Preparation and application of immunosensor and detector for prostate antigen detection

Abstract

This invention belongs to the field of photoelectrochemical biosensors, specifically relating to the preparation and application of an immunosensor and detector for prostate antigen detection. Based on the principle of photoelectrochemistry (PEC), it involves the combination of three materials: nitrogen-sulfur co-doped carbon quantum dots (N,S-CQDs), MoS2, and Bi2S3, to selectively measure prostate-specific antigen (PSA), a biomarker for prostate cancer, in clinical serum and urine samples. To facilitate analyte detection, an integrated portable detection instrument is designed, with its shell designed using 3D printing technology. This instrument offers advantages such as simple operation and portability, significantly aiding in the rapid detection of PSA and other biomarkers required for clinical applications. This method is crucial for the early and effective diagnosis and treatment of prostate cancer, overcoming the difficulties of existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of photoelectrochemical biosensors, specifically relating to a method for preparing a MoS2 / N,S-CQDs / Bi2S3 nanocomposite photoelectrochemical sensor and the application of an integrated photoelectrochemical portable sensor based on this sensor in the detection of prostate antigens. Background Technology

[0002] In recent years, cancer has become one of the leading causes of death worldwide. Although the mortality rate of cancer is high, early diagnosis and treatment intervention can significantly improve the cure rate for cancer patients. Prostate-specific antigen (PSA) is a biomarker for prostate cancer, a glycoprotein produced by prostate epithelial cells and ducts. Its concentration in the serum of healthy men is usually below 4.0 ng / mL. Abnormally elevated serum PSA levels make PSA the most reliable tumor marker for diagnosing prostate cancer at different stages. Therefore, rapid and sensitive detection of PSA in human serum and urine may provide an effective strategy for the diagnosis and treatment of prostate cancer.

[0003] Photoelectrochemical analysis (PEC), as a technology integrating photochemistry and electrochemistry, has broad application prospects and significant application value. The main characteristics of PEC technology include high sensitivity, fast response speed, low background response, and simple operation, making it widely applicable in fields such as protein detection, DNA detection, and biomarker detection. In photoelectrochemical detection, when a light source shines on a photoactive material, photon energy is absorbed and photogenerated electrons and holes are generated. The separation and transfer of these photogenerated charge carriers (electrons and holes) are key steps in the generation of photocurrent. Photogenerated electrons and holes can migrate to the surface or interface of the material, and then react with the analyte in solution through redox reactions, causing the photogenerated charge carriers to recombine and generate a current as a detection signal. To date, researchers have developed various PSA measurement techniques, including mass spectrometry immunoassay, electrochemical methods, and enzyme-linked immunosorbent assay (ELISA). Although these methods are mature, they still suffer from drawbacks such as complex sample pretreatment, expensive instruments, and low sensitivity. Compared to these methods, photoelectrochemical (PEC) immunoassay, based on the specific reaction between antigen and matched antibody, exhibits superior characteristics such as simple equipment, low background interference, high sensitivity, strong selectivity, and good stability.

[0004] This invention utilizes MoS2 / N,S-CQDs / Bi2S3 as the base material and employs a layer-by-layer assembly method to construct a PEC immunosensor for detecting PSA in serum and urine. To facilitate the portability and rapid detection of biomarkers using an optochemical workstation, this invention also proposes and designs an integrated portable optochemical detector. This research provides a novel approach for constructing PEC immunosensor platforms, demonstrating significant potential for clinical and other disease diagnostic applications. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sensitive, rapid, portable and low-cost photoelectrochemical immunosensor and its integrated portable detector to achieve ultrasensitive detection of the cancer biomarker prostate-specific antigen.

[0006] This invention provides a method for preparing a photoelectrochemical immunosensor using highly photoactive MoS2 / N,S-CQDs / Bi2S3 nanocomposite materials, comprising the following steps:

[0007] Step 1: Preparation of N,S-CQDs:

[0008] L-cysteine ​​and thiourea were added to ultrapure water and mixed well. The mixture was then transferred to a polytetrafluoroethylene-lined autoclave and heated at 200°C for 10 hours to obtain an N,S-CQDs solution. The solution was then freeze-dried to form pure N,S-CQDs solid.

[0009] Step 2: Preparation of MoS2 / N,S-CQDs / Bi2S3 nanocomposites:

[0010] Sodium molybdate dihydrate was dissolved in ultrapure water, stirred, and then an aqueous solution of N,S-CQDs was added. Stirring continued, and finally bismuth nitrate pentahydrate was added and stirred to allow it to react fully to obtain a homogeneous mixed solution of sodium molybdate dihydrate and N,S-CQDs.

[0011] The obtained homogeneous mixed solution was transferred to a polytetrafluoroethylene-lined autoclave and heated to 200°C. After cooling to room temperature, it was centrifuged to obtain a black precipitate, which was then washed and dried to obtain a photoactive black composite material, labeled as MoS2 / N,S-CQDs / Bi2S3.

