Method for screening tumor immune checkpoint inhibitor based on electrochemical luminescence sensing

Abstract

The embodiment of the invention discloses a method for screening a tumor immune checkpoint inhibitor based on electrochemical luminescence sensing. According to the invention, an electrochemiluminescence sensor is established, and resonance energy transfer between luminol (an electrochemiluminescence donor) and a PD-L1-targeting metal cluster (an electrochemiluminescence receptor) is utilized; the electrochemical luminescence sensor capable of rapidly, sensitively, simply, conveniently and quantitatively analyzing the equilibrium dissociation constant between the non-antibody immune checkpoint inhibitor and the PD-L1 expressed on the tumor cell membrane at low cost is established. The efficiency of blocking the PD-1 / PD-L1 by the inhibitor is evaluated through quantitative analysis of the constant, so that the potential non-antibody immune checkpoint inhibitor is effectively screened.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biological detection technology, and specifically to a method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing. Background Art

[0002] PD-L1 is a transmembrane protein typically expressed on the surface of antigen-presenting cells and tumor cells. PD-L1 expressed on tumor cells can bind to the programmed cell death receptor-1 (PD-1) on the surface of T lymphocytes, thereby inhibiting the killing effect of T lymphocytes and leading to tumor immune escape. One solution to this problem is to develop corresponding non-antibody immune checkpoint inhibitors to block the PD-1 / PD-L1 pathway. Currently, there are various methods for screening non-antibody immune checkpoint inhibitors that recognize PD-1 / PD-L1, such as enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence polarization, and nuclear magnetic resonance spectroscopy. However, these methods generally require expensive equipment and specialized personnel to operate.

[0003] Electrochemiluminescence (ECL) detection technology has attracted widespread attention in areas such as disease prescreening, single-cell analysis, food safety analysis, and environmental protection due to its simplicity, rapidity, and high sensitivity. ECL does not require an additional light source during detection and offers higher analytical sensitivity due to its low background noise.

[0004] Metal clusters are aggregates of metal atoms consisting of a few to several hundred metal atoms. They exhibit excellent photoelectrochemical properties and good biocompatibility. Their high surface-to-volume ratio provides a large number of binding sites for catalysis or chemical sensing. In recent years, metal clusters have been applied to various fields of electroanalytical chemistry due to their unique electrochemical and electrocatalytic properties. Summary of the Invention

[0005] To this end, an embodiment of the present invention provides a method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing, the method comprising:

[0008] (1) Preparing a polypeptide-metal cluster that specifically recognizes cell membrane PD-L1; wherein the polypeptide-metal cluster contains a targeting polypeptide that binds to the cell membrane PD-L1;

[0009] (2) Peptide-metal clusters compete with non-antibody immune checkpoint inhibitors for binding to PD-L1 on tumor cell membranes;

[0010] (3) Use electrochemical sensors to detect the ECL signals of tumor cells.

[0011] Furthermore, the preparation method of the polypeptide-metal cluster includes:

[0012] (1) Dissolve the polypeptide with specific recognition function in ultrapure water to prepare a 1 mM to 2 mM polypeptide solution;

[0013] (2) Add 25 mM HAuCl4 solution or AgNO3 solution to the polypeptide solution and stir vigorously at room temperature; wherein the molar ratio of polypeptide to HAuCl4 or AgNO3 is 1:1 to 1:2;

[0014] (3) In a light-proof environment, 0.5 M NaOH solution was added dropwise to the mixed solution obtained in step (2) until the pH of the system reached 9, and the mixture was reacted at 37° C. for 22 to 48 hours. The peptide-metal clusters were then purified by ultrafiltration.

[0015] Furthermore, the targeting polypeptide is a polypeptide sequence targeting PD-L1:

[0016] H2N-FPNWSLRPMNQMKKKKKYCC-COOH; or

[0017] H2N-MRNRERYPKPYY-COOH.

