Preparation method of FRET response near-infrared fluorescent gold nanoprobe and application thereof in urine detection

Abstract

The application discloses a preparation method of FRET response near-infrared fluorescent gold nanoprobe and application of the nanoprobe in urine detection. 3+ AuNCs are synthesized, specific antibodies are coupled to the surface of the nanoclusters by a chemical coupling method to form a fluorescent nanoprobe, and MnO2 NS is synthesized by a reduction method as a quencher. max The maximum energy transfer efficiency E of FRET is up to 89.5%. After mixing AuNCs with MnO2 NS, the fluorescence of the nanoprobe is quenched through FRET effect, the target antigen or cells to be detected are added into the system, and the nanoprobe is specifically recognized and combined with the target antigen or cells, so that the nanoprobe is separated from the quencher, the fluorescence is turned on, and the target object can be detected in 10-15 min. The detection range of the method for nuclear matrix protein 22 (NMP-22) in urine is 10 ag mL ‑1 ~1 μg mL ‑1 , and the detection limit in a real sample is 1.68 ag mL ‑1 ; the detection range of the method for T cells in urine is 10-100 cells / 100 μL, and the detection limit is as low as 1 cell / 100 μL.

Description

Technical Field

[0001] This invention relates to kits for detecting biomarkers of bladder cancer in urine, and particularly to kits for detecting nuclear matrix protein NMP-22 and T cells in urine. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, with high global incidence and mortality rates (European Urology, 2025, 12011). Early-stage bladder cancer has a high 5-year survival rate with standard treatment, but once it progresses to muscle-invasive bladder cancer, the survival rate drops significantly. Therefore, early diagnosis is crucial to reducing mortality in bladder cancer patients (Urologic Oncology: Seminars and Original Investigations, 2022, 40:410.e19-410.e27). However, current bladder cancer diagnosis faces many challenges. Cystoscopy, considered the "gold standard," suffers from low patient compliance and is prone to postoperative complications due to its invasiveness. Therefore, non-invasive testing is of great significance for early screening and diagnosis of bladder cancer.

[0003] Non-invasive testing primarily involves bodily fluids such as urine and blood. Urine, being directly related to the bladder, is low-cost, readily available, and easy to administer, making it the preferred choice for non-invasive bladder cancer testing (Nature Reviews Disease Primers, 2023, 9: 58). Urine is rich in bladder cancer-related tumor markers, which can be categorized into three main types: proteins, nucleic acids, and cells. Each type of marker has different diagnostic value (Nature Reviews Urology, 2023, 20: 597-614). Protein markers are currently the most widely studied and clinically mature urinary markers for bladder cancer. Nuclear matrix protein-22, a bladder cancer-specific protein marker of significant clinical interest, shows significantly elevated levels in patient urine (Urologic Oncology, 2015, 33: 66.e25-66.e31). The abundance and phenotypic changes of exfoliated cells in urine, especially T cells, are mainly related to the immune response of the bladder cancer tumor microenvironment, and are gradually becoming important indicators reflecting the local immune microenvironment and anti-tumor immune response of the bladder (Annals of Internal Medicine, 2015, 163: e12). Currently, the detection of various biomarkers in urine still faces significant problems and challenges: First, the detection sensitivity is low, especially for low-grade, early-stage bladder cancer, where biomarker abundance is often extremely low, making trace detection difficult; second, existing detection methods are easily affected by the urine environment, resulting in high background signals and insufficient sensitivity, making accurate quantitative detection difficult; third, the detection methods are relatively cumbersome, with some methods being costly and requiring stringent conditions, and separation and purification techniques are complex. Traditional ultracentrifugation is cumbersome and has low recovery rates, hindering widespread clinical application (Journal of Clinical Pathology, 2002, 55: 641-647).

[0004] Various methods for detecting biomarkers in urine have been developed, including optical, electrochemical, and immunological platforms. Among these, fluorescence immunoassay shows greater promise due to its flexible sensing mechanism and ease of operation. However, existing urine detection methods suffer from insufficient sensitivity and low specificity due to low biomarker abundance, complex urine environment, and high background signal, making it difficult to meet the needs of early diagnosis. To address these issues, there is an urgent need to develop a fluorescent probe that can avoid interference from urine's own fluorescence, reduce background signal, and adapt to complex environments for the detection of bladder cancer in urine.