[0012] Step 3: Fabrication of the photoelectrochemical (PEC) sensor:

[0013] The FTO glass electrode was washed sequentially with acetone, ethanol and ultrapure water. The washed FTO glass electrode was coated with a MoS2 / N,S-CQDs / Bi2S3 suspension and dried to obtain an electrode labeled FTO / MoS2 / N,S-CQDs / Bi2S3.

[0014] Thioglycolic acid (TGA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), prostate antibody (Anti-PSA) and bovine serum albumin (BSA) were sequentially added to the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode to block non-specific sites, resulting in an electrode labeled FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA;

[0015] The FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode was co-incubated with PSA solution for 1–3 hours. The incubated electrode was then rinsed with ultrapure water before photocurrent measurement experiments were performed.

[0016] In step 1, the volume of ultrapure water is 10-50 mL;

[0017] In step 1, the molar ratio of L-cysteine ​​to thiourea is 4:6.

[0018] In step 1, the freeze-drying temperature is -50 to -80°C;

[0019] In step 2, the stirring temperature is 40℃ and the stirring time is 2-4 hours.

[0020] In step 2, the centrifugation speed is 4200 rpm and the time is 10-20 min;

[0021] In step 2, the drying temperature is 60℃ and the time is 10-15 hours.

[0022] In step 2, the N,S-CQDs concentration is 10–15 mg / mL;

[0023] In step 3, the FTO glass electrode is cut into a circular thin slice and ultrasonically rinsed with acetone, ethanol and ultrapure water for 30-60 minutes in sequence.

[0024] In step 3, the mass concentration of the MoS2 / N,S-CQDs / Bi2S3 suspension is 10-15 mg / mL, and the application amount is 10-20 μL.

[0025] In step 3, the drying temperature is 80–120°C and the drying time is 12–20 hours.

[0026] In step 3, the molar concentrations of TGA, EDC, and NHS are 5–10 μmol / L, the amount added is 5–10 μL, and the reaction time is 1–3 h.

[0027] In step 3, the mass concentrations of Anti-PSA and BSA are 0–10 μg / mL, the amount added is 5–10 μL, and the reaction time is 1–3 h.

[0028] The preparation of an integrated portable detector specifically includes the following steps:

[0029] The integrated portable testing instrument includes: a microcontroller, a socket, a resistor array, a sensor, a transistor, a crystal oscillator, a capacitor, a universal board, and an LCD screen.

[0030] Step 1: The display, detector, light source, power supply and electrolytic cell are integrated into one system. All microprocessors are fixed to the circuit board by soldering and placed in the sealed housing of the photoelectrochemical workstation.

[0031] Step 2: The photoelectrochemical immunosensor prepared in this invention is reset by the controller connected to the microcontroller. After receiving the reset signal, the sensor will send back an existence pulse. After the reset process is completed, the controller pulls the bus of the data unit high and receives the existence pulse.

[0032] Step 3: After receiving the pulse, the controller sends an operation command to the sensor, reads the current intensity data, and inputs it into the microcontroller. In the microcontroller, the relationship table between current magnitude and concentration is stored in advance. The controller searches for the PSA concentration value corresponding to the input current intensity value in the relationship table, inputs it into the sensor chip, and displays it.

[0033] In step 1, the sealed shell of the photoelectrochemical workstation is made of environmentally friendly material (resin material) using 3D printing technology.

[0034] In step 2, when the sensor receives the reset signal, it will send back a presence pulse after 15 to 60 μs. The presence pulse is a low-level signal of 60 to 240 μs.

[0035] In step 3, the operation instructions are 8 bits long and consist of 6 instructions.

[0036] Application of integrated portable testing devices in prostate antigen detection:

[0037] A three-electrode system was employed: the prepared PEC immunosensor was used as the working electrode, the platinum wire electrode as the control electrode, the Ag / AgCl electrode as the reference electrode, and the FTO sensor coated with MoS2 / N,S-CQDs / Bi2S3 composite material was used as the working electrode. Ascorbic acid (AA) was added to the PBS buffer solution as an electron donor.

[0038] Step 1: Centrifuge the blood and urine samples, filter and dilute the supernatant for later use;

[0039] Step 2: The diluted sample is combined with the MoS2 / N,S-CQDs / Bi2S3 composite material and attached to the working electrode of the sensor. The sample is placed in a PBS buffer solution containing ascorbic acid and irradiated with a xenon lamp to generate photocurrent.

[0040] Step 3: Obtain the PSA concentration using the conversion table between photocurrent and PSA concentration, and then display the final PSA concentration on the detector's screen.

[0041] In step 1, the blood and urine samples are centrifuged at 8000-12000 rpm for 10-20 min.