[0018] Furthermore, the process of the metal clusters competing with non-antibody immune checkpoint inhibitors for binding to PD-L1 on the tumor cell membrane is as follows:

[0019] After culture, PBS washing, digestion, and cell fixation, tumor cells were incubated with bovine serum albumin to block nonspecific active sites on the cell surface. After incubation, the supernatant was removed by centrifugation and the cells were thoroughly washed with PBS to obtain cell samples.

[0020] Competitively incubating the cell sample with a 12.5 μM peptide-metal cluster solution and a 5-60 μM immune checkpoint inhibitor;

[0021] The cells were washed extensively with PBS and counted for subsequent analysis.

[0022] Furthermore, the assembly process of the electrochemical sensor is as follows:

[0023] (1) Apply 6 μL of Luminol at a concentration of 1-6 mg / mL to the electrode surface and allow to dry at room temperature.

[0024] (2) Tumor cells that have been combined with peptide-metal clusters and immune checkpoint inhibitors and a certain number are evenly added to the electrode surface using a microinjector, and then ECL analysis is performed using Luminol as a substrate.

[0025] Furthermore, the test buffer used in the ECL analysis is a PBS buffer solution containing 20-50 mM H2O2, the pH of the PBS buffer solution is 7.0-10.0, and it is prepared with 0.1 M Na2HPO4 and 0.1 M KH2PO4.

[0026] Furthermore, the steps of detecting the ECL signal are as follows:

[0027] (1) Connect the modified screen-printed electrode to the dark box of the chemiluminescence detector, and connect the electrochemical workstation and the chemiluminescence detector together;

[0028] (2) The high-voltage parameters of the photomultiplier tube were set to ×1 gain mode, and the scanning potential window was optimized to 0.1–1.5 V to ensure the best acquisition sensitivity of the electrochemiluminescence signal;

[0029] (3) The ECL signal size of a series of tumor cells that recognize metal clusters and a certain number of immune checkpoint inhibitors is measured, and a nonlinear relationship between the ECL signal and different immune checkpoint inhibitor incubation concentrations is established to evaluate the equilibrium dissociation constant between the immune checkpoint inhibitors and PD-L1 expressed on the cell membrane.

[0030] like Figure 1 As shown, the present invention will build an ECL sensor, using the resonance energy transfer between luminol (ECL donor) and metal clusters targeting PD-L1 (ECL receptors), to build an ECL sensor that can quickly, sensitively, simply and at low cost quantitatively analyze the equilibrium dissociation constant between non-antibody immune checkpoint inhibitors and PD-L1 expressed on tumor cell membranes. Specifically, the metal clusters compete with non-antibody immune checkpoint inhibitors for binding to PD-L1 on tumor cell membranes. As the binding of non-antibody immune checkpoint inhibitors to PD-L1 increases, the ECL signal increases. The analysis results are used to quantify the equilibrium dissociation constant between immune checkpoint inhibitors and PD-L1 expressed on tumor cell membranes, and the efficiency of the inhibitors in blocking PD-1 / PD-L1 is evaluated by quantitative analysis of the constant, thereby effectively screening potential non-antibody immune checkpoint inhibitors.

[0031] The embodiments of the present invention have the following advantages:

[0032] (1) The present invention uses a peptide-metal cluster that targets cell membrane PD-L1 to achieve efficient sensor assembly and precise analysis of the interaction between inhibitors and PD-L1. This design not only improves the analytical specificity of the sensor, but also simplifies the preparation process and reduces the analysis cost.

[0033] (2) The present invention utilizes the principle of ECL resonance energy transfer between Luminol (ECL donor) and metal clusters targeting cell membrane PD-L1 (ECL receptor) to quantitatively analyze the equilibrium dissociation constant between non-antibody immune checkpoint inhibitors and PD-L1 expressed on tumor cell membranes.

[0034] (3) The sensor of the present invention can operate under conditions that simulate near-physiological conditions, thereby more accurately measuring the equilibrium dissociation constant between non-antibody inhibitors and cell membrane PD-L1. This precise detection capability is of great significance for understanding the interaction mechanism between drugs and targets.

