CN119639902A

CN119639902A - Probe set for detecting soluble PD-L1 in peripheral blood and application thereof

Info

Publication number: CN119639902A
Application number: CN202510037386.1A
Authority: CN
Inventors: 彭兰稂; 陈维贤; 丁小娟
Original assignee: Second Affiliated Hospital of Chongqing Medical University
Current assignee: Second Affiliated Hospital of Chongqing Medical University
Priority date: 2025-01-09
Filing date: 2025-01-09
Publication date: 2025-03-18

Abstract

The present application relates to a probe set for detecting soluble PD-L1 in peripheral blood and its application, the probe set includes a recognition probe and a substrate probe, the recognition probe is formed by hybridization of an aptamer and an activator ssDNA, the substrate probe includes modified carboxyl magnetic beads and horseradish peroxidase modified with streptavidin, and the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino groups and biotin. The present application innovatively connects HRP-ssDNA signal probes on the surface of modified carboxyl magnetic beads, and a large number of HRP-ssDNA signal reporter probes are loaded on the surface of modified carboxyl magnetic beads. The activated Cas12a enzyme cyclically shears the HRP-ssDNA signal probes on the surface of modified carboxyl magnetic beads. The modified carboxyl magnetic beads can greatly increase the concentration of reporter probes in the confined area, overcome the problem of low reaction efficiency caused by free diffusion, and significantly improve the shearing efficiency of the Cas12a enzyme, realizing single target input and large product output. The chemiluminescent sensor made of the probe of the present application is used to detect sPD-L1 in serum, which has high sensitivity and strong specificity, making up for the shortcomings of the prior art in terms of sensitivity.

Description

Probe set for detecting soluble PD-L1 in peripheral blood and application thereof

Technical Field

The application relates to the technical field of biology, in particular to a probe set for detecting soluble PD-L1 in peripheral blood and application thereof.

Background

The rise of tumor immunotherapy, in particular Immune Checkpoint (ICB) therapy, significantly improves the therapeutic prospects in patients with advanced tumors. To date, anti-apoptosis ligand 1 (PD-L1) immunohistochemical detection (IHC) on tissue sections is the only validated companion diagnostic in the first line immunotherapy of advanced and metastatic cancers, especially non-small cell lung cancer (NSCLC). However, this approach has the limitations of originality, tumor tissue heterogeneity, and difficulty in dynamic monitoring. Therefore, the exploration of new biomarkers and detection techniques is critical to achieving personalized accurate predictions of immunotherapy.

The soluble PD-L1 (sPD-L1) in peripheral blood is derived from a metalloprotease cleavage and splice variant of the membrane-bound PD-L1, so that the PD-1 binding domain is reserved, and the polypeptide has an immunosuppressive function, can interfere with the PD-1 on T cells, inhibit immune response and further promote immune escape of tumor cells. In addition, sPD-L1 can also act as a "bait", mediating resistance of PD-L1 blocking antibodies, and thus is considered a key biomarker for predicting the efficacy of immune checkpoint inhibitors. Studies have shown that sPD-L1 levels are significantly elevated in various tumor patients compared to healthy individuals and are associated with poor survival rates for various tumor types (e.g., malignant melanoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, biliary tract cancer, renal cell carcinoma, colorectal cancer).

In the related art, ELISA method is generally used for detecting soluble PD-L1 (sPD-L1) in peripheral blood. However, ELISA method for detecting soluble PD-L1 (sPD-L1) in peripheral blood has the defects that the detection limit is ng/mL, detection of low-abundance sPD-L1 in early clinical or drug remission is difficult to realize, and meanwhile, the antibody adopted by ELISA method is expensive and is unfavorable for preservation.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a probe for detecting soluble PD-L1 in peripheral blood, and a preparation method and application thereof, so as to solve the technical problems that the detection limit of the soluble PD-L1 (sps d-L1) in peripheral blood by ELISA method is ng/mL, it is difficult to detect low abundance sps d-L1 in early clinical or drug remission period, and the antibody adopted by ELISA method is expensive and is unfavorable for preservation.