[0042] In step 1, a nylon filter (0.45 μm pore size) is used for filtration;

[0043] In step 2, a 500W xenon lamp is used as the excitation source, and the applied potential is 0V throughout the experiment.

[0044] The beneficial effects of this invention are:

[0045] 1. A PEC immunosensor based on nanocomposite materials was successfully constructed for the first time to detect prostate antigen in urine and serum samples.

[0046] 2. The photoelectrochemical immunosensor can be prepared using a one-step solvent method to construct a highly photoactive MoS2 / N,S-CQDs / Bi2S3 nanocomposite material, which is simple to operate. Compared with traditional photoelectrochemical detectors, the integrated portable detector of this invention adopts an integrated detection device and equipment, concentrating the display, detector, light source, power supply, electrolytic cell, and other components into one system, facilitating the detection of analytes.

[0047] 3. In the bio-preparation process of the photoelectrochemical immunosensor, the specific binding of prostate antibody (Anti-PSA) to different concentrations of PSA gives the sensor good specificity. Ultimately, within the range of 0.001 ng / mL to 50 ng / mL, the photocurrent intensity and PSA exhibit a good logarithmic linear relationship, with the linear equation being I = -4.822logC. PSA +19.03(R 2 =0.9916), and according to the calculation method in the literature, the detection limit is 0.35 pg / mL. Compared with previously reported PSA detection methods, the PEC immunosensor designed in this invention has a more satisfactory linear range and a lower detection limit, higher sensitivity than traditional sensors, and good specificity, reproducibility, and stability.

[0048] 4. This invention utilizes resin materials as raw materials and combines them with 3D printing technology to construct an integrated portable testing instrument. This instrument offers advantages such as small size, portability, good stability, non-toxicity, and environmental friendliness. In the detection of prostate-specific antigen (PSA), only a PBS solution needs to be prepared separately. The working electrode of the PEC portable testing instrument is immersed in the PBS solution, and the surface of the working electrode is illuminated by the built-in light source to obtain the PSA concentration on the portable testing instrument. Furthermore, this portable testing instrument can not only be used for the detection of PSA as described in this invention, but also for the detection of other biomarkers. Attached Figure Description

[0049] Figure 1 TEM images of N,S-CQD. The inset shows images of N,S-CQD under natural light (yellow) and a 365nm ultraviolet lamp (blue).

[0050] Figure 2 SEM images of MoS2 / N,S-CQDs / Bi2S3.

[0051] Figure 3 EDS element mapping diagrams for C, N, O, S, Mo, and Bi.

[0052] Figure 4 XPS spectra of MoS2 / N,S-CQDs / Bi2S3

[0053] Figure 5 XRD patterns of MoS2, Bi2S3, and MoS2 / N,S-CQDs / Bi2S3.

[0054] Figure 6 The effect of the amount of N,S-CQDs on photocurrent.

[0055] Figure 7 The effect of the amount of MoS2 / N,S-CQDs / Bi2S3 on photocurrent.

[0056] Figure 8 The effect of AA concentration on photocurrent.

[0057] Figure 9 The effect of pH on photocurrent.

[0058] Figure 10 The photocurrent response for different concentrations of PSA.

[0059] Figure 11 Logarithmic calibration curves for PEC sensors detecting different concentrations of PSA.

[0060] Figure 12To assess the specificity and anti-interference capability of the PEC sensor in detecting PSA. (a) Blank, (b) Blank + 10 ng / mL CEA, (c) Blank + 10 ng / mL AFP, (d) Blank + 10 ng / mL HSA, (e) Blank + 10 ng / mL BSA, (f) Blank + 10 ng / mL PSA. (g) 10 ng / mL PSA + 100 ng / mL CEA, (h) 10 ng / mL PSA + 100 ng / mL AFP, (i) 10 ng / mL PSA + 100 ng / mL HSA, (j) 10 ng / mL PSA + 100 ng / mL BSA.

[0061] Figure 13 The photocurrent response of the PEC sensor under 10 on / off illumination cycles is given by the photocurrent response of the PEC sensor under 400-second on / off illumination cycles (CPSA = 10 ng / mL). Detailed Implementation

[0062] The principles and features of the present invention are described below with reference to the accompanying drawings. The descriptions below are merely preferred embodiments of the present invention and are used only to explain the invention, not to limit its scope. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0063] Unless otherwise specified, all experimental materials used in this invention can be prepared by conventional methods in the field or obtained through commercial purchases.

[0064] The instruments and reagents required in the following examples are as follows:

[0065] Instruments: The photoelectrochemical detection process was performed on a photoelectrochemical workstation (CHI-660E, China). The light source was a 500W xenon lamp (CEL-HXF300, Beijing, China). An electronic analytical balance (Satorius, Germany) was used. A KQ5200 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.) was used. A scanning electron microscope (FEI Helios Nanolab 600iSEM, USA) was used. Transmission electron microscopes and high-resolution transmission electron microscopes (JEOL, Japan) were used. An energy-dispersive X-ray spectrometer (EDS) was used. An X-ray photoelectron spectroscopy (XPS) was used. X-ray diffraction (XRD) was performed using a D8 Advance diffractometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALAB 250Xi X-ray photoelectron spectroscopy.