[0035] (4) The sensor of the present invention can perform in situ detection at the cellular level, without destroying the cell membrane structure to extract the target protein. This feature enables the sensor to more realistically reflect the physiological state of the cell membrane PD-L1 protein, providing an ideal tool for cell function research and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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 the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0037] Figure 1 A diagram showing the construction process of the electrochemiluminescence immunosensor provided by the present invention;

[0038] Figure 2 The fluorescence excitation and emission spectra of the polypeptide-gold cluster provided by the present invention;

[0039] Figure 3 The fluorescence excitation and emission spectra of the polypeptide-silver cluster provided by the present invention;

[0040] Figure 4 This is a graph showing the relationship between the ECL response intensity and the concentration of the small molecule inhibitor IN3 provided by the present invention;

[0041] Figure 5 A linear relationship diagram between the reciprocal of the ECL change rate and the reciprocal of the concentration of the small molecule inhibitor IN3 provided by the present invention;

[0042] Figure 6 This is a graph showing the relationship between the ECL response intensity and the concentration of the small molecule inhibitor IN23 provided by the present invention;

[0043] Figure 7 The linear relationship between the reciprocal of the ECL change rate and the reciprocal of the concentration of the small molecule inhibitor IN23 provided by the present invention;

[0044] Figure 8 A graph showing the relationship between the ECL response intensity and the concentration of the peptide inhibitor 22 provided by the present invention;

[0045] Figure 9 A linear relationship diagram between the reciprocal of the ECL change rate and the reciprocal of the peptide concentration of the polypeptide inhibitor 22 provided by the present invention;

[0046] Figure 10 A graph showing the relationship between the ECL response intensity and the concentration of the peptide inhibitor 5 provided by the present invention;

[0047] Figure 11 The linear relationship between the reciprocal of the ECL change rate and the reciprocal of the peptide inhibitor 5-peptide concentration provided by the present invention;

[0048] Figure 12 The Jurkat cells provided by the present invention were activated by CD3 and CD28, and then co-cultured with A549 cells stimulated with 1 ng / mL IFN-γ and 100 nM of the small molecule inhibitor IN-3 or IN-23. The relative viability of the Jurkat cells was assessed using a CCK-8 kit (n=3).

[0049] Figure 13 The Jurkat cells provided by the present invention were activated by CD3 and CD28 and then co-cultured with A549 cells stimulated with 1 ng / mL IFN-γ and 100 nM of the polypeptide inhibitor 22-peptide or 5-peptide. The IFN-γ content in the cell supernatant of each group was determined using an ELISA kit (n=3). DETAILED DESCRIPTION

[0050] The present invention will be described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can understand and implement the present invention and further recognize the advantages of the present invention.

[0051] Unless otherwise defined in this specification, all technical terms herein are used according to conventional definitions commonly used and understood by those skilled in the art. The experimental methods described in the following examples are conventional methods unless otherwise noted; the reagents and materials described are commercially available unless otherwise noted.

[0052] Example 1

[0053] In this example, a peptide-gold cluster that specifically recognizes PD-L1 was first prepared, and then an ECL sensor was constructed based on this gold cluster. The specific steps are as follows:

[0054] (1) Preparation of peptide-gold clusters that specifically recognize PD-L1

[0055] (a) A peptide with a specific recognition function (peptide sequence: H2N-FPNWSLRPMNQMKKKKKYCC-COOH) was dissolved in ultrapure water and stirred for 3 minutes to prepare a 1 mM peptide solution.