[0005] Fluorescent nanoprobes are a class of fluorescent labels based on nanomaterials, attracting widespread attention in the field of single-molecule detection due to their superior photophysical properties. Compared with traditional organic dyes and fluorescent proteins, fluorescent nanoprobes exhibit higher photostability, longer fluorescence lifetime, and stronger fluorescence intensity, enabling highly sensitive detection at the single-molecule level (Journal of Materials Chemistry B, 2017, 5: 6701-6727). Compared with traditional quantum dots, organic dyes, and rare-earth nanoparticles, gold nanoclusters have significant advantages in fluorescence detection, especially suitable for fields such as biosensing and bioimaging (Nano Letter, 2019, 19: 4527-34). Gold nanoclusters possess large Stokes shifts, their fluorescence exhibits size dependence and tunable emission wavelength, good photostability and biosafety, and are simple and safe to synthesize, with easily functionalized surfaces.

[0006] Fluorescence resonance energy transfer (FRET) is a nonradiative energy transfer process in which an excited-state fluorescent donor molecule transfers energy to a neighboring ground-state fluorescent acceptor molecule through long-distance dipole-dipole interactions. Its mechanism is illustrated in the figure below (Trends in Analytical Chemistry, 2023, 167: 117271). Unlike other mechanisms, FRET is an energy transfer process achieved through spatial interactions, without involving charge transfer or photon reabsorption. Therefore, it is extremely sensitive to changes in the distance between the donor and acceptor. Furthermore, the resulting low background signal improves the signal-to-noise ratio, making it an ideal tool for enhancing detection sensitivity (ACS Nano, 2019, 13: 505–514).

[0007] In this invention, we synthesized a gold nanocluster conjugated specific antibody for ultrasensitive and rapid detection of nuclear matrix protein (NMP-22) and urinary T cells. First, using bovine serum albumin as a template, Au was reduced under alkaline conditions. 3+Gold nanoclusters were synthesized, and specific antibodies were chemically coupled to their surface to form fluorescent nanoprobes (Au-Ab). Manganese dioxide nanosheets (MnO2 NS) were synthesized via a reduction method as a quencher, quenching the probe's fluorescence through a highly efficient FRET effect. Spectroscopic analysis and theoretical calculations confirmed a significant spectral overlap and efficient FRET effect between AuNCs and MnO2 NS. Time-resolved fluorescence lifetime testing further validated that this system is a dynamic non-radiative energy transfer process, with a maximum energy transfer efficiency (Emax) of 89.5%. After the target antigen specifically recognizes and binds to the antibody, the probe detaches from the quencher, achieving fluorescence "on," and detection of the target analyte can be achieved within 10–15 minutes. The detection range for NMP-22 using this method is 10 ag / mL. -1 ~1 μg mL -1 The detection limit in real samples is 1.68 ag mL. -1 The detection range for urinary T cells is 10~100 cells / 100 μL, with a detection limit as low as 1 cell / 100 μL, enabling rapid and ultrasensitive detection of NMP-22 and urinary T cells. Summary of the Invention

[0008] The purpose of this invention is to provide a kit for detecting NMP-22 and T cells in urine from bladder cancer patients. The NMP-22 kit comprises phosphate buffer, human NMP-22 standard, gold nanoparticles conjugated with NMP-22 antibody (Au-NMP), MnO2 NS, and at least one 96-well plate. The T cell detection kit comprises phosphate buffer, gold nanoparticles conjugated with CD3 antibody (Au-CD3), MnO2 NS, and at least one 96-well plate.

[0009] A method for preparing gold nanoclusters coupled with anti-NMP-22 antibody fluorescent nanoprobes, comprising 50 mg mL -10.5 mL of concentrated BSA solution was stirred until completely dissolved, and the centrifuge tube was preheated in a 37 ℃ constant temperature metal bath. HAuCl4·4H2O was weighed and dissolved in ultrapure water to prepare a 0.5 mL 20 mM aqueous solution. This solution was added to the centrifuge tube containing the BSA solution and mixed at 37 ℃ for 5 min. 0.05 mL of a 1 M NaOH solution was prepared and added to the preheated centrifuge tube. The reaction was carried out at 37 ℃ in the dark for 24 h to synthesize AuNCs. The AuNCs were washed and purified by centrifugation at 13000 rpm for 5 min using a 10 kDa ultrafiltration tube. The final AuNCs were dispersed in PBS buffer (0.01 M, pH=7.4) and stored at 4 ℃ in the dark. At an excitation wavelength of 395 nm, the AuNCs solution exhibited strong fluorescence emission at 795 nm.

