A Composite Structure Optical Fiber Sensor Based on Gold Nanosea Urchins and Its Application in PD-L1 Detection

Abstract

The present invention discloses a method for detecting tumor marker PD-L1 using a composite structure optical fiber sensor based on gold nano-urchins, comprising the following steps: preparing a pSC4 solution, immersing the NS-LSPR-FOS in the pSC4 solution for 12 hours; washing to remove unbound pSC4 molecules; immersing the sensor end face in a PBS solution containing 20 μg / mL PD-L1 antibody for 1 hour to form a specific recognition layer; immersing the sensor end face in a BSA solution for 20 minutes; successively immersing the sensor end face in PD-L1 antigen solutions of different concentrations, with each group being immersed for 45 minutes, from low to high concentration; recording the reflection spectrum every 15 seconds through a spectrometer to track the movement of the resonance valley wavelength and detect the PD-L1 concentration. Compared with liquid biopsy methods such as ELISA, IFA, and microfluidic chips, this application has many advantages such as small size, label-free, real-time detection, and in-vivo detection, and is expected to overcome the two major challenges of low-concentration detection and spatio-temporal heterogeneity characterization of tumor marker PD-L1, playing an important role in cancer research and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of fiber optic sensors, and particularly relates to a composite structure fiber optic sensor based on gold nano-urchins and its application in PD-L1 detection. Background Art

[0002] In the study of laboratory cell models, traditional detection of target proteins mostly uses endpoint methods (such as enzyme-linked immunosorbent assay, western blotting, flow cytometry). These methods require cell lysis or fixation, which may change the protein state (such as phosphorylation level) and cannot obtain continuous measurement results. In order to preserve the physiological state of living cells and monitor the dynamics of cell responses, cell heterogeneity, and the timeliness of drug responses under the condition of minimal sample interference, methods capable of real-time detection of protein molecule expression are needed to dynamically observe the concentration of some target proteins. Therefore, there is an urgent need to design highly sensitive and real-time monitorable biosensors to track the dynamic changes of key protein molecules in cell models, optimize the spatio-temporal laws of cell responses, and accurately understand the changes in cell functions.

[0003] Real-time detection technology needs to meet the characteristics of high time resolution, non-destructiveness, and high sensitivity. Currently, there are three core defects in common detection methods: First, the detection equipment is bulky and the operation is complex. For example, for nanoparticle probes, the dedicated equipment is very large and it is difficult to meet the requirement of being placed in a cell culture environment for detection. Second, the sensitivity is limited by technology. Especially when detecting protein markers, fluorescence labeling is often required and the anti-interference ability is weak. Fluorescence labeling imaging of living cells cannot take into account the detection of free proteins. Third, it is difficult to obtain environmental-related physical and chemical parameters while monitoring protein concentration.

[0004] Fiber optic sensors provide a revolutionary solution to break through the bottleneck of existing technologies. Its unique advantages are reflected in: 1) High sensitivity and accuracy, single-molecule level detection can be achieved based on the local surface plasmon resonance (LSPR) effect of nanostructures; 2) Strong anti-electromagnetic interference ability, suitable for complex body fluid environments; 3) Strong real-time monitoring ability, miniaturization (<125μm) can be placed in a cell culture container for continuous dynamic tracking; 4) Potential for multi-parameter detection, parameters such as the concentration of target proteins, pH value of the culture environment, glucose, and ion concentration can be synchronously monitored through wavelength / intensity multimodal analysis; 5) Equipment integration advantages, the fiber optic sensor system has characteristics such as light weight, small size, flexibility, and long transmission distance, which is convenient for developing application devices in an incubator. Currently, the feasibility of fiber optic sensors based on nanostructured LSPR has been verified in the detection of prostate-specific antigen, but the sensitivity still needs to be improved.

[0005] Therefore, in order to overcome the problem of real-time monitoring of protein concentration in the existing life science research process, and in response to the challenges of low detection sensitivity and inability to perform real-time monitoring of fiber optic sensors based on nanostructured LSPR, there is an urgent need for a sensitive, micron-scale, and easy-to-operate detection strategy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a composite structure fiber optic sensor based on gold nano-urchins and its application in PD-L1 detection, so as to solve the problems of low sensitivity, inability to perform real-time monitoring, complex operation, and high cost existing in the existing protein PD-L1 detection technology.