[0056] (b) adding 25 mM HAuCl4 solution to the polypeptide solution of step (a), and vigorously stirring at room temperature to obtain a polypeptide-HAuCl4 mixed solution; wherein the molar ratio of polypeptide to HAuCl4 is 1:1;

[0057] (c) In a dark environment, 0.5M NaOH solution was added dropwise to the mixture of step (b) until the pH of the system was 9, and the reaction was continued at 37°C for 22 hours. After the reaction, ultrafiltration (molecular weight cut-off of 10KD) was used for purification to obtain polypeptide-gold clusters with good fluorescence properties ( Figure 2 ).

[0058] (2) Metal clusters compete with small molecule immune checkpoint inhibitors for binding to PD-L1 on tumor cell membranes

[0059] Remove the A549 cell culture dish that has been cultured for 24 hours from the incubator and gently remove the culture medium. Subsequently, wash the A549 cells three times with phosphate buffered saline (PBS) to remove residual culture medium and impurities. Next, add PBS-EDTA solution to digest the cells until the cells are completely detached and collected in a 15mL centrifuge tube. Add 1mL of 4% cell fixative to the centrifuge tube and fix the cells at room temperature for 30 minutes. After fixation, remove the fixative by centrifugation (2000rpm; 5min) and wash the cells three times with PBS to remove the residual fixative. Subsequently, add 2mL of 3% bovine serum albumin (BSA) solution to the centrifuge tube and incubate at room temperature for 30 minutes to block the nonspecific active sites on the cell surface. After incubation is complete, centrifuge to remove the supernatant and discard the BSA solution. Wash the cells thoroughly 5 times with PBS, removing excess PBS after each centrifugation to ensure that the nonspecific sites on the cell surface are fully blocked. Finally, the washed cell samples were competitively incubated with a peptide-gold cluster solution and varying concentrations of the small molecule inhibitors IN3 (CAS No.: 2953044-29-4) or IN23 (CAS No.: 2597056-04-5) for half an hour at room temperature. Following incubation, the cells were washed thoroughly with PBS to remove any unbound clusters or inhibitors. Following washing, the cells were counted for subsequent analysis.

[0060] (3) Construction process of electrochemical sensor

[0061] (a) 10 μL of 1 mg / mL Luminol was added dropwise to the electrode surface and allowed to dry at room temperature.

[0062] (b) A certain number of tumor cells that have been identified with the gold clusters and small molecule inhibitors are evenly added to the electrode surface using a microinjector to allow them to adhere evenly to the electrode. Subsequently, ECL analysis is performed using Luminol as a substrate.

[0063] Example 2

[0064] In this example, a peptide-silver cluster that specifically recognizes PD-L1 was first prepared, and then an ECL sensor was constructed based on this silver cluster. The specific preparation steps are as follows:

[0065] (1) Preparation of peptide-silver clusters that specifically recognize PD-L1

[0066] (a) Dissolve peptide P2 with a purity greater than 98% in ultrapure water to prepare a 1 mM to 2 mM peptide solution, and stir for 3 minutes. The targeting peptide sequence is H2N-MRNRERYPKPYY-COOH.

[0067] (b) Add 25 mM AgNO3 solution to the polypeptide solution and stir vigorously at room temperature to obtain a polypeptide-AgNO3 mixed solution, wherein the molar ratio of polypeptide to AgNO3 is 1:1.2.

[0068] (c) In a dark environment, 0.5M NaOH solution was added dropwise to the mixed solution of step (b) and the reaction was continued for 48 hours. After the reaction was completed, the resulting solution was stirred at room temperature and then purified by ultrafiltration to obtain polypeptide-silver clusters with good fluorescence properties ( Figure 3 ).