[0066] Reagents: Prostate antibody (Anti-PSA) and prostate antigen (PSA) were purchased from Shanghai Lingchao Biotechnology Co., Ltd. L-cysteine, thiourea, sodium molybdate dihydrate, bismuth nitrate pentahydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and ascorbic acid (AA) were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Thioglycolic acid (TGA) was purchased from Hebei Bailingwei Ultrafine Materials Co., Ltd. Bovine serum albumin (BSA) was purchased from Hefei BOSF Biotechnology Co., Ltd. Alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) standards were purchased from Shanghai Zeye Biotechnology Co., Ltd. Human serum albumin (HSA) was purchased from Tianjin Bailuns Biochemical Co., Ltd.

[0067] This invention provides a method for preparing a photoelectrochemical immunosensor based on MoS2 / N,S-CQDs / Bi2S3 nanocomposite materials, the specific steps of which are as follows:

[0068] Step 1: Preparation of N,S-CQDs:

[0069] N,S-CQDs were prepared using a one-step hydrothermal method. L-cysteine ​​and thiourea were added to 20 mL of ultrapure water in a molar ratio of 4:6 to prepare a homogeneous aqueous solution. This solution was then transferred to a polytetrafluoroethylene-lined autoclave and heated at 200 °C for 10 h. Finally, the obtained N,S-CQDs solution was freeze-dried at -70 °C to form pure N,S-CQDs solids.

[0070] The prepared N,S-CQDs were characterized by transmission electron microscopy (TEM), such as... Figure 1 As shown in the figure, the prepared N,S-CQDs have a near-spherical morphology and uniform size distribution. The inset shows that the aqueous solution of N,S-CQDs is yellow under natural light and blue under 365nm ultraviolet light, indicating that N,S-CQDs were successfully prepared and have a certain absorption capacity for visible light.

[0071] Step 2: Preparation on MoS2 / N, S-CQDs / Bi2S3 nanomaterials

[0072] Sodium molybdate dihydrate (0.7259 g) was dissolved in 25 mL of ultrapure water, transferred to a 100 mL Erlenmeyer flask, and stirred at a constant temperature of 40 °C for 3 h using a magnetic stirrer. Then, 15 mL of N,S-CQDs (10 mg / mL) was added and stirring was continued for 3 h. Finally, bismuth nitrate pentahydrate (0.517 g) was added and stirring was continued for 3 h to allow the reaction to proceed fully and the mixture to become homogeneous.

[0073] Subsequently, the obtained homogeneous solution was transferred to a polytetrafluoroethylene-lined autoclave and gradually heated to 200°C (20 h). After cooling to room temperature, the black suspension was centrifuged (4200 rpm, 10 min) to obtain a black precipitate, washed three times with water and ethanol, and then dried in an oven at 60°C for 12 h to obtain a black powder. The obtained photoactive black composite material was labeled as MoS2 / N,S-CQDs / Bi2S3;

[0074] Scanning electron microscopy (SEM) analysis of MoS2 / N, S-CQDs / Bi2S3 nanocomposites, such as... Figure 2 As shown in the figure, it can be observed that not only Bi2S3 nanorods exist on and around the surface of the MoS2 spherical nanomaterials, but also N,S-CQDs. The reason for this phenomenon may be that the added N,S-CQDs are in excess, and when combined with Mo... 4+ and Bi 3+ Even after the reaction is complete, a small amount of N,S-CQDs remain attached to the surface and surrounding area of ​​MoS2 and Bi2S3.

[0075] To investigate the success of the prepared MoS2 / N,S-CQDs / Bi2S3 composite material, its energy dispersive diffraction (EDS) pattern was studied. Figure 3 As shown in the figure, the MoS2 / N,S-CQDs / Bi2S3 composite material contains six elements: C, N, O, S, Mo, and Bi. The above characterization indicates the successful formation of the MoS2 / N,S-CQDs / Bi2S3 photoactive composite material.

[0076] To better describe the elemental composition and valence state of the MoS2 / N,S-CQDs / Bi2S3 nanocomposite materials, X-ray photoelectron spectroscopy (XPS) was used for in-depth analysis. Figure 4 Bi was observed in 4f and S 2p The XPS results show that the two fitted peaks at 158.6 and 163.9 eV are respectively related to Bi 3+ 4f 7 / 2 and 4f 5 / 2 The weak peak at 161.4 eV between the two main peaks is attributed to S. 2p Based on this result, the successful preparation of MoS2 / N,S-CQDs / Bi2S3 is further confirmed.