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

[0008] A composite structure fiber optic sensor based on gold nano-urchins, which is prepared by the following method:

[0009] (1) Prepare a 10 mM solution of 4-sulfonylcalix[4]arene hydrate (pSC4), and place NS-LSPR-FOS in the above pSC4 solution and soak for 12 hours; wherein, the photon cavity thickness of NS-LSPR-FOS is 340 - 460 nm, and the size and spacing of the gold nano-urchins are 50 - 80 nm;

[0010] (2) After the pSC4 modification is completed, wash the fiber optic end face with deionized water to remove unbound pSC4 molecules on the surface; immerse the fiber optic sensor end face in a PBS solution containing 20 μg / mL PD-L1 antibody and soak for 1 hour to form a PD-L1 specific recognition layer on the surface of the gold nano-urchins; wherein, during the connection process of the PD-L1 antibody and the pSC4 monolayer, the environmental temperature is maintained at 20 ± 0.5 °C;

[0011] (3) 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 and soak for 20 minutes to block the active sites on the surface that are not bound to PD-L1, so as to obtain a composite structure fiber optic sensor for detecting PD-L1 concentration.

[0012] An application of a composite structure fiber optic sensor based on gold nano-urchins in PD-L1 detection, including the following steps:

[0013] (1) Preparation of a localized surface plasmon resonance fiber optic sensor (NS-LSPR-FOS) for PD-L1 specific detection: Prepare a 10 mM solution of 4-sulfonylcalix[4]arene hydrate (pSC4). Immerse the NS-LSPR-FOS in the above pSC4 solution for 12 hours to form a monolayer of pSC4 on the surface of the gold nanosea urchins through Au-S bonds.

[0014] (2) Connection of PD-L1 antibody to the pSC4 monolayer: After the pSC4 modification, wash the fiber end face with deionized water to remove unbound pSC4 molecules on the surface. Immerse the fiber sensor end face 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 nanosea urchins. At the same time, record the reflection spectrum of the fiber optic sensor with a spectrometer.

[0015] (3) BSA blocking: Rinse the sensor end face with PBS and immerse the fiber 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 and avoid non-specific binding.

[0016] (4) PD-L1 antigen detection:

[0017] (41) Immerse the fiber sensor end face successively in PD-L1 antigen solutions of different concentrations. Each group is immersed for 45 minutes, from low to high concentration, and wash with PBS to remove unbound PD-L1 antigen specifically.

[0018] (42) Record the reflection spectrum of the fiber optic sensor every 15 seconds with a spectrometer. By tracking the wavelength shift of the resonance valley, quantitative detection of PD-L1 protein at different concentrations is achieved.

[0019] (43) Based on the antigen detection calibration curve measured for the fiber optic sensor previously, calculate the PD-L1 concentration according to the measured wavelength shift value of the resonance valley.

[0020] Preferably, in the above technical solution, in step (1), the photon cavity thickness of the NS-LSPR-FOS is 340 - 460 nm, and the size and spacing of the gold nanosea urchins are 50 - 80 nm.

[0021] Preferably, in the above technical solution, in step (2), during the connection process of the PD-L1 antibody to the pSC4 monolayer, the ambient temperature is maintained at 20 ± 0.5 °C.

[0022] Preferably, in the above technical solution, in step (41), the concentration of the PD-L1 antigen solution is 50-4000 ng / ml. Before and after soaking in antigen solutions of different concentrations, washing is performed in PBS solution to remove un-specifically bound PD-L1 antigen.

[0023] Preferably, in the above technical solution, step (42) is specifically as follows: First, establish a baseline with the measured value in the PBS solution environment after BSA blocking and before detection; immerse it in a solution containing PD-L1 antibody. After antigen-antibody binding, the wavelength corresponding to the resonance valley undergoes a red shift; then, wash away the impurities that have not bound to the PD-L1 antibody with PBS buffer. The resonance wavelength reaches the binding point of the antibody and PD-L1 and remains stable, and the wavelength shift value of the resonance valley is measured.

[0024] Preferably, in the above technical solution, step (4) further includes:

[0025] After completing the PD-L1 antigen solution of known concentration, immerse the fiber optic sensor in a solution of SIBP-modified gold nanoparticles (AuNPs-SIBP) and react for 45 minutes to allow SIBP to bind to the PD-L1 antigen. Record the reflection spectrum of the fiber optic sensor every 15 seconds to track the wavelength shift of the resonance valley, so as to amplify the detection signal of PD-L1.

