical fiber in a plasma cleaner for 1 minute;

[0072] (8) Immerse the fiber end face in a 0.05 mg / ml PS-b-P4VP THF solution for 10 minutes for functionalization;

[0073] (9) After cleaning with deionized water, place the optical fiber in a fume hood to dry for 30 minutes, and then soak it in a gold nano-urchin solution (0.5 mg / ml, size 50-80 nm) for 12 hours to make the gold nano-urchin particles evenly distributed on the tip of the optical fiber;

[0074] (10) Finally, the end face of the optical fiber sensor modified with gold nano-sea urchin was quickly cleaned with deionized water and dried for 30 min to obtain NS-LSPR-FOS for use.

[0075] Of course, the present application is not limited thereto, and those skilled in the art may also select optical fiber sensors of the prior art.

[0076] 2. PD-L1 antibody connected to pSC4 monolayer: After pSC4 modification, the fiber end face was cleaned with deionized water to remove unbound pSC4 molecules on the surface, and then the fiber end face was immersed in a PBS solution containing 20 μg / mL PD-L1 antibody for 1 h to form a PD-L1 specific recognition layer on the surface of the gold nanourchin. At the same time, from this step on, the ambient temperature was maintained at 20±0.5℃, and the reflectance spectrum of the sensor was recorded every 15 s by a spectrometer to dynamically monitor the PD-L1 antibody molecule modification and subsequent antigen-antibody interaction process.

[0077] 3. BSA blocking: After rinsing the sensor end surface with PBS, immerse it in a 10 mg / ml BSA solution for 20 minutes to block the active sites on the surface that are not bound to PD-L1.

[0078] 4. PD-LI antigen detection: See the following detection steps for details.

[0079] (1) During PD-L1 detection, the end face of the optical fiber sensor was immersed in centrifuge tubes containing solutions of different concentrations of PD-L1 antigen (50-4000 ng / ml) in sequence, with each group lasting 45 minutes, from small to large concentrations. In order to reduce the impact of nonspecific binding on the measurement results during the detection process, the sensor end face was rinsed with PBS buffer before and after the measurement of different concentrations of PD-L1 protein to remove nonspecifically adsorbed PD-L1 protein to avoid errors in the experimental results.

[0080] (2) The reflectance spectrum of the optical fiber sensor is recorded every 15 seconds by a spectrometer, and the quantitative detection of PD-L1 protein at different concentrations is achieved by tracking the wavelength shift of the resonance valley.

[0081] Specifically: first, a baseline is established using the environmental measurement values ​​of the PBS solution after BSA blocking and before detection; then, the sample is immersed in a solution containing PD-L1 antibodies, and after antigen-antibody binding, the wavelength corresponding to the resonance valley undergoes a red shift; then, PBS buffer is used to wash away impurities that are not bound to the PD-L1 antibody, and the resonance wavelength reaches the binding point between the antibody and PD-L1 and remains stable, and the wavelength shift value of the resonance valley is measured.

[0082] (3) Based on the antigen detection calibration curve (50-4000 ng / ml) previously measured by the optical fiber sensor, the PD-L1 concentration was calculated according to the measured resonance valley wavelength shift value.

[0083] Result analysis:

[0084] 1. PD-L1 detection results of NS-LSPR-FOS are as follows Figure 2 As shown:

[0085] Figure 2 Figure (a) shows the change in the resonance valley wavelength during the entire detection process, which is mainly based on the change in refractive index caused by antibody and antigen binding. First, the pSC4 solution used to bind the antibody was removed with PBS to establish a baseline. The gold nanourchin particles were then modified with PD-L1 antibodies for subsequent antigen-antibody binding. After washing with PBS, the sites that were not modified with pSC4 were blocked with BSA solution to avoid subsequent interference and nonspecific adsorption.

[0086] The results showed that after adding antibodies and BSA, the wavelength shifted to a certain extent due to the change in the refractive index of the environment, and returned to stability after PBS washing. In addition, the red shift caused by antibodies (from 877.1 nm to 881.4 nm) was significantly greater than that of BSA (very small), indicating that pSC4 effectively bound to antibodies in the presence of a small amount of blocking BSA. After washing the BSA remaining on the fiber end face with PBS, the sensor was immersed in a PD-L1 antigen solution with a concentration of 4 μg / ml. As expected, the resonance peak wavelength was red-shifted, which means that the presence of PD-L1 antigen was successfully detected. Finally, PBS solution was used to rinse off any impurities that were not bound to the antibody, allowing the sensor to accurately measure the PD-L1 concentration, shifting from 881.06 nm to 881.97 nm. At this point, the real-time specific quantitative detection of PD-L1 was achieved through the resulting wavelength shift.