[0077] Figure 5 The X-ray diffraction (XRD) patterns of MoS2, Bi2S3, and MoS2 / N,S-CQDs / Bi2S3 are shown. It can be clearly seen that N,S-CQDs differ from Bi... 3+The Bi₂S₃ formed by the reaction corresponds to the (020), (120), (130), (211), (221), (431), and (351) planes at 15.8°, 17.6°, 24.9°, 28.6°, 31.8°, 46.5°, and 52.6°, respectively (JCPDS No. 17-0320). This result indicates that N,S-CQDs can provide a sulfur source and react with Bi. 3+ Bi₂S₃ was successfully reacted and prepared. N,S-CQDs were reacted with Mo... 4+ The resulting MoS2 exhibits three distinctive diffraction peaks in the pattern, located at 10.3°, 32.7°, and 58.3°, which coincide with the (002), (100), and (110) planes (JCPDS No. 37-1492). Similar to the preparation method of Bi2S3, this result also demonstrates that N,S-CQDs can react with Mo... 4+ MoS2 was successfully reacted and prepared. It was observed that the peak value on the (002) crystal plane shifted compared to the peak value on the normal MoS2 (002) crystal plane, which is likely caused by oxygen doping. Mo... 4+ ,N,S-CQDs and Bi 3+ After combination, the characteristic diffraction peaks of Bi2S3 and some characteristic diffraction peaks of MoS2 were clearly observed in the resulting MoS2 / N,S-CQDs / Bi2S3 composite material. This phenomenon may be due to the poor crystallinity of MoS2, causing its characteristic diffraction peaks to be covered by the characteristic peaks of Bi2S3. Furthermore, the crystal structure of the prepared sample did not change significantly, indicating that the MoS2 / N,S-CQDs / Bi2S3 photoactive material was successfully constructed and possesses good dispersibility.

[0078] Step 3: Fabrication of the PEC sensor

[0079] Following the layer-by-layer assembly method, first assemble the material with a geometric area of ​​0.64 cm². 2FTO conductive glass (2.5cm × 2.5cm) was continuously sonicated in acetone, ethanol, and ultrapure water for 30 min, and then transferred to an oven at 60°C for drying. A 20 μL suspension of MoS2 / N,S-CQDs / Bi2S3 (10 mg / mL) was coated onto the exposed FTO conductive glass, and the electrode was dried in an oven at 120°C for 12 h. The resulting electrode was labeled FTO / MoS2 / N,S-CQDs / Bi2S3. Subsequently, 5 μL of 10 μmol / L TGA was added to the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode, allowed to stand for 30 min, and then washed with PBS (pH 7.2–7.4) buffer solution. Then, an EDC / NHS (10 μmol / L / 2 μmol / L) solution (5 μL) was added to the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA electrode for 30 min to activate the -COOH group of TGA. Excess TGA was then washed away to obtain the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode. Subsequently, 10 μL of a 10 μg / mL Anti-PSA solution was further added to the surface of the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA. Subsequently, 10 μL of BSA (10 ng / mL) was dropped onto the modified electrode surface and reacted at room temperature for 1 h to block nonspecific sites, and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA. Finally, the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode was incubated at room temperature with 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 10 ng / mL, and 50 ng / mL PSA solutions (5 μL) for 1 h, respectively. The electrode was then washed with ultrapure water and kept at 4°C for subsequent photocurrent measurement experiments.

[0080] Application of an integrated portable detector for prostate antigen detection based on a photoelectrochemical immunosensor made of MoS2 / N,S-CQDs / Bi2S3 nanocomposite materials. Specific operation is as follows:

[0081] A three-electrode system was employed: the prepared PEC immunosensor was used as the working electrode; a platinum wire electrode was used as the counter electrode; an Ag / AgCl electrode was used as the reference electrode; and an FTO sensor coated with MoS2 / N,S-CQDs / Bi2S3 composite material was used as the working electrode. Ascorbic acid (AA) was added to PBS buffer solution as an electron donor.

[0082] Blood and urine samples were centrifuged at 12,000 rpm for 20 min. The supernatant was filtered through a nylon filter (pore size 0.45 μm) and diluted 100 times with pure water. It was then combined with MoS2 / N,S-CQDs / Bi2S3 composite material and attached to the working electrode of the sensor. The electrode was placed in PBS buffer solution containing ascorbic acid and irradiated with a 500W xenon lamp to generate photocurrent (the applied potential was 0V throughout the experiment). The PSA concentration was obtained from the conversion table of photocurrent and PSA concentration, and the final PSA concentration was obtained from the display screen of the detector.

[0083] The Ag / AgCl electrode, used as a reference electrode, ensured the reproducibility of the experiment, while the platinum electrode, used as the counter electrode, ensured the working electrode... Current Unobstructed flow is ensured to guarantee that the studied reaction occurs at the working electrode. Adding ascorbic acid to the PBS buffer solution as an electron donor facilitates the timely consumption of electron and hole vacancies on the electrode during the reaction, promoting its continuation. Adopting these parameters contributes to obtaining better photocurrent performance.