[0026] Preferably, in the above technical solution, AuNPs-SIBP is prepared by the following method:

[0027] Prepare a 100 μg / mL SIBP solution and a 10 nm gold nanoparticle solution with a mass fraction of 0.25 mg / mL;

[0028] Mix the SIBP solution and the gold nanoparticle solution at a volume ratio of 1:1 and let it stand at 4 °C for 12 hours to form an Au-S bond between the thiol group (-SH) of cysteine in the polypeptide and the gold nanoparticles, obtaining AuNPs-SIBP.

[0029] Preferably, in the above technical solution, it further includes: washing the end face of the fiber optic sensor with PBS solution to remove unbound AuNPs-SIBP.

[0030] The above technical solution of the present invention has the following beneficial effects:

[0031] (1) High-sensitivity detection: The present application realizes high-sensitivity detection of PD-L1 by designing a nanostructure localized surface plasmon resonance fiber optic sensor (NS-LSPR-FOS). Its sensitivity is significantly better than traditional detection methods, such as traditional fiber optic sensors based on gold nanoparticles (70 nm / RIU), and the sensitivity of the present application can reach 386.3 nm / RIU.

[0032] (2) Real-time dynamic monitoring: The fiber optic sensor of this application can monitor the change of PD-L1 concentration in real time and dynamically. By recording the reflection spectrum every 15 seconds with a spectrometer, the antigen-antibody binding process can be tracked in real time, helping doctors adjust the treatment plan in a timely manner.

[0033] (3) Label-free detection: The detection method of this application does not require the use of fluorescent or radioactive labels, avoiding the errors and background interference that may be introduced during the labeling process. It simplifies the detection process and improves the accuracy and reliability of the detection.

[0034] (4) Strong anti-interference ability: The fiber optic sensor is based on optical signal transmission and is not affected by electromagnetic interference, and can work stably in complex biological samples and medical environments. In addition, by optimizing the nanostructure design, the sensor can effectively exclude the interference of other components in the complex body fluid background and focus on the target analyte.

[0035] (5) Miniaturization and flexibility: The fiber optic sensor has the characteristics of small size, light weight and easy bending; the miniaturized design is convenient for detection.

[0036] (6) Multi-parameter detection ability: The fiber optic sensor of this application can simultaneously detect 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 detection.

[0037] (7) Simple operation and low cost: The detection method of this application is simple to operate and does not require complex sample pretreatment. At the same time, the preparation and use costs of the fiber optic sensor are relatively low, which is suitable for large-scale production and laboratory use.

[0038] (8) Signal amplification: By introducing SIBP-modified gold nanoparticles in this application to form a sandwich structure, the detection signal is significantly improved, making the fiber optic sensor feasible for quantitative detection and heterogeneity characterization of trace, real-time, label-free tumor markers.

[0039] In summary, the fiber optic sensor proposed in this application has many advantages such as small size, label-free, and real-time detection compared with liquid biopsy methods such as ELISA, IFA, and microfluidic chips. It is expected to overcome the two major challenges of low concentration and spatio-temporal heterogeneity characterization in PD-L1 detection and play an important role in related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings incorporated in and constituting a part of this specification illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0041] Figure 1Schematic diagram of the functionalization and detection steps for PD-L1 detection of NS-LSPR-FOS.

[0042] Figure 2 For the real-time detection of PD-L1 by a PPR fiber optic sensor based on gold nano-urchins, where: (a) Real-time reflection spectral response for PD-L1 detection; (b) Relationship between the resonance valley wavelength shift and different concentrations of PD-L1.

[0043] Figure 3 For the sensitivity of the fiber optic sensor, where: (a) Reflection spectra of the sensor in aqueous glucose solutions with different concentrations; (b) Real-time responses of the sensor in different concentrations of glucose solutions. Inset: Relationship between the resonance valley wavelength and the corresponding refractive index.

[0044] Figure 4 For detecting signals through the sandwich structure method, where: a) Schematic diagram of signal amplification; b) Wavelength shift for PD-L1 detection and signal amplification. Detailed implementation manners

[0045] Now, various exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be noted that: Unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions, and numerical values set forth in these embodiments do not limit the scope of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials and reagents used, unless otherwise specified, can all be obtained from commercial sources. The equipment used in the experiments, unless otherwise specified, are all well-known to those skilled in the art.