[0087] Figure 2 Figure (b) shows the wavelength shift corresponding to different PD-L1 concentrations in the range of 50 ng / ml to 4000 ng / ml. In order to quantify the biosensing performance of the fiber optic sensor, three repeated measurements were performed for each different concentration. Based on the Langmuir equation, formula (3-5), fitting the above data points yields:

[0088] Where a = 1.093 ± 0.097 nm -1 , b = 395.8±93.72 μg·(ml·nm) -1 , c = 0, fitting coefficient R 2 =0.969. Substituting three times the standard deviation of the wavelength shift at the measured concentration into the above linear fitting formula, the detection limit of this sensor is calculated to be 28.1 ng / ml. According to existing literature reports, the detection limit of 28.1 ng / ml is relatively higher than the clinical peripheral blood sample PD-L1 standard (1.92-25 ng / ml), and is also higher than existing SERS, ELISA and other technologies.

[0089] 2. Sensitivity verification data and results:

[0090] The sensing performance and temporal response of the sensor were evaluated using glucose solution. Figure 3 Figure (a) records the reflectance spectra of glucose solutions with concentrations ranging from 0% to 40%, and the corresponding refractive index (RI) changes from 1.333 to 1.384. As the glucose concentration increases, the wavelength of the resonance valley red-shifts. In the range of glucose concentration from 0% to 40%, the wavelength of the resonance valley shifts by up to 18.4 nanometers, and the response time of adjacent concentrations is less than 10 seconds. Figure 3Figure (b) further shows the relationship between the resonance valley wavelength and the refractive index of the solution, and a linear fit is performed. According to the definition of wavelength sensitivity

[0091] Where Δλ is the wavelength shift of the resonance valley and Δn is the change in the refractive index of the solution. The sensitivity of the fiber optic sensor reached 386.3 nm / RIU. This sensitivity is significantly better than that of the traditional fiber optic sensor based only on gold nanoparticles (AuNPs) (70 nm / RIU).

[0092] Example 2 Signal amplification strategy for PD-L1 detection based on nanostructured localized surface plasmon resonance fiber sensor (NS-LSPR-FOS)

[0093] Peptides have attracted interest in many fields due to their small size, easy modification without damaging their affinity, and the ability to produce a strong binding effect. They can also be flexibly fixed on gold nanoparticles, showing their great potential in promoting the development of sensing. Therefore, with the help of the specific intracellular binding peptide (SIBP) designed by Hu JJ et al., the PD-L1 detection signal was amplified by forming a sandwich sensing platform.

[0094] Existing PD-L1 aptamers and antibodies are both extracellular region binding molecules with overlapping binding sites, which severely limits the construction of biosensors. Specific intracellular binding peptide (SIBP) is a unique PD-L1 intracellular differentiation probe. It binds to PD-L1 antibodies and aptamers extracellularly and has overlapping binding sites. It can eliminate spatial obstacles and bind to PD-L1 cells. This method mainly prepares SIBP-modified AuNPs by forming an Au-S bond between the thiol group (-SH) of cysteine ​​in the peptide and AuNPs, and then achieves the purpose of amplifying the SPR signal through the specific binding between AuNPs-SIBP and PD-L1 antigen. Figure 4 As shown in a), a sandwich structure of spherical gold nanoparticles (AuNPs) functionalized with PD-L1 antibody, PD-L1 antigen, and SIBP was modified at the tip of the optical fiber to amplify the signal.

[0095] Experimental Materials:

[0096] Experimental steps (this experiment differs from Example 1 in that a sandwich structure is added, such as Figure 4 a) of the figure):

[0097] The preliminary experimental steps are similar to those in Example 1 and will not be repeated here. It is also necessary to prepare a SIBP solution to form a sandwich structure:

[0098] 1. Prepare 100 μg / ml SIBP solution and 0.25 mg / ml gold nanosphere solution with a diameter of 10 nm, mix the two in a volume ratio of 1:1, and let it stand at 4°C for 12 hours to form Au-S bonds between the thiol group (-SH) of cysteine ​​in the polypeptide and the spherical gold nanoparticles (AuNPs).

[0099] 2. After all experiments were completed, that is, after measuring the known concentration of PD-L1 antigen, the fiber optic sensor was washed with PBS solution and immersed in the prepared AuNPs-SIBP solution for 45 minutes to allow SIBP to bind to the PD-L1 antigen. From this step on, the reflection spectrum of the fiber optic sensor was recorded every 15 seconds.

[0100] 3. Clean the sensor end surface with PBS solution to remove unbound AuNPs-SIBP.