[0010] The concentration was prepared as 1 mg / mL. -1 Add 80 μL of AuNCs solution to the reaction flask, then add 10 μL of a 10 mg / mL solution. -1 EDC, 10 μL concentration is 10 mg / mL -1 The NHS solution was stirred in the dark for 20 min. After the reaction was complete, the product was washed in a 10 kDa ultrafiltration tube to remove excess EDC and NHS. 100 μL of a 120 μg mL solution was then added to the reaction flask. -1 The anti-NMP-22 antibody was added and the reaction was stirred for 2 h. Then 100 μL of a 5 mg / mL solution was added. -1 An ovalbumin (OVA) solution was used as a blocking agent, and the reaction was stirred for 1 h. The final product was purified by washing with a 300 kDa ultrafiltration tube to obtain the Au-NMP fluorescent probe.

[0011] The preparation method of manganese dioxide nanosheets involves adding 3.5 mL of anhydrous ethanol, 3 mL of 0.5 M sodium dodecyl sulfate (SDS), 0.25 mL of 0.1 M H₂SO₄, and 25 mL of ultrapure water to separate round-bottom reaction flasks. The flasks are placed in an oil bath and heated to 95 °C. Then, 2 mL of a pre-prepared 0.025 M KMnO₄ solution is added, and the mixture is stirred for 40 min. After the reaction is complete, the mixture is cooled to room temperature. The liquid in the flask is removed and centrifuged at 10,000 rpm for 5 min. The precipitate is washed twice with anhydrous ethanol and once with ultrapure water to remove other impurities, resulting in a brownish-black precipitate. The precipitate is resuspended in 2 mL of ultrapure water and then freeze-dried to obtain a brownish-black powder.

[0012] The kit of the present invention is used to detect biomarkers for bladder cancer, namely NMP-22 and urinary T cells.

[0013] The kit of the present invention is used for early diagnosis of bladder cancer, monitoring of treatment effects, or prognostic assessment.

[0014] Using the kit of the present invention can improve the early diagnosis rate of bladder cancer and predict prognostic outcomes and treatment effects, providing effective guidance for better and more specific treatment.

[0015] The advantages of this kit are: it introduces a novel FRET-responsive near-infrared fluorescent nanoprobe, which offers higher sensitivity, simpler operation, shorter processing time, and easier widespread adoption compared to traditional ELISA methods. The kit only requires urine collection for testing, causing no harm to patients and facilitating real-time monitoring of their condition.

[0016] The obtained FRET-responsive fluorescent nanoprobes have the following characteristics:

[0017] (1) The nanoprobes have a uniform size with a particle size of approximately 26.7 nm;

[0018] (2) The nanoprobes have good near-infrared fluorescence intensity;

[0019] (3) The nanoprobe has a good effect of avoiding interference from urine's own fluorescence;

[0020] (4) MnO2 NS has good fluorescence quenching properties and avoids interference from urine's own fluorescence;

[0021] (5) The test kit has good detection performance, with a detection range of 10 ag / mL for NMP-22 antigen. -1 Up to 1 μg mL -1 The limit of detection is 1.53 ag / mL. -1 The detection range for T cells in urine is 10 cells / 100 μL to 100 cells / 100 μL, with a detection limit of 1 cell / 100 μL. Attached Figure Description

[0022] Figure 1 : Fluorescence emission pattern of AuNCs in this invention.

[0023] Figure 2 The particle size distribution of AuNCs and Au-NMP, and the transmission electron microscope image of AuNCs in this invention.

[0024] Figure 3 Comparison of the quenching effects of four groups of MnO2 NS in this invention.

[0025] Figure 4 The ultraviolet absorption spectrum and transmission electron microscopy image of MnO2 NS in this invention.

[0026] Figure 5 The standard curve for detecting NMP-22 in this invention.

[0027] Figure 6 The standard curve for detecting Jurkat cells in this invention. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments. For example, the concentration and volume of the solution used can be adjusted as needed.