[0047] English names and abbreviations of terms

[0048] ICIs: immune checkpoint inhibitors, ICIs, immune checkpoint inhibitors

[0049] PD-L1: Programmed Death-Ligand 1, Programmed Death-Ligand 1

[0050] LSPR: Localized Surface Plasmon Resonance, Localized Surface Plasmon Resonance

[0051] LSPR-FOS: Fiber Optic Sensor based on Localized Surface Plasmon Resonance, Fiber Optic Sensor based on Localized Surface Plasmon Resonance

[0052] NS-LSPR-FOS: Fiber Optic Sensors Based on Nanostructure-Localized Surface Plasmon Resonance, Nanostructure-Localized Surface Plasmon Resonance-Based Fiber Optic Sensors

[0053] PPR: Plasomonic-Photonic Resonance

[0054] APTES: 3-Aminopropyltriethoxysilane

[0055] PS-b-P4VP: Poly(styrene)-b-poly(4-vinyl pyridine)

[0056] THF: Tetrahydrofuran

[0057] pSC4: 4-Sulfonylcalix[4]arene hydrate

[0058] SIBP: Specific Intracellular Binding Peptide

[0059] AuNP: Gold Nanoparticles

[0060] AuNU: Gold Nanourchins

[0061] SERS: Surface-Enhanced Raman Scatterin

[0062] ELISA: enzyme-linked immunosorbent assay, Example 1 Real-time Detection of PD-L1 by Nanostructure-based Localized Surface Plasmon Resonance Fiber Optic Sensor (NS-LSPR-FOS)

[0063] Experimental Materials:

[0064]

[0065] Experimental Procedures (see Figure 1 , where (a) is the start):

[0066] 1. Preparation of NS-LSPR-FOS for PD-L1 specific detection: Prepare a 10 mM solution of 4-sulfonylcalix[4]arene hydrate (pSC4). Place the prepared NS-LSPR-FOS (localized surface plasmon resonance fiber optic sensor, photon cavity thickness: 340 - 460 nm, gold nanosea urchin size and spacing 50 - 80 nm) in the above solution for 12 hours to form a pSC4 monolayer on the surface of the gold nanosea urchin, which is connected through strong Au-S bonds. Calixarene in pSC4, as a commonly used protein linker, can maintain the natural activity of antibodies through several soft non-covalent bonds.

[0067] Among them, the preparation process of NS-LSPR-FOS (localized surface plasmon resonance fiber optic sensor) is specifically as follows:

[0068] (1). Cut a 10-cm long optical fiber and remove the 2-cm long coating at the tip of the optical fiber with a stripping pliers.

[0069] (2). To obtain a smooth and clean optical fiber end face, cut the tip of the optical fiber leaving only the cladding and the core with an optical fiber cutter for effective sensing.

[0070] (3). Hydroxylate the optical fiber in a plasma cleaner for 1 minute.

[0071] (4). Prepare a solution with a volume ratio of water:isopropanol:APTES of 100:100:1 and let it stand for 2 hours.

[0072] (5). Immerse the tip of the optical fiber in the APTES solution and let it stand for 15 minutes. After silanization, wash it with deionized water for 1 minute to remove the residual liquid left on the optical fiber end face.

[0073] (6). Chemically vapor deposit Parylene-C on the surface of the optical fiber tip to form a photon cavity with a thickness of 340 - 460 nm.

[0074] (7). Hydroxylate the optical fiber in a plasma cleaner for 1 minute.

[0075] (8). Immerse the end face of the optical fiber in a 0.05 mg / ml THF solution of PS-b-P4VP for 10 minutes for functionalization.

[0076] (9). After washing with deionized water, place the optical fiber in a fume hood to dry for 30 minutes, and then immerse it in a gold nanosea urchin solution (0.5 mg / ml, size 50 - 80 nm) for 12 hours to make the gold nanosea urchin particles evenly distributed at the tip of the optical fiber.

[0077] (10). Finally, quickly wash the end face of the gold nanosea urchin-modified optical fiber sensor with deionized water and dry it for 30 min to obtain the NS-LSPR-FOS for standby.

[0078] Of course, the present application is not limited thereto, and those skilled in the art can also select existing fiber optic sensors.