In order to achieve the above object, the present application is as follows:

In a first aspect, the present invention provides a probe set for detecting soluble PD-L1 in peripheral blood, the probe set comprising a recognition probe hybridized by an aptamer with an activator ssDNA and a substrate probe (mb@ssdna-HRP) comprising a modified carboxyl magnetic bead modified by ssDNA modified with an amino group (-NH ₂) and Biotin (Biotin) and horseradish peroxidase (Horseradish Peroxidase, HRP) modified with streptavidin.

Optionally, the amino group is modified at the 5' end of the ssDNA, and the amino group of the ssDNA is bound to the carboxyl magnetic bead in a reaction.

Optionally, the biotin is modified at the 3' end of the ssDNA.

Alternatively, the aptamer may specifically recognize soluble anti-apoptosis ligand 1 (sPD-L1).

Alternatively, the activator ssDNA can activate Cas12a enzyme.

Optionally, the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the activator ssDNA is shown as SEQ ID NO. 2.

Alternatively, the nucleotide sequence of the ssDNA is shown as SEQ ID NO. 4.

Optionally, the recognition probe is prepared by a method comprising the steps of:

mixing the aptamer, the activator ssDNA and a buffer solution, incubating, and cooling to prepare the recognition probe;

Alternatively, the molar ratio of the aptamer to the activator ssDNA is 1-2:1-2.

Alternatively, the buffer is a PBS buffer containing magnesium chloride and having a pH of 7.2-7.5.

Optionally, the concentration of magnesium chloride in the PBS buffer is 0.5-0.6mM.

Optionally, the temperature of the co-incubation is 90-100 ℃, and the duration of the co-incubation is 4-6min.

Optionally, the temperature is reduced to room temperature at a rate of 0.05-0.2 ℃ per minute.

Alternatively, the substrate probe is prepared by a method comprising the steps of:

Adding a carbodiimide hydrochloride solution and an N-hydroxysuccinimide (NHS) solution into a carboxyl magnetic bead solution, activating, magnetically separating, adding a biotinylation reagent into the obtained precipitate, wherein the biotinylation reagent comprises amino and biotin, reacting, magnetically separating for the second time, adding a blocking solution into the obtained precipitate, incubating, magnetically separating for the third time, adding streptavidin coupled with horseradish peroxidase into the obtained precipitate, magnetically separating for the second time, and magnetically separating for the fourth time to obtain the substrate probe.

Alternatively, the carbodiimide hydrochloride is selected from 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC).

Optionally, the mass ratio of the carbodiimide hydrochloride to the N-hydroxysuccinimide is 8-12:8-12.

Optionally, the activation temperature is 20-30 ℃, and the activation time is 0.5-1.5h.

Optionally, during the activation, the rotational speed is 500-1000rpm.

Optionally, the mass ratio of the carboxyl magnetic beads to the biotinylation reagent is 8-12;0.05-0.06.

Optionally, the mass ratio of the carboxyl magnetic beads to the horseradish peroxidase-conjugated streptavidin is 8-12:0.0005-0.001.

Alternatively, the temperature of the reaction is 20-30 ℃, and the duration of the reaction is 0.5-1.5h.

Optionally, the rotational speed is 500-1000rpm during the reaction.

Optionally, the incubation is at a temperature of 20-30 ℃ and the incubation period is 0.5-1.5h.

Optionally, the temperature of the secondary reaction is 20-30 ℃, and the duration of the secondary reaction is 20-40min.

Optionally, the rotation speed is 500-1000rpm in the secondary reaction process.

In a second aspect, the application also provides a chemiluminescent sensor for detecting soluble anti-apoptotic ligand 1 in peripheral blood comprising a probe set as described above.

Optionally, the chemiluminescent sensor further comprises a Cas12a enzyme.