[0029] Example 1: Synthesis and Screening of an AuNCs Fluorescent Material

[0030] Prepare 50 mg mL -1 0.5 mL of BSA solution was stirred until completely dissolved, and the centrifuge tube was preheated in a 37 ℃ constant temperature metal bath. Different weights of HAuCl4·4H2O were weighed and dissolved in ultrapure water to prepare 0.5 mL aqueous solutions of different concentrations (8, 10, 12, 14, 16, 18, 20 mM). These solutions were added to centrifuge tubes containing BSA solution and mixed at 37 ℃ for 5 min. A 1 M NaOH solution was prepared, and 0.05 mL of this solution was added to the preheated centrifuge tube. The reaction was carried out at 37 ℃ in the dark for 24 h to synthesize AuNCs. The AuNCs were washed and purified using a 10 kDa ultrafiltration tube and centrifuged at 13000 rpm for 5 min. The final AuNCs were dispersed in PBS buffer (0.01 M, pH=7.4) and stored in the dark at 4 ℃.

[0031] The fluorescence emission of AuNCs synthesized under different conditions in artificial urine was detected using a fluorescence spectrophotometer. AuNCs prepared with 20 mM HAuCl4·4H2O exhibited the highest fluorescence intensity at 795 nm; therefore, this synthesis condition was selected for further optimization. The fluorescence emission spectrum of this nanocluster is shown below. Figure 1 As shown, the emission peak is in the near-infrared region, at 795 nm, which can minimize interference from the urine's own fluorescence. Figure 2 As shown, the dynamic light scattering particle size of AuNCs is 12.8 nm, and the PDI is 0.204.

[0032] Example 2: Preparation of Au-NMP probe

[0033] The concentration was prepared as 1 mg / mL. -1 80 μL of AuNCs solution was added, followed by 10 μL of a 10 mg / mL solution to each reaction flask. -1 EDC and 10 μL concentration is 10 mg / mL-1 The NHS solution was stirred in the dark for 20 min. After the reaction was complete, the product was washed in a 10 kDa ultrafiltration tube to remove excess EDC and NHS. 100 μL of a 120 μg mL solution was added to the reaction flask. -1 The bladder cancer-specific antibody Anti-NMP22 was added and reacted with stirring for 2 h. Then 100 μL of a 5 mg / mL solution was added. -1 An ovalbumin (OVA) solution was used as a blocking agent, and the reaction was stirred for 1 h. The final product was purified using a 300 kDa ultrafiltration tube to obtain Au-NMP. Figure 2 As shown, the particle size of the Au-NMP probe was measured to be 26.7 nm.

[0034] Example 3 Synthesis and Screening of MnO2 NS Quencher

[0035] 3.5 mL of anhydrous ethanol, 3 mL of 0.5 M SDS, 0.25 mL of 0.1 M H₂SO₄, and 25 mL of ultrapure water were added to separate round-bottom reaction flasks. The flasks were placed in an oil bath and heated to 95 °C. Then, 2 mL of KMnO₄ solutions of different concentrations (0.025, 0.05, 0.1, and 0.15 M) were added, and the mixture was stirred for 40 min. The samples were labeled MnO₂⁻1, MnO₂⁻2, MnO₂⁻3, and MnO₂⁻4, from lowest to highest KMnO₄ concentration. After the reaction, the flasks were removed and cooled to room temperature. The samples were then centrifuged twice with anhydrous ethanol and once with ultrapure water at 10,000 rpm for 10 min to remove impurities, resulting in a brownish-black precipitate. The precipitate was resuspended in 2 mL of ultrapure water and then freeze-dried to obtain a brownish-black powder.

[0036] The quenching effect of different concentrations of MnO2NS under different synthesis conditions on AuNCs was determined. Equal volumes and concentrations of AuNCs were added to black 96-well plates, and equal volumes of four different concentrations of MnO2NS were added to each well. Three replicates were set up for each group, and the fluorescence emission intensity after quenching was measured. Figure 3 As shown, the MnO2-1 group has the best quenching efficiency, so this group was selected as the quencher for subsequent use.

[0037] like Figure 4 As shown, the UV-Vis absorption spectra of the selected MnO2 group were measured and characterized by transmission electron microscopy. The surface of the MnO2 group has a typical wrinkled morphology of nanosheet structure.

[0038] Spectroscopic analysis and theoretical calculations confirmed a significant spectral overlap and efficient FRET effect between AuNCs and MnO2 NS. Time-resolved fluorescence lifetime measurements further verified that this system is a dynamic nonradiative energy transfer process, with a FRET critical distance R0 of 4.90 nm, combined with the constant k. a = 9.78×10 4 M -1 Maximum energy transfer efficiency E max Up to 89.5%.