[0079] 2. Connection of PD-L1 antibody to the pSC4 monolayer: After the pSC4 modification is completed, the fiber optic end face is washed with deionized water to remove unbound pSC4 molecules on the surface, and then the fiber optic end face is 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 nanosea urchin. Meanwhile, starting from this step, the ambient temperature is maintained at 20 ± 0.5 °C, and the reflection spectrum of the sensor is recorded every 15 s by a spectrometer to dynamically monitor the process of PD-L1 antibody molecule modification and subsequent antigen-antibody interaction.

[0080] 3. BSA blocking: After rinsing the sensor end face with PBS, it is immersed 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.

[0081] 4. Detection of PD-LI antigen: See the following detection steps for details.

[0082] (1) During the PD-L1 detection, the fiber optic sensor end face is successively immersed in centrifuge tubes containing PD-L1 antigen solutions with different concentrations (50 - 4000 ng / ml), with each group lasting for 45 minutes, from low concentration to high concentration. To reduce the influence of non-specific binding during the detection on the measurement results, the sensor end face is rinsed with PBS buffer before and after measuring PD-L1 proteins with different concentrations to remove non-specifically adsorbed PD-L1 proteins, so as to avoid errors in the experimental results.

[0083] (2) The reflection spectrum of the fiber optic sensor is recorded every 15 s by a spectrometer, and quantitative detection of PD-L1 proteins with different concentrations is achieved by tracking the wavelength shift of the resonance valley.

[0084] Specifically: First, a baseline is established using the measured value in the PBS solution environment after BSA blocking and before detection; after immersing in the solution containing PD-L1 antibody and after antigen-antibody binding, the wavelength corresponding to the resonance valley undergoes a red shift; then, the impurities that are not bound to the PD-L1 antibody are washed away with PBS buffer, and the resonance wavelength reaches the binding point of the antibody and PD-L1 and remains stable, and the wavelength shift value of the resonance valley is measured.

[0085] (3) Based on the antigen detection calibration curve (50 - 4000 ng / ml) measured in advance for the fiber optic sensor, the PD-L1 concentration is calculated according to the actually measured wavelength shift value of the resonance valley.

[0086] Result analysis:

[0087] 1. The PD-L1 detection results of NS-LSPR-FOS are as follows: Figure 2 as shown in:

[0088] Figure 2 (a) of Figure 2 shows the change in the resonance valley wavelength during the entire detection process, mainly based on the refractive index change caused by antibody-antigen binding. First, the pSC4 solution used for antibody binding was removed with PBS to establish a baseline. Then, the gold nanosea urchin particles were modified with PD-L1 antibody for subsequent antigen-antibody binding. After washing with PBS, the unmodified pSC4 sites were blocked with BSA solution to avoid subsequent interference and non-specific adsorption.

[0089] The results show that after adding the antibody and BSA, the wavelength shifted to a certain extent due to the change in the environmental refractive index and returned to stability after washing with PBS. In addition, the red shift caused by the antibody (red shifted from 877.1 nm to 881.4 nm) was significantly greater than that caused by BSA (negligible), indicating that pSC4 effectively binds to the antibody in the presence of a small amount of blocking BSA. After washing the residual BSA 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 showed a red shift, which means the presence of PD-L1 antigen was successfully detected. Finally, any impurities not bound to the antibody were rinsed off with PBS solution, enabling the sensor to accurately measure the PD-L1 concentration, which shifted from 881.06 nm to 881.97 nm. At this time, through the obtained wavelength shift, real-time specific quantitative detection of PD-L1 was achieved.

[0090] Figure 2 (b) of Figure 2 shows the corresponding wavelength shifts for different PD-L1 concentrations in the range of 50 ng / ml to 4000 ng / ml. 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 gives:

[0091] Y = 1(a + b·Xc-1) / (4 - 1)

[0092] where a = 1.093 ± 0.097 nm -1 , b = 395.8 ± 93.72 μg·(ml·nm) -1 , c = 0, and the 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 was calculated to be 28.1 ng / ml. According to the existing literature reports, the detection limit of 28.1 ng / ml is relatively higher than the PD-L1 standard for clinical peripheral blood samples (1.92 - 25 ng / ml) and also higher than existing technologies such as SERS and ELISA.

[0093] 2. Sensitivity verification data and results:

[0094] 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 moves up to 18.4 nanometers, and the response time of adjacent concentrations is less than 10 seconds. Figure 3 Figure (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

[0095]

[0096] 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 386.3nm / RIU. This sensitivity is significantly better than the traditional fiber optic sensor based only on gold nanoparticles (AuNPs) (70nm / RIU).