In a third aspect, the application also provides the use of a probe set as described above or a chemiluminescent sensor as described above for the preparation of a kit for detecting soluble anti-apoptotic ligand 1 in peripheral blood.

In a fourth aspect, the application also provides a kit for detecting soluble anti-apoptotic ligand 1 in peripheral blood, said kit comprising a probe set as described above or a chemiluminescent sensor as described above.

As described above, the probe set for detecting soluble PD-L1 in peripheral blood and the application thereof of the application have the following beneficial effects:

The application designs a probe set for detecting a Soluble anti-apoptosis ligand 1 (sPD-L1) in peripheral blood, wherein the probe set comprises a substrate probe (MB@ssDNA-HRP) and a recognition probe, the substrate probe comprises a modified carboxyl magnetic bead and a streptomycin-modified horseradish peroxidase (Horseradish Peroxidase, HRP), wherein the modified carboxyl magnetic bead is obtained by modifying the carboxyl magnetic bead by amino (-NH ₂) and Biotin (Biotin) modified ssDNA, the substrate probe can serve as a non-specific cleavage substrate of Cas12a protein (namely Cas12a enzyme), the recognition probe is a nucleic acid probe capable of recognizing the Soluble PD-L1, the nucleic acid aptamer (Apt) capable of specifically recognizing the Soluble PD-L1 is complementary with an ' activator ' single-stranded nucleic acid (activator ssDNA) of Cas12a protein, when the Soluble (namely Solubside) PD-L1 exists, the Apt recognizes sPD-L1 and then undergoes a conformational change, the ' HRP-is released, the ' activator DNA ' is chemically amplified to release a fluorescent signal, and the fluorescent signal is released from the fluorescent signal of the fluorescent protein (namely, the fluorescent protein) is chemically amplified by the fluorescent protein (HRP) and the fluorescent protein) is released to the fluorescent protein (namely Cas12a protein), and the fluorescent protein is quantitatively released by the fluorescent protein (namely, the fluorescent protein) and the fluorescent protein is bound to the fluorescent protein (In) and the fluorescent protein) is released by the fluorescent protein (Inactive protein) and the fluorescent protein.

According to the application, the HRP-ssDNA signal probes are innovatively connected to the surface of the modified carboxyl magnetic beads, a large number of HRP-ssDNA signal reporting probes are loaded on the surface of the modified carboxyl magnetic beads, and the activated Cas12a enzyme circularly shears the HRP-ssDNA signal probes on the surface of the magnetic beads, meanwhile, the modified carboxyl magnetic beads can greatly improve the concentration of the reporting probes in a limited area, overcome the problem of low reaction efficiency caused by free diffusion, remarkably improve the shearing efficiency of the Cas12a enzyme, simply realize single target input and output a large number of products.

The chemiluminescent sensor prepared by the probe provided by the application detects sPD-L1 in serum, has high sensitivity and strong specificity, overcomes the defects in the sensitivity of the detection technology in the prior art, is beneficial to the personalized treatment of tumor patients, and can effectively monitor the prognosis of immunotherapy.

Compared with an antibody, the nucleic acid aptamer is low in cost, easy to modify and store, and can be used for recognizing a target with high specificity and high affinity.

The substrate probe MB@ssDNA-HRP of the application gathers the output signal in a limited range, and the activated Cas12a protein can circularly shear the ssDNA around the probe to release more HRP, so that the output signal is further enhanced.

Unlike detecting PD-L1 on tissue section, the chemiluminescent sensor prepared with the probe of the present application detects sPD-L1 in serum, and has no wound, no minimally invasive, high patient acceptance, and capacity of effectively and dynamically monitoring blood sample during treatment.

In the prior art, a ELSIA method is adopted to detect sPD-L1 in serum, the detection time is about 165min, and a chemiluminescent sensor prepared by the probe is adopted to detect sPD-L1 in serum, the detection time is about 50min, so that the reaction time is obviously shortened, and the detection efficiency is improved.