[0084] The N,S-CQDs prepared by the above method can not only serve as charge bridges, but also promote the growth of Mo 4+ and Bi 3+ Co-sensitized charge separation and charge transfer can also be used for Mo 4+ and Bi 3 It provides a sulfur source and has advantages such as good sensing performance and stability, no pollution, harmless to the human body and the environment, simple preparation and easy availability of materials.

[0085] I. Investigation of the Dosage of N,S-CQDs

[0086] Example 1

[0087] Sodium molybdate dihydrate (0.7259 g) was dissolved in 25 mL of ultrapure water and transferred to a 100 mL Erlenmeyer flask. The mixture was stirred at a constant temperature of 40 °C for 3 h using a magnetic stirrer. Then, 5 mL of N,S-CQDs (10 mg / mL) was added and stirring continued for another 3 h. The sodium molybdate dihydrate and N,S-CQDs were mixed, and finally, bismuth nitrate pentahydrate (0.517 g) was added and stirring continued for another 3 h to ensure complete reaction and homogenization. The resulting homogeneous solution was then transferred to a polytetrafluoroethylene-lined autoclave and gradually heated to 200 °C (20 h). After cooling to room temperature, the black suspension was centrifuged (4200 rpm, 10 min) to obtain a black precipitate. The precipitate was washed three times with water and ethanol and then dried in an oven at 60 °C for 12 h to obtain a black powder. The obtained photoactive black composite material was labeled MoS2 / N,S-CQDs / Bi2S3. FTO conductive glass (2.5cm × 2.5cm) was continuously sonicated in acetone, ethanol, and ultrapure water for 30 minutes, and then transferred to an oven at 60°C for drying. First, an exposed geometric area of ​​0.64cm² was... 2 A 20 μL suspension of MoS2 / N,S-CQDs / Bi2S3 (10 mg / mL) was coated onto an FTO conductive glass substrate and dried in an oven at 120 °C for 12 h. The resulting electrode was labeled FTO / MoS2 / N,S-CQDs / Bi2S3, and the photocurrent signal at this time was 32 μA.

[0088] Example 2 differs from Example 1 in that the amount of N,S-CQDs added at a concentration of 10 mg / mL is 10 mL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 128 μA.

[0089] Example 3 differs from Example 1 in that the amount of N,S-CQDs added at a concentration of 10 mg / mL is 15 mL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 180 μA.

[0090] Example 4 differs from Example 1 in that the amount of N,S-CQDs added at a concentration of 10 mg / mL is 20 mL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 130 μA.

[0091] Example 5 differs from Example 1 in that the amount of N,S-CQDs added at a concentration of 10 mg / mL is 25 mL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 64 μA.

[0092] Example 6 differs from Example 1 in that the amount of N,S-CQDs added at a concentration of 10 mg / mL is 30 mL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 4 μA.

[0093] Experimental results:

[0094] The effects of different amounts of N,S-CQDs added in Examples 1-6 on the photocurrent signal of the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode are as follows: Figure 6 As shown, it can be seen that with the increase of N,S-CQDs dosage, the photocurrent intensity first increases and then decreases, and the photoelectric signal is optimal at 15mL.

[0095] II. Investigation of the drop volume of MoS2 / N,S-CQDs / Bi2S3 composite solution

[0096] Example 7

[0097] FTO conductive glass (2.5cm × 2.5cm) was continuously sonicated in acetone, ethanol, and ultrapure water for 30 minutes, and then transferred to an oven at 60°C for drying. First, an exposed geometric area of ​​0.64cm² was... 2 A 5 μL suspension of MoS2 / N,S-CQDs / Bi2S3 with a concentration of 10 mg / mL was coated onto an FTO conductive glass substrate and dried in an oven at 120 °C for 12 h. The resulting electrode was labeled as FTO / MoS2 / N,S-CQDs / Bi2S3, and the photocurrent signal at this time was 150 μA.

[0098] Example 8 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 10 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 170 μA.

[0099] Example 9 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 15 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 190 μA.

[0100] Example 10 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 20 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 200 μA.

[0101] Example 11 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 25 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 150 μA.

[0102] Example 12 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 30 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 125 μA.

[0103] Example 13 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 35 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 100 μA.

[0104] Example 14 differs from Example 7 in that the drop volume of the 10 mg / mL MoS2 / N,S-CQDs / Bi2S3 suspension is 40 μL, and the resulting FTO / MoS2 / N,S-CQDs / Bi2S3 electrode photocurrent signal is 80 μA.