[0039] Example 4: FRET-responsive Au-NMP probe for detecting bladder cancer marker NMP-22 in urine.

[0040] The fluorescent probe synthesized in Example 2 and the quencher screened in Example 3 were used for quenching.

[0041] 50 μL of Au-NMP probe (0.1 mg / mL) and 50 μL of MnO2 NS (2 mg / mL) quencher were added to each well of a black 96-well plate. Then, 50 μL of NMP-22 antigen at different concentrations was added to each of the quenched groups. After incubation for 10 min, the fluorescence recovery of the probe in each group was detected. A linear relationship between fluorescence intensity at 795 nm and NMP-22 concentration was established to obtain a standard curve. Figure 5 As shown, the detection range is from 10 ag mL -1 Up to 1 μg mL -1 The standard curve relationship is y = 0.570x + 1.135 (R²). 2 = 0.989), indicating good linearity, and the calculated detection limit was 1.68 ag mL. -1 .

[0042] NMP-22 was added to urine samples from healthy individuals to prepare solutions of 0.1, 0.5, and 1 pg / mL using the standard addition method. -1 For samples with varying concentrations, the concentration of NMP-22 antigen in the test sample can be obtained from the fluorescence intensity using a standard curve. The recovery rate of NMP-22 was calculated for each group, and the relative standard deviation was also calculated. The recovery rates for the three groups of samples were 98.7%–103.7%, indicating good detection accuracy; the RSDs were 1.40%–2.11%, all <5%, meeting the "excellent" evaluation level. This demonstrates that the constructed fluorescent nanoprobe has high precision, strong reliability, good repeatability, and excellent detection performance for antigen detection in real urine samples.

[0043] Example 5: FRET-responsive Au-CD3 probe for detecting T cells in urine

[0044] Using Jurkat cells as a T cell model, an antibody specific to cell surface antigen CD3, Anti-CD3, was coupled to the surface of AuNCs to synthesize Au-CD3 probes. The synthesis steps were the same as in Example 2, and the quenchers selected in Example 3 were used for quenching.

[0045] 50 μL of Au-CD3 probe (0.1 mg / mL) and 50 μL of MnO2 NS quencher (2 mg / mL) were added to each well of a black 96-well plate. The initial fluorescence intensity and the fluorescence intensity after quenching were recorded. Then, 50 μL of Jurkat cells at different concentrations were added to each of the quenched groups. After incubation for 15 min, the fluorescence recovery of the probe in each group was detected at room temperature. The fluorescence intensity at the point of maximum emission was recorded, and a linear fitting relationship between fluorescence intensity and Jurkat cell concentration was established to obtain a standard curve. Figure 6 As shown, the detection range is from 10 cells / 100 μL to 100 cells / 100 μL, and the standard curve relationship is y = 105.81x + 3699.20 (R²). 2 = 0.992), indicating good linearity, and the calculated detection limit was 1 cell / 100 μL.

[0046] Jurkat cells were added to urine samples from healthy individuals to prepare samples with concentrations of 35, 55, and 75 cells / 100 μL using a standard addition method. The concentration of Jurkat cells in the sample was obtained from the fluorescence intensity using a standard curve. The recovery rate of Jurkat cells for each group was calculated, and the relative standard deviation was also calculated. The recovery rates for the three groups of samples were 95.48%–102.60%, indicating good detection accuracy; the RSDs were 1.60%–3.13%, all <5%, meeting the "excellent" evaluation level. This indicates that the constructed fluorescent nanoprobe has high precision, strong reliability, good reproducibility, and good detection effect for the detection of T cells in real urine samples.

Claims

1. A method for preparing FRET-responsive near-infrared fluorescent gold nanoprobes, characterized in that, Gold nanoclusters (AuNCs) were synthesized using bovine serum albumin (BSA) as a template. Specific antibodies were then chemically coupled to the surface of the nanoclusters to form fluorescent nanoprobes. Manganese dioxide nanosheets (MnO2 NS) were synthesized via a reduction method as quenchers. After mixing AuNCs with MnO2 NS, the fluorescence of the nanoprobes was quenched through the FRET effect. Once the target antigen or T cells specifically recognize and bind to the nanoprobes, the probes detach from the quenchers, thus enabling fluorescence "on" detection.