Drawings

FIG. 1 is a schematic diagram of the present application;

FIG. 2 is a flow chart of a process for preparing a substrate probe according to the present application;

FIG. 3 is a graph of the colorimetric characterization results of the substrate probes of the present application;

FIG. 4 is a schematic diagram of an identification probe in a chemiluminescent sensor of the present application, MFE proxy structure representing a minimum free energy proxy structure, FREE ENERGY of the secondary structure representing the free energy of a secondary structure;

FIG. 5 is a graph of chemiluminescent signals in a feasibility test, fold indicating a multiple, without indicating no, and width indicating no, CL INTENSITY indicating the intensity of chemiluminescent signals, and the same applies;

FIG. 6 is a graph of the results of chemiluminescent signal analysis for samples of different concentrations in a sensitivity test, the Wavelength being shown by Wavelength;

FIG. 7 is a graph (i.e., a linear fit) of calibration of various concentrations of sPD-L1 versus chemiluminescent signal values;

FIG. 8 is a graph showing the results of the specificity test, and Blank represents a Blank group.

Detailed Description

The present invention will be further described with reference to the following specific examples, but it should be noted that the specific material ratios, process conditions, results, etc. described in the embodiments of the present invention are only for illustrating the present invention, and are not intended to limit the scope of the present invention, and all equivalent changes or modifications according to the spirit of the present invention should be included in the scope of the present invention.

As shown in fig. 1, the principle of the chemiluminescent sensor of the present application is as follows:

The application designs a probe set for detecting a Soluble anti-apoptosis ligand 1 (sPD-L1) in peripheral blood, which comprises a substrate probe (MB@ssDNA-HRP) and a recognition probe, wherein the substrate probe comprises a modified carboxyl magnetic bead and a streptomycin-modified horseradish peroxidase (Horseradish Peroxidase, HRP), the modified carboxyl magnetic bead is obtained by modifying the carboxyl magnetic bead by amino (-NH ₂) and Biotin (Biotin) modified ssDNA, the substrate probe can serve as a non-specific cleavage substrate of Cas12a protein (namely Cas12a enzyme), the recognition probe is a nucleic acid probe capable of recognizing the Soluble PD-L1, the nucleic acid aptamer (Apt) capable of specifically recognizing the Soluble PD-L1 is complementarily composed of an activator (activator ssDNA) capable of specifically recognizing the Soluble PD-L1, when the Soluble (namely Soluble) PD-L1 exists, the conformation change is generated after the AN_SNt recognizes sPD-L1, the activator DNA is released, the activator DNA is chemokines-activated, and the fluorescent dye (HRP) is released, and the fluorescent dye (HRP) is chemically separated from the fluorescent dye-RNA is released, and the fluorescent dye (HRP) is bound to a fluorescent dye-activated by the fluorescent dye, and the fluorescent dye (HRP) is released, and the fluorescent dye is released.

The present invention will be described in detail with reference to specific exemplary examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, as many insubstantial modifications and variations are within the scope of the invention as would be apparent to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.

Construction of (one) MB@ssDNA-HRP (i.e., substrate probe)

The construction of MB@ssDNA-HRP (i.e., substrate probe) was performed according to the procedure shown in FIG. 2, and specifically comprises the following steps:

1.1 washing 100. Mu.L of carboxyl magnetic beads (i.e.MB-COOH) solution (commercially available, at a concentration of 1. Mu.M) with 200. Mu.L of MEST solution (commercially available, containing 100mM MES and 0.05% Tween-20, pH 5.0) 2 times, magnetically separating for 1min each time using a magnetic rack, and discarding the supernatant;

1.2 to the washed carboxyl magnetic beads were added 100. Mu.L of EDC (i.e., 1-ethyl- (3-dimethylaminopropyl)) solution (prepared using MEST solution) at a concentration of 10mg/mL and 100. Mu.L of NHS (i.e., N-hydroxysuccinimide) solution (prepared using MEST solution) at a concentration of 10mg/mL, and activated for 1h at 800rpm in a 25℃metal bath;