[0105] Experimental results:

[0106] The effect of drop volume of different MoS2 / N,S-CQDs / Bi2S3 composite material solutions on the photocurrent of the materials in Examples 7-14 is as follows: Figure 7 As shown, the photocurrent intensity initially increases and then decreases with increasing drop volume of the MoS2 / N,S-CQDs / Bi2S3 composite solution, reaching its peak at a drop volume of 20 μL. This is mainly because a drop volume greater than 20 μL may lead to a decrease in visible light absorption. Therefore, 20 μL was chosen as the minimum drop volume for the MoS2 / N,S-CQDs / Bi2S3 composite solution.

[0107] III. Examination of Ascorbic Acid (AA) Concentration During Measurement

[0108] Example 15

[0109] Photocurrent measurement conditions: A three-electrode system of a CHI-660E electrochemical workstation was used in the photocurrent study, including a platinum wire electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and an FTO sensor with MoS2 / N and S-CQDs / Bi2S3 composite material attached as the working electrode. The photocurrent was plotted by adding 0.025 mol / L ascorbic acid (AA) to a PBS buffer solution at pH 7.5 as the electron donor. The light source was a 500W xenon lamp, and the applied potential was 0V throughout the experiment.

[0110] Example 16 differs from Example 15 in that 0.05 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0111] Example 17 differs from Example 15 in that 0.1 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0112] Example 18 differs from Example 15 in that 0.2 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0113] Example 19 differs from Example 15 in that 0.3 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0114] Example 20 differs from Example 15 in that 0.4 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0115] Example 21 differs from Example 15 in that 0.5 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0116] Example 22 differs from Example 15 in that 1.0 mol / L ascorbic acid (AA) is added to the PBS buffer solution.

[0117] Example 23 differs from Example 15 in that ascorbic acid (AA) is not added to the PBS buffer solution.

[0118] Experimental results:

[0119] The effect of ascorbic acid (AA) concentration on the measurement of photocurrent in Examples 15-23 is as follows: Figure 8 As shown, the photocurrent intensity initially increases and then decreases with increasing ascorbic acid (AA) concentration, reaching a peak at a concentration of 0.1 mol / L. This can be attributed to electron donor saturation when the ascorbic acid (AA) concentration exceeds 0.1 mol / L. Therefore, 0.1 mol / L is chosen as the optimal concentration of ascorbic acid (AA).

[0120] IV. pH value assessment

[0121] Example 24

[0122] Photocurrent measurement conditions: A three-electrode system of a CHI-660E electrochemical workstation was used in the photocurrent study, including a platinum wire electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and an FTO sensor with MoS2 / N and S-CQDs / Bi2S3 composite material attached as the working electrode. The photocurrent was plotted by adding 0.1 mol / L ascorbic acid (AA) to a PBS buffer solution at pH 4.0 as an electron donor. The light source was a 500W xenon lamp, and the applied potential was 0V throughout the experiment.

[0123] Example 25 differs from Example 24 in that the pH of the PBS buffer is 4.5.

[0124] Example 26 differs from Example 24 in that the pH of the PBS buffer is 5.0.

[0125] Example 27 differs from Example 24 in that the pH of the PBS buffer is 5.5.

[0126] Example 28 differs from Example 24 in that the pH of the PBS buffer is 6.0.

[0127] Example 29 differs from Example 24 in that the pH of the PBS buffer is 6.5.

[0128] Example 30 differs from Example 24 in that the pH of the PBS buffer is 7.0.

[0129] Example 31 differs from Example 24 in that the pH of the PBS buffer is 7.5.

[0130] Example 32 differs from Example 24 in that the pH of the PBS buffer is 8.0.

[0131] Example 33 differs from Example 24 in that the pH of the PBS buffer is 8.5.

[0132] Example 34 differs from Example 24 in that the pH of the PBS buffer is 9.0.

[0133] Experimental results:

[0134] The effect of pH value on the measured photocurrent in Examples 24-34 is as follows: Figure 9As shown in the figure, the photocurrent intensity increases with increasing pH within the range of 4–5.5. However, when the pH value is higher than 5.5, the photocurrent intensity does not change significantly. Considering that the physiological pH value of the human body is between 7.35 and 7.45, which is suitable for fixing protein activity, 7.35–7.45 is selected as the optimal pH value.

[0135] V. Study on PSA Concentration of Modified PEC Electrode

[0136] The following experiments were conducted using the preferred results from Examples 1-34 above.