2. The preparation method according to claim 1, characterized in that, The preparation method of AuNCs includes the following steps: 1) dissolving BSA in ultrapure water; 2) adding multiple groups of chloroauric acid tetrahydrate (HAuCl4·4H2O) solution to the above solution at 37 °C and reacting for 5 min; 3) adding 1 M NaOH solution to the above solution and reacting in the dark for 24 h to synthesize AuNCs, purifying them by ultrafiltration using a 10 kDa ultrafiltration tube, dispersing them in PBS solution, and storing them in the dark at 4 °C. The molar ratio of HAuCl4 : BSA in the reaction of multiple groups of AuNCs was 11:1-29:

1.

3. The preparation method according to claim 1, characterized in that, The preparation method of the fluorescent nanoprobe includes the following steps: 1) Using AuNCs as the final material, prepare 80 mL and 1 mg mL solutions with PBS. -1 1) Add 10 mL of the above solution and 10 mg of the solution to the above solution. -1 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mL, 10 mg / mL -1 N-hydroxysuccinimide (NHS) was stirred in the dark for 20 min; 3) 120 μg mL of N-hydroxysuccinimide was added to the reaction flask. -1 Specific antibody, stir and react for 2 h; 4) Add 5 mg mL -1 5) Use OVA solution as a blocking agent and react for 1 h; 6) Purify and remove impurities using a 300 kDa ultrafiltration tube, and redisperse the fluorescent probe in PBS solution and store it in the dark at 4 °C.

4. The preparation method according to claim 1, characterized in that, The preparation method of the manganese dioxide nanosheets (MnO2 NS) includes the following steps: 1) Anhydrous ethanol, 0.5 M sodium dodecyl sulfate (SDS), 0.1 M H2SO4 and ultrapure water are added to a round-bottom reaction flask and heated to 95 °C; 2) 0.025-0.15 M KMnO4 solution of different concentrations are added and the mixture is stirred for 40 min; 3) After the reaction is completed, the reaction flask is removed and cooled to room temperature. Under the conditions of 10000 rpm and 10 min, the mixture is centrifuged and washed twice with anhydrous ethanol and once with ultrapure water to remove impurities and obtain a brown-black precipitate.

5. A FRET-responsive near-infrared fluorescent gold nanoprobe was prepared by the preparation method described in claim 1.

6. The detection method of the FRET-responsive near-infrared fluorescent probe detection kit for nuclear matrix protein (NMP-22) in urinary urine of bladder cancer as described in claim 5; characterized in that, The NMP-22 kit contains 0.01 M phosphate buffer, 10 mg / mL human NMP-22 standard, 1 mg / mL gold nanoprobe conjugated with NMP-22 antibody (Au-NMP), 20 mg MnO2, and a black 96-well plate. 50 μL of Au-NMP probe at a concentration of 0.05–0.2 mg / mL and 50 μL of MnO2 quencher at a concentration of 1–3 mg / mL are added to each well of the black 96-well plate. Then, 50 μL of urine NMP-22 antigen at different concentrations is added to each of the quenched wells. After incubation for 10 min, the fluorescence recovery of the probe in each well is detected. The fluorescence intensity at 795 nm is used to establish a linear relationship between fluorescence intensity and NMP-22 concentration, obtaining a standard curve. The test samples are treated with the same concentration of Au-NMP probe and MnO2, and the fluorescence intensity is detected. The concentration is quantified using the standard curve.

7. The detection method of the kit for detecting T cells in urine of bladder cancer using a FRET-responsive near-infrared fluorescent probe as described in claim 5; characterized in that, The T-cell kit contains 0.01 M phosphate buffer, 1 mg / mL gold nanoparticle probe conjugated with CD3 antibody (Au-CD3), 20 mg MnO2, and a black 96-well plate. 50 μL of Au-CD3 probe at a concentration of 0.05–0.2 mg / mL and 50 μL of MnO2 quencher at a concentration of 1–3 mg / mL are added to each well of the black 96-well plate. Then, 50 μL of urine T cells at different concentrations are added to each of the quenched groups. After incubation at 37 ℃ for 15 min, the fluorescence recovery of the probe in each group is detected. The fluorescence intensity at 795 nm is used to establish a linear relationship between fluorescence intensity and T-cell concentration, obtaining a standard curve. The test samples use the same concentration of Au-CD3 probe and MnO2, and the concentration is quantified using the standard curve.

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