1.3 magnetic separation after 1min the supernatant was discarded, 55. Mu.L of 5'NH ₂ C6-ssDNA-3' Biotin (commercially available) solution at a concentration of 100. Mu.M was added to the resulting precipitate and reacted in a 25℃metal bath at 800rpm for 1h;

1.4 magnetic separation for 1min, discarding the supernatant, adding 200 μl of blocking solution (specifically 1% BSA in PBS), and incubating at 25deg.C for 1 hr to obtain a mixture;

1.5 magnetic separation of the mixture for 1min, discarding the supernatant, adding 40. Mu.L of HRP-conjugated streptavidin (i.e., streptavidin-modified horseradish peroxidase) solution with a concentration of 2. Mu.g/mL to the obtained precipitate, and reacting for 30min twice in a metal bath at 25 ℃ and at 800 rpm;

1.6 magnetic separation after 1min, discarding supernatant, washing the pellet with 200. Mu.L of PBST (PBS, 0.05% Tween-20) for 8 times to obtain MB@ssDNA-HRP;

1.7 MB@ssDNA-HRP was resuspended in 1mL stock solution (0.5 mM Tris-HCl, pH 8.0) and stored at 4℃for later use.

Colorimetric characterization of the (two) Probe MB@ssDNA-HRP

2.1 Taking 5uL MB-COOH (commercially available, at a concentration of 10 mg/mL) solution, 5uL of supernatant obtained by magnetic separation in step 1.6, namely MB@ssDNA-HRP separation supernatant (MB@ssDNA-HRP supernatant), and 5uL of MB@ssDNA-HRP liquid (MB@ssDNA-HRP) in step 1.7, respectively adding the three liquids into an enzyme-free EP tube;

2.2 tubes were each filled with 35uL PBS buffer (0.01M, pH 7.4) to make up 40uL, and 40uL TMB (i.e., 3', 5' -tetramethylbenzidine) was added to each of the tubes, and reacted for 15min in the absence of light, visually inspected and photographed, as shown in FIG. 3.

As shown in FIG. 3, the MB@ssDNA-HRP liquid showed a clear color after 15min of reaction in the dark. The result shows that the MB@ssDNA-HRP is successfully constructed, and the introduced HRP still has strong catalytic activity.

(III) construction of hybridization probes

TABLE 1 nucleotide sequence listing

Note that all nucleotide sequences were synthesized by biological companies.

Mu.L of Apt solution (nucleotide sequence shown in Table 1 and prepared by using enzyme-free PBS buffer) with 100nM concentration, 2 mu.L of activator ssDNA solution (nucleotide sequence shown in Table 1 and prepared by using enzyme-free PBS buffer) with 100nM concentration and 16 mu.L of hybridization buffer (specifically PBS buffer containing 0.55mM MgC ₂ and pH 7.4) are uniformly mixed, and then incubated in a 95 ℃ water bath for 5min, and then cooled slowly to room temperature at a cooling rate of 0.1 ℃ per second to form hybridization probes, and the prepared hybridization probes (shown in FIG. 4) are stored at 4 ℃ for later use.

(IV) feasibility verification

4.1 Preparing a sPD-L1 (-) system and a sPD-L1 (+) system, wherein;

The sPD-L1 (-) system is prepared by uniformly mixing 10 mu L of hybridization probe solution with the concentration of 10nM and 10 mu L of hybridization buffer solution (the same as above) to obtain the sPD-L1 (-) system;

the sPD-L1 (+) system is prepared by uniformly mixing 10 mu L of hybridization probe solution with the concentration of 10nM and 10 mu L of sPD-L1 solution with the concentration of 1ng/mL to obtain the sPD-L1 (+) system;

4.2, respectively incubating the sPD-L1 (-) system and the sPD-L1 (+) system at 37 ℃ for 20min to obtain an incubation product;