[0101] Signal amplification results of NS-LSPR-FOS for PD-L1 detection:

[0102] like Figure 4 As shown in b), after forming a sandwich structure of AuNP functionalized with PD-L1 antibody, PD-L1 antigen and SIBP, a significant amplification effect was obtained, with the original displacement distance increasing from 0.35 nm to 2.42 nm, and the wavelength shift increased by more than 5 times, which proved the applicability of this sandwich structure-based signal amplification strategy for sensitive detection of PD-L1 in this method, and also showed the potential of using this sandwich structure signal amplification strategy to reduce the detection limit of PD-L1 in this method.

[0103] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for detecting tumor marker PD-L1 based on a composite structure optical fiber sensor of gold nano-sea urchin, characterized in that: The following steps are involved: (1) Preparation of localized surface plasmon resonance fiber sensor (NS-LSPR-FOS) for specific detection of PD-L1: Prepare a 10 mM 4-sulfonylcaliphatic [4]arene hydrate (pSC4) solution, place the NS-LSPR-FOS in the pSC4 solution and soak for 12 hours; (2) Connection of PD-L1 antibody to pSC4 monolayer: After pSC4 modification, the fiber end face was cleaned with deionized water to remove unbound pSC4 molecules on the surface; The end face of the optical fiber sensor was immersed in a PBS solution containing 20 μg / mL PD-L1 antibody for 1 hour to form a PD-L1 specific recognition layer on the surface of the gold nano-sea urchin; (3) BSA blocking: rinse the sensor end face with PBS, and immerse the fiber optic sensor end face in a 10 mg / mL bovine serum albumin (BSA) solution for 20 minutes to block the active sites on the surface that are not bound to PD-L1; (4) PD-L1 antigen detection: (41) The end face of the optical fiber sensor was immersed in PD-L1 antigen solutions of different concentrations in sequence, with each group immersed for 45 minutes, from small to large concentrations, and then washed with PBS to remove the non-specifically bound PD-L1 antigen; (42) The reflectance spectrum of the optical fiber sensor was recorded every 15 seconds by a spectrometer, and the wavelength shift of the resonance valley was tracked to achieve quantitative detection of different concentrations of PD-L1 protein; (43) Based on the antigen detection calibration curve obtained by the optical fiber sensor in the early stage, the PD-L1 concentration was calculated according to the measured resonance valley wavelength shift value.

2. The method for detecting the tumor marker PD-L1 by the composite structure optical fiber sensor based on gold nano-sea urchin according to claim 1, characterized in that: In step (1), the photon cavity thickness of NS-LSPR-FOS is 340-460 nm, and the size and spacing of gold nanourchins are 50-80 nm.

3. The method for detecting the tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 1, characterized in that: In step (2), during the connection process between the PD-L1 antibody and the pSC4 monolayer, the ambient temperature was maintained at 20±0.5°C.

4. The method for detecting the tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 1, characterized in that: In step (41), the concentration of the PD-L1 antigen solution is 50 to 4000 ng / ml. Before and after immersion in antigen solutions of different concentrations, the samples are washed with a PBS solution to remove non-specifically bound PD-L1 antigens.

5. The method for detecting tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 1, characterized in that: Step (42) is specifically as follows: first, a baseline is established by measuring the environmental value of the PBS solution after BSA blocking and before detection; immersing in a solution containing PD-L1 antibodies, and after antigen-antibody binding, the wavelength corresponding to the resonance valley is red-shifted; Afterwards, PBS buffer was used to wash away impurities that were not bound to the PD-L1 antibody. The resonance wavelength reached the binding point between the antibody and PD-L1 and remained stable, and the wavelength shift value of the resonance valley was measured.

6. The method for detecting tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 1, characterized in that: Step (4) also includes: After preparing a PD-L1 antigen solution of known concentration, the optical fiber sensor was immersed in a SIBP-modified gold nanoparticle (AuNPs-SIBP) solution for 45 minutes to allow SIBP to bind to the PD-L1 antigen. The reflectance spectrum of the optical fiber sensor was recorded every 15 seconds to track the wavelength movement of the resonance valley to achieve amplification of the PD-L1 detection signal.

7. The method for detecting tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 6, characterized in that: AuNPs-SIBP was prepared by the following method: Prepare 100 μg / mL SIBP solution and 0.25 mg / mL 10 nm gold nanoparticle solution; The SIBP solution and the gold nanoparticle solution were mixed in a volume ratio of 1:1 and allowed to stand at 4°C for 12 h to obtain AuNPs-SIBP.

8. The method for detecting tumor marker PD-L1 based on the composite structure optical fiber sensor of gold nano-sea urchin according to claim 6, characterized in that: Also includes: The end face of the optical fiber sensor was cleaned with PBS solution to remove unbound AuNPs-SIBP.

Images