[0069] (2) Metal clusters compete with peptide immune checkpoint inhibitors for binding to PD-L1 on tumor cell membranes

[0070] Remove the A549 cell culture dish that has been cultured for 24 hours from the incubator and gently remove the culture medium. Subsequently, wash the A549 cells three times with phosphate buffered saline (PBS) to remove residual culture medium and impurities. Next, add PBS-EDTA solution to digest the cells until the cells are completely detached and collected in a 15mL centrifuge tube. Add 1mL of 4% fixative to the centrifuge tube and fix the cells at room temperature for 30 minutes. After fixation, remove the fixative by centrifugation and wash the cells three times with PBS to remove the residual fixative. Subsequently, add 2mL of 3% bovine serum albumin (BSA) solution to the centrifuge tube and incubate at room temperature for 30 minutes to block the nonspecific active sites on the cell surface. After incubation is complete, centrifuge to remove the supernatant and discard the BSA solution. Wash the cells thoroughly 5 times with PBS, removing excess PBS after each centrifugation to ensure that the nonspecific sites on the cell surface are fully blocked. Finally, the washed cell samples were competitively incubated with a 12.5 μM peptide-silver cluster solution and 2–60 nM peptide inhibitors, either a 22-peptide (H2N-SGQYASYHCWCWRDPGRSGGSK-COOH) or a 5-peptide (H2N-RVYSF-COOH). Following incubation, the cells were washed thoroughly with PBS to remove any unbound clusters or peptide inhibitors. After washing, the cells were counted for subsequent analysis.

[0071] (3) Assembly process of electrochemical sensors

[0072] (a) 10 μL of 1 mg / mL Luminol was applied to the electrode surface and allowed to dry at room temperature.

[0073] (b) A certain number of tumor cells that have recognized silver clusters and inhibitors are evenly added to the electrode surface using a microinjector and uniformly adhere to the electrode. Subsequently, ECL analysis is performed using Luminol as a substrate.

[0074] Example 3

[0075] In this example, ECL was used to analyze the binding ability of non-antibody (including small molecules and peptides) immune checkpoint inhibitors to PD-L1 expressed on tumor cell membranes. The specific steps are as follows:

[0076] (1) Connect the modified screen-printed electrode to the dark box of the chemiluminescence detector, and connect the electrochemical workstation and the chemiluminescence detector together;

[0077] (2) Chemiluminescence detector parameter settings: The high voltage of the photomultiplier tube is set to ×1, and the scanning potential range is 0.1 to 1.5 V;

[0078] (3) The electrochemical test buffer solution is a 30 mM H2O2 PBS buffer solution, wherein the PBS buffer solution has a pH of 7.5 and is composed of 0.1 M Na2HPO4 and 0.1 M KH2PO4;

[0079] (4) The ECL signal size of a series of tumor cells that recognize metal clusters (gold clusters or silver clusters) and non-antibody inhibitors (including small molecules and peptides) is measured, and a nonlinear relationship between the ECL signal and the incubation concentration of different inhibitors is established to evaluate the binding ability of the inhibitors to PD-L1 expressed on the cell membrane.

[0080] Example 4

[0081] In this example, ECL was used to analyze the equilibrium dissociation constant between a small molecule immune checkpoint inhibitor and PD-L1 expressed on the tumor cell membrane. Specifically, the sensor constructed in Example 1 was used to detect specific small molecule inhibitors IN3 or IN23 according to the detection method of Example 3. According to the changes in the incubation concentration of the PD-L1 small molecule inhibitor, the signal change curve of the immobilized cell ECL sensor was plotted (see Figure 4 、 Figure 6 ), there is a hyperbolic relationship between the two, and the saturation effect can be described by the Lineweaver-Burk formula. Then the double reciprocal fitting method is used to fit the small molecule inhibitor concentration and the signal change rate of the ECL sensor, as shown in Figure 5 、 Figure 7 As shown, for IN3 and IN23, the correlation results are: 1 / ΔECL=7.5×10 -6 ×1 / C+5.1×10 -5 (R 2 =0.99) and 1 / ΔECL=3.7×10 -6 ×1 / C+5.9×10 -4 (R 2=0.99). The calculated equilibrium dissociation constants (Kd) for the small molecule inhibitors IN3 and IN23 with PD-L1 were 146 nM and 6.29 nM, respectively. These results closely matched previously reported Kd values ​​for IN3 and IN23 molecules with PD-L1, demonstrating the excellent detection performance of the constructed sensor.