4.3 mixing 40. Mu.L of Cas12a protein solution with concentration of 10. Mu.M, 40. Mu.L of crRNA solution with concentration of 10. Mu.M (nucleotide sequence is shown in table 1 and is prepared by adopting enzyme-free PBS buffer solution) and 920. Mu.L of DEPC water uniformly, and then incubating for 30min at 37 ℃ to obtain an incubation product;

4.4 mixing 40. Mu.L of the liquid obtained in step 1.7, 20. Mu.L of the incubation product obtained in step 4.2 by the sPD-L1 (-) system, 10. Mu.L of the incubation product obtained in step 4.3, 10. Mu.L of NEBuffer r2.1 (10X) and 60. Mu. LDEPC of water uniformly, and then incubating at 37 ℃ for 30min to activate trans-cleavage activity of cas12a, thereby cleaving MB@ssDNA-HRP substrate to obtain an incubation product;

4.5 mixing 40. Mu.L of the liquid obtained in step 1.7, 20. Mu.L of the sPD-L1 (+) system in step 4.2, 10. Mu.L of the incubation product obtained in step 4.3, 10. Mu.L of NEBuffer r2.1 (10X) and 60. Mu. LDEPC of water uniformly, and then incubating at 37 ℃ for 30min to activate trans-cleavage activity of cas12a, thereby cleaving MB@ssDNA-HRP substrate to obtain an incubation product;

4.6 after magnetically separating the incubation products obtained in step 4.4 and step 4.5 by using a magnetic rack for 1min, transferring 80 μl of the supernatant to a 96-well black microplate, setting chemiluminescent detection on a SpectraMax iD3 multifunctional microplate reader, detecting signal values once every 40s at a wavelength of 430nm, monitoring for 4min in total, adding luminol solution (final concentration of 5 mM) and hydrogen peroxide solution (final concentration of 75 mM) to the supernatant after setting the procedure and position, and immediately detecting chemiluminescent signal values on the SpectraMax iD3 multifunctional microplate reader, wherein the result is shown in fig. 5.

As shown in fig. 5, a significantly amplified chemiluminescent signal appears in the presence of the target, and the chemiluminescent signal is almost negligible in the absence of the target. The results show that the chemiluminescent sensor prepared by the probe set provided by the application has feasibility for detecting the soluble anti-apoptosis ligand 1 in peripheral blood.

(Fifth) sensitivity test

Six experimental groups were set, the concentrations of the sPD-L1 solutions of each group were respectively 0, 2pg/mL, 5pg/mL, 10pg/mL, 100pg/mL and 1000pg/mL, and the treatment was performed in the same treatment manner as the sPD-L1 (+) system In (IV), and the chemiluminescent signal values were detected, the results were shown in FIG. 6, and the results were shown in FIG. 7, which were obtained by performing linear fitting using Prism software and calculating the detection limits according to the blank average +3 times the blank standard deviation.

As shown in FIG. 6, in the range of 0-1000pg/mL, the chemiluminescent intensity increases gradually as the target concentration increases.

As shown in fig. 7, the chemiluminescent signal is strongly linear with respect to the logarithmic value of the concentration of sps-L1, ranging from 2pg/mL to 1000pg/mL, and the linear equation fitted is y=5716 IgC-116.2 (r2=0.989), where Y is the chemiluminescent intensity signal and C is the concentration of sps-L1. Blank samples were tested 3 times in duplicate, and the limit of detection was calculated to be 1.46pg/mL. The result shows that the chemiluminescence sensor prepared from the probe set provided by the application detects the soluble anti-apoptosis ligand 1 in peripheral blood, and has high sensitivity.

(Six) specificity test

Four experimental groups were set up, each of which was used to detect common proteins and target proteins in serum, and was divided into four groups, group A was a blank group, the target proteins were replaced with equal volumes of PBS buffer, group B was tumor necrosis factor- α (TNF-. Alpha., 1 ng/mL), group C was myeloperoxidase (myeloperoxidase, 1 ng/mL), group D was sPD-L1 (1 ng/mL), and the results were shown in FIG. 8, which were processed in the same manner as the sPD-L1 (+) system in group (IV), and chemiluminescent signal values were detected.