[0097] Example 2 Signal amplification strategy for PD-L1 detection based on nanostructured localized surface plasmon resonance fiber sensor (NS-LSPR-FOS)

[0098] 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. 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.

[0099] Existing PD-L1 aptamers and antibodies are 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. Unlike PD-L1 antibodies and aptamers, it binds to PD-L1 cells outside the cell with overlapping binding sites, eliminating the obstacles of spatial position. 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 4As shown in Figure a), a sandwich structure with spherical gold nanoparticles (AuNPs) modified with PD-L1 antibody, PD-L1 antigen, and SIBP functionalization was fabricated at the tip of the optical fiber to amplify the signal.

[0100] Experimental materials:

[0101]

[0102] Experimental procedure (the difference between this experiment and Example 1 is the addition of the sandwich structure, as shown in Figure a)): Figure 4 of Figure a)):

[0103] The previous experimental procedure is similar to that of Example 1 and will not be elaborated here. It is also necessary to prepare an SIBP solution to form a sandwich structure:

[0104] 1. Prepare a 100 μg / ml SIBP solution and a 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 groups (-SH) of cysteine in the polypeptide and the spherical gold nanoparticles (AuNPs).

[0105] 2. After all experiments are completed, that is, after measuring the PD-L1 antigen with a known concentration, wash it with PBS solution, and immerse the optical fiber sensor in the prepared AuNPs-SIBP solution for reaction for 45 minutes to allow SIBP to bind to the PD-L1 antigen. From this step on, record the reflection spectrum of the optical fiber sensor every 15 seconds.

[0106] 3. Wash the end face of the sensor with PBS solution to remove the unbound AuNPs-SIBP.

[0107] Signal amplification results of NS-LSPR-FOS for PD-L1 detection:

[0108] As Figure 4 shown in Figure b), after forming the sandwich structure of PD-L1 antibody, PD-L1 antigen, and SIBP-functionalized AuNP, a significant amplification effect was obtained, with the displacement distance increasing from the original 0.35 nm to 2.42 nm, and the wavelength shift increasing by more than 5 times. This proves the applicability of this sandwich structure-based signal amplification strategy for the sensitive detection of PD-L1 in this method, and also shows the potential of using this sandwich structure to amplify the signal strategy to reduce the detection limit of PD-L1 in this method.

[0109] Although the present invention has been disclosed as above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

1. A composite structure optical fiber sensor based on gold nano-urchins, characterized in that, The composite structure fiber optic sensor is prepared by the following method: (1) Prepare a 10 mM solution of 4-sulfonylcalix[4]arene hydrate (pSC4). Immerse NS-LSPR-FOS in the above pSC4 solution for 12 hours. Among them, the photon cavity thickness of NS-LSPR-FOS is 340 - 460 nm, and the size and spacing of the gold nano-urchins are 50 - 80 nm. (2) After the pSC4 modification, wash the fiber optic end face with deionized water to remove unbound pSC4 molecules on the surface. Immerse the fiber optic sensor end face in a PBS solution containing 20 μg / mL of PD-L1 antibody for 1 hour to form a PD-L1 specific recognition layer on the surface of the gold nano-urchins. Among them, during the connection process of the PD-L1 antibody and the pSC4 monolayer, the ambient temperature is maintained at 20 ± 0.5 °C. (3) Rinse the sensor end face with PBS. 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, and obtain a composite structure fiber optic sensor for detecting the concentration of PD-L1.

2. The composite structure optical fiber sensor based on gold nano-urchins according to claim 1, wherein The NS-LSPR-FOS is prepared by the following method: (1) Cut a 10-cm-long fiber optic and remove the 2-cm-long coating at the tip of the fiber optic with a stripping pliers. (2) To obtain a smooth and clean fiber optic end face, cut the tip of the fiber optic with only the cladding and the core left using a fiber optic cutter for effective sensing. (3) Hydroxylate the fiber optic in a plasma cleaner for 1 minute. (4) Prepare a solution with a volume ratio of water:isopropanol:APTES of 100:100:1, and let it stand for 2 hours. (5) Immerse the tip of the fiber optic in the APTES solution and let it stand for 15 minutes. After silanization, wash it with deionized water for 1 minute to remove the residual liquid remaining on the fiber optic end face. (6) Chemically vapor deposit Parylene-C on the surface of the fiber optic tip to form a photon cavity with a thickness of 340 - 460 nm. (7) Hydroxylate the fiber optic in a plasma cleaner for 1 minute. (8) Immerse the fiber optic end face in a 0.05 mg / ml THF solution of PS-b-P4VP for 10 minutes for functionalization. (9) After washing with deionized water, place the fiber optic in a fume hood to dry for 30 minutes, and then immerse it in a gold nano-urchin solution for 12 hours to evenly distribute the gold nano-urchin particles at the tip of the fiber optic. (10) Finally, quickly wash the end face of the gold nano-urchin-modified fiber optic sensor with deionized water and dry it for 30 min to obtain NS-LSPR-FOS for standby.