[0082] Example 5

[0083] In this example, ECL was used to analyze the equilibrium dissociation constant between a polypeptide immune checkpoint inhibitor and PD-L1 expressed on the tumor cell membrane. Using the sensor constructed in Example 2, a specific polypeptide inhibitor was detected according to the detection method of Example 4. According to the changes in the incubation concentration of the PD-L1 polypeptide inhibitor, the signal change curve of the immobilized cell ECL sensor was plotted (see Figure 8 、 Figure 10 ), there is a hyperbolic relationship between the two, and the saturation effect can be described by the Lineweaver-Burk formula. Then the double reciprocal fitting method was used to fit the peptide inhibitor concentration and the signal change rate of the ECL sensor, as shown in Figure 9 、 Figure 11 As shown, for 22 peptides and 5 peptides, the correlation results are: 1 / ΔECL=2.0×10 -5 ×1 / C+1.5×10 -4 (R 2 =0.99) and 1 / ΔECL=4.6×10 -5 ×1 / C+3.5×10 -4 (R 2 =0.99). The calculated binding constants (Kd) for the peptide inhibitors 22-peptide or 5-peptide with PD-L1 were 133 nM or 1.31 μM, respectively. These results closely matched previously reported Kd values ​​for 22-peptide or 5-peptide inhibitors with PD-L1, demonstrating the excellent detection performance of the constructed sensor.

[0084] Example 6

[0085] Under simulated physiological conditions, the effectiveness of small molecule inhibitors targeting PD-L1 to block immune checkpoints was verified. The specific steps are as follows:

[0086] In this embodiment, if Figure 12As shown, Jurkat cells in the experimental group were first stimulated with CD3 and CD28 antibodies to activate the T cell receptor signaling pathway. This resulted in a significant increase in cell viability, indicating a well-activated state. Subsequently, the activated Jurkat cells were co-cultured with A549 cells. The results showed that Jurkat cell viability decreased significantly under co-culture conditions, suggesting that A549 cells may affect Jurkat cell activity through an immunosuppressive mechanism. This decrease in Jurkat cell viability is attributed to the interaction between PD-1 and PD-L1, which inhibits Jurkat cell proliferation. To test this hypothesis, the small molecule inhibitors IN3 or IN23 were added to block PD-1 / PD-L1 binding. The results showed that cell viability increased to 61.69% and 115.24%, respectively, demonstrating that small molecule inhibitors can effectively antagonize the interaction between PD-1 and PD-L1, blocking the negative regulatory signals transmitted by immune checkpoints. This example verified the successful establishment of the immunosuppressive microenvironment model through changes in Jurkat cell activity and confirmed that the small molecule inhibitor IN23 has a higher effectiveness in blocking the PD-1 / PD-L1 signaling pathway than IN3, which is consistent with the smaller Kd value of IN23 measured by ECL than IN3.

[0087] Example 7

[0088] Under simulated physiological conditions, the effectiveness of peptide inhibitors targeting PD-L1 in blocking immune checkpoints was verified. The specific steps are as follows:

[0089] In this example, the concentration of interferon-γ (IFN-γ) in the supernatant of each group of Jurkat cells was quantitatively analyzed. Figure 13 As shown in the experiment, enzyme-linked immunosorbent assay (ELISA) revealed a significant increase in IFN-γ secretion when Jurkat cells were activated with CD3 and CD28 antibodies, indicating that activation of the T cell receptor (TCR) signaling pathway can effectively induce the secretion of these two cytokines. As PD-L1 expression in A549 cells increased, IFN-γ secretion by Jurkat cells decreased significantly, suggesting that the interaction between PD-L1 and PD-1 may inhibit Jurkat cell activation and thereby reduce cytokine secretion. However, after the addition of the peptide inhibitors 22-peptide or 5-peptide, IFN-γ levels rebounded to a higher level, indicating that the peptide inhibitors can block the PD-1 / PD-L1 signaling pathway, restore Jurkat cell activation, and promote cytokine secretion. The 22-peptide peptide demonstrated greater efficacy in blocking the PD-1 / PD-L1 signaling pathway than the 5-peptide peptide, consistent with the smaller Kd value of the 22-peptide compared to the 5-peptide peptide as measured by ECL.