As shown in FIG. 8, other proteins produced chemiluminescent signals comparable to the blank results, which were negligible. The result shows that the chemiluminescence sensor prepared by the probe set provided by the application detects the soluble anti-apoptosis ligand 1 in peripheral blood, is not interfered by other common proteins, has better anti-interference capability, and can specifically detect sPD-L1.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A probe group for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that the probe group includes a recognition probe and a substrate probe, the recognition probe is formed by hybridization of an aptamer and an activator ssDNA, the substrate probe includes modified carboxyl magnetic beads and horseradish peroxidase modified with streptavidin, and the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino groups and biotin.

2. The probe set according to claim 1, characterized in that the amino group is modified at the 5' end of the ssDNA, and the amino group of the ssDNA reacts and binds to the carboxyl magnetic beads;

And/or, the biotin is modified at the 3' end of the ssDNA.

3. The probe set according to claim 1, wherein the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the ssDNA is shown as SEQ ID NO.2;

And/or, the nucleotide sequence of the ssDNA is shown as SEQ ID NO.4.

4. The probe set according to claim 1, wherein the recognition probe is prepared by a method comprising the following steps:

The aptamer is mixed with the activator ssDNA and a buffer, and then incubated together, and cooled to obtain the recognition probe;

And/or, the substrate probe is prepared by a method comprising the following steps:

A carbodiimide hydrochloride solution and an N-hydroxysuccinimide solution are added to a carboxyl magnetic bead solution for activation and magnetic separation, and then a biotinylation reagent is added to the obtained precipitate, wherein the biotinylation reagent includes an amino group and biotin, and then a reaction is performed, and magnetic separation is performed twice. A blocking solution is added to the obtained precipitate, and the mixture is incubated and magnetically separated three times. Streptavidin coupled with horseradish peroxidase is added to the obtained precipitate, and the reaction is continued and magnetic separation is performed four times to obtain the substrate probe.

5. The probe set according to claim 4, characterized in that the molar ratio of the aptamer to the activator ssDNA is 1-2:1-2;

And/or, the buffer solution is a PBS buffer solution containing magnesium chloride and having a pH of 7.2-7.5;

And/or, the co-incubation temperature is 90-100° C., and the co-incubation time is 4-6 min;

and/or, cooling to room temperature at a cooling rate of 0.05-0.2°C/min.

6. The probe set according to claim 4, characterized in that the carbodiimide hydrochloride is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

and/or, the mass ratio of the carbodiimide hydrochloride to the N-hydroxysuccinimide is 8-12:8-12;

And/or, the activation temperature is 20-30° C., and the activation time is 0.5-1.5 h;

and/or, the mass ratio of the carbodiimide hydrochloride to the biotinylation reagent is 8-12:0.05-0.06;

and/or, the mass ratio of the carbodiimide hydrochloride to the streptavidin coupled with horseradish peroxidase is 8-12::0.0005-0.001;

And/or, the reaction temperature is 20-30°C, and the reaction time is 0.5-1.5h;

And/or, the incubation temperature is 20-30° C., and the incubation time is 0.5-1.5 h;

And/or, the temperature of the secondary reaction is 20-30° C., and the duration of the secondary reaction is 20-40 min.

7. A chemiluminescent sensor for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that the chemiluminescent sensor comprises the probe set according to any one of claims 1 to 6.

8. chemiluminescent sensor as claimed in claim 7, characterized in that the chemiluminescent sensor also includes Cas12a enzyme.

9. Use of the probe set according to any one of claims 1 to 6 or the chemiluminescent sensor according to claim 7 or 8 in the preparation of a kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood.

10. A kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that the kit comprises the probe set according to any one of claims 1 to 6 or the chemiluminescent sensor according to claim 7 or 8.