3. Use of the composite structure optical fiber sensor based on gold nanosea urchins according to any one of claims 1-2 in the detection of PD-L1, characterized in that, It includes the following steps: (1) Preparation of a local surface plasmon resonance fiber optic sensor (NS-LSPR-FOS) for specific detection of PD-L1: Prepare a 10 mM solution of 4-sulfonylcalix[4]arene hydrate (pSC4). Immerse NS-LSPR-FOS in the above pSC4 solution for 12 hours. (2) Connection of PD-L1 antibody to the pSC4 monolayer: After the pSC4 modification was completed, the end face of the optical fiber was washed 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 nanosea urchins; (3) BSA blocking: The end face of the sensor was rinsed with PBS, and the end face of the optical fiber sensor was immersed in a 10 mg / mL bovine serum albumin (BSA) solution for 20 minutes to block the active sites on the surface that were not bound to PD-L1; (4) Detection of PD-L1 antigen: (41) The end face of the optical fiber sensor was successively immersed in PD-L1 antigen solutions of different concentrations. Each group was immersed for 45 minutes, with the concentration increasing from low to high. Then, it was washed with PBS to remove unbound PD-L1 antigen specifically; (42) The reflection spectrum of the optical fiber sensor was recorded every 15 seconds by a spectrometer. By tracking the wavelength shift of the resonance valley, quantitative detection of PD-L1 protein at different concentrations was achieved; (43) Based on the antigen detection calibration curve measured for the optical fiber sensor in advance, the PD-L1 concentration was calculated according to the measured wavelength shift value of the resonance valley.

4. The application according to claim 3, wherein In step (1), the photon cavity thickness of NS-LSPR-FOS is 340 - 460 nm, and the size and spacing of the gold nanosea urchins are 50 - 80 nm.

5. The application according to claim 3, wherein In step (2), during the connection process of the PD-L1 antibody to the pSC4 monolayer, the ambient temperature was maintained at 20 ± 0.5 °C.

6. The application according to claim 3, characterized in that, In step (41), the concentration of the PD-L1 antigen solution is 50 - 4000 ng / ml. Before and after immersion in antigen solutions of different concentrations, it was washed with PBS solution to remove unbound PD-L1 antigen specifically.

7. The application according to claim 3, characterized in that, Step (42) is specifically as follows: First, a baseline was established using the measured value in the PBS solution environment after BSA blocking and before detection; After immersion in the solution containing PD-L1 antibody and after antigen-antibody binding, the wavelength corresponding to the resonance valley underwent a red shift; After that, impurities that were not bound to the PD-L1 antibody were washed away with PBS buffer, and the resonance wavelength reached the binding point of the antibody and PD-L1 and remained stable, and the wavelength shift value of the resonance valley was measured.

8. The application according to claim 3, wherein Step (4) also includes: After the reaction with PD-L1 antigen solutions of known concentrations was completed, the optical fiber sensor was immersed in a solution of SIBP-modified gold nanoparticles (AuNPs-SIBP) for 45 minutes to allow SIBP to bind to the PD-L1 antigen. The reflection spectrum of the optical fiber sensor was recorded every 15 seconds to track the wavelength shift of the resonance valley, so as to amplify the PD-L1 detection signal.

9. The application according to claim 8, wherein AuNPs-SIBP was prepared by the following method: Prepare a 100 μg / mL SIBP solution and a 10 nm gold nanoparticle solution with a mass fraction of 0.25 mg / mL; The SIBP solution was mixed with the gold nanoparticle solution at a volume ratio of 1:1 and left to stand at 4 °C for 12 hours to obtain AuNPs-SIBP.

10. The application according to claim 8, characterized in that It also includes: The end face of the optical fiber sensor was washed with PBS solution to remove unbound AuNPs-SIBP.

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