[0090] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing, characterized in that: The method comprises: (1) Preparing a polypeptide-metal cluster that specifically recognizes cell membrane PD-L1; wherein the polypeptide-metal cluster contains a targeting polypeptide that binds to the cell membrane PD-L1; (2) Peptide-metal clusters compete with non-antibody immune checkpoint inhibitors for binding to PD-L1 on tumor cell membranes; (3) Use electrochemical sensors to detect the ECL signals of tumor cells.

2. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 1, characterized in that: The preparation method of the polypeptide-metal cluster comprises: (1) Dissolve the polypeptide with specific recognition function in ultrapure water to prepare a 1 mM to 2 mM polypeptide solution; (2) Add 25 mM HAuCl4 solution or AgNO3 solution to the polypeptide solution and stir vigorously at room temperature; wherein the molar ratio of polypeptide to HAuCl4 or AgNO3 is 1:1 to 1:2; (3) In a light-proof environment, 0.5 M NaOH solution was added dropwise to the mixed solution obtained in step (2) until the pH of the system reached 9, and the mixture was reacted at 37° C. for 22 to 48 hours. The peptide-metal clusters were then purified by ultrafiltration.

3. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 1, characterized in that: The targeting polypeptide is a polypeptide sequence targeting PD-L1: H2N-FPNWSLRPMNQMKKKKKYCC-COOH; or H2N-MRNRERYPKPYY-COOH.

4. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 1, characterized in that: The process of the metal clusters competing with the non-antibody immune checkpoint inhibitors for binding to PD-L1 on the tumor cell membrane is as follows: After culture, PBS washing, digestion, and cell fixation, tumor cells were incubated with bovine serum albumin to block nonspecific active sites on the cell surface. After incubation, the supernatant was removed by centrifugation and the cells were thoroughly washed with PBS to obtain cell samples. Competitively incubating the cell sample with a 12.5 μM peptide-metal cluster solution and a 5-60 μM immune checkpoint inhibitor; The cells were washed extensively with PBS and counted for subsequent analysis.

5. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 1, characterized in that: The assembly process of the electrochemical sensor is as follows: (1) Apply 6 μL of Luminol at a concentration of 1-6 mg / mL to the electrode surface and allow to dry at room temperature. (2) Tumor cells that have been combined with peptide-metal clusters and immune checkpoint inhibitors and a certain number are evenly added to the electrode surface using a microinjector, and then ECL analysis is performed using Luminol as a substrate.

6. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 5, characterized in that: The test buffer used in the ECL analysis is a PBS buffer solution containing 20-50 mM H2O2, the pH of the PBS buffer solution is 7.0-10.0, and it is prepared with 0.1 M Na2HPO4 and 0.1 M KH2PO4.

7. The method for screening tumor immune checkpoint inhibitors based on electrochemiluminescence sensing according to claim 1, characterized in that: The detection steps of the ECL signal are as follows: (1) Connect the modified screen-printed electrode to the dark box of the chemiluminescence detector, and connect the electrochemical workstation and the chemiluminescence detector together; (2) The high-voltage parameters of the photomultiplier tube were set to ×1 gain mode, and the scanning potential window was optimized to 0.1–1.5 V to ensure the best acquisition sensitivity of the electrochemiluminescence signal; (3) The ECL signal size of a series of tumor cells that recognize metal clusters and a certain number of immune checkpoint inhibitors is measured, and a nonlinear relationship between the ECL signal and different immune checkpoint inhibitor incubation concentrations is established to evaluate the equilibrium dissociation constant between the immune checkpoint inhibitors and PD-L1 expressed on the cell membrane.

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