[0137] Example 35

[0138] Following the layer-by-layer assembly method, first assemble the material with a geometric area of ​​0.64 cm². 2FTO conductive glass (2.5cm × 2.5cm) was continuously sonicated in acetone, ethanol, and ultrapure water for 30 min, and then transferred to an oven at 60°C for drying. A 20 μL suspension of MoS2 / N,S-CQDs / Bi2S3 (10 mg / mL) was coated onto the exposed FTO conductive glass, and the electrode was dried in an oven at 120°C for 12 h. The resulting electrode was labeled FTO / MoS2 / N,S-CQDs / Bi2S3. Subsequently, 5 μL of 10 μmol / L TGA was added to the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode, allowed to stand for 30 min, and then washed with PBS (pH 7.2–7.4) buffer solution. Then, an EDC / NHS (10 μmol / L / 2 μmol / L) solution (5 μL) was added to the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA electrode for 30 min to activate the -COOH group of TGA. Excess TGA was then washed away to obtain the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode. Subsequently, 10 μL of a 10 μg / mL Anti-PSA solution was further added to the surface of the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA. Subsequently, 10 μL of BSA (10 ng / mL) was dropped onto the modified electrode surface and reacted at room temperature for 1 h to block nonspecific sites, and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA. Finally, the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode was incubated at room temperature with 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 10 ng / mL, and 50 ng / mL PSA solutions (5 μL) for 1 h, respectively. The electrode was then washed with ultrapure water and kept at 4°C for subsequent photocurrent measurement experiments.

[0139] Example 36 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 0.005 ng / mL.

[0140] Example 37 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 0.01 ng / mL.

[0141] Example 38 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 0.05 ng / mL.

[0142] Example 39 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 0.1 ng / mL.

[0143] Example 40 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 0.5 ng / mL.

[0144] Example 41 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 5 ng / mL.

[0145] Example 42 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 10 ng / mL.

[0146] Example 43 differs from Example 35 in that the concentration of the PSA solution co-incubated with the electrode is 50 ng / mL.

[0147] Experimental results:

[0148] From the photocurrent curve ( Figure 10 As observed, with the specific binding of prostate antibody (Anti-PSA) to different concentrations of PSA, the photocurrent response on the PEC sensor decreases with increasing PSA concentration. This is mainly because PSA and Anti-PSA form an inert barrier layer of immune complexes on the electrode surface, hindering electron transfer across the PEC electrode. This further demonstrates the successful fabrication of a label-free PEC sensor for PSA detection. Furthermore, within the range of 0.001 ng / mL to 50 ng / mL, the photocurrent intensity exhibits a good logarithmic linear relationship with PSA. Figure 11 As shown, the fitted linear equation is I = -4.822logC PSA +19.03(R 2 =0.9916), and according to the calculation method in the literature, the limit of detection is 0.35 pg / mL. Compared with most previously reported PSA detection methods, the PEC immunosensor designed in this experiment has a more satisfactory linear range and a lower limit of detection.

[0149] VI. Specificity and anti-interference ability of the method using photocurrent intensity measurement

[0150] Specificity and interference are important criteria for evaluating the detection of PEC immunosensors. To demonstrate that the photocurrent response originates from the specific binding of PSA and Anti-PSA, the PEC immunosensor prepared in Example 35 was used to perform selectivity and interference tests on some representative interfering proteins, including carcinoembryonic antigen (CEA), bovine serum albumin (BSA), human serum albumin (HSA), and alpha-fetoprotein (AFP). The results are as follows: Figure 12As shown in the figure, these interfering proteins do not cause significant changes in the photocurrent signal, indicating that PSA detection has satisfactory selectivity and anti-interference capabilities.

[0151] VII. Repeatability and stability of this method by using photocurrent intensity determination

[0152] Repeatability and stability are also important characteristics of PEC immunosensors, and are of great significance for the development and application of biosensors. The PEC immunosensor prepared in Example 35 was used, and the photocurrent response of the PEC immunosensor with 10 ng / mL PSA added was recorded over 400 s in 10 on / off illumination cycles. The reaction results are as follows: Figure 13 As shown in the figure, the photocurrent did not change significantly over time, and the relative standard deviation (RSD) of the PEC measurement was 1.9%, indicating that the prepared PEC immunosensor has good stability.

[0153] 8. Determination of PSA concentration in serum samples using photocurrent intensity

[0154] The feasibility and applicability of the PEC immunosensor prepared by this method in determining PSA levels in human serum and urine were investigated using the method described in Example 35. Unlike Example 35, the analytes incubated on the PEC immunosensor were serum and urine samples containing different concentrations of PSA. Fresh blood and urine samples were centrifuged at 12,000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm nylon filter and diluted 100-fold with pure water. To study the recovery rate, low, medium, and high concentrations of PSA solutions were introduced into the diluted serum and urine samples, and the analytes were quantitatively analyzed using the standard addition method. The experimental results are shown in Table 1, with relative standard deviations ranging from 1.54% to 5.53% and spike recoveries ranging from 97.4% to 103.0%. The results indicate that the designed biosensor is feasible for the determination of PSA in serum. This result also demonstrates the potential of the PEC biosensor in practical sample analysis.

[0155] Table 1. Determination of PSA in human serum and urine samples (n=6)

[0156]

Claims

1. A method for preparing an immunosensor for prostate antigen detection, characterized by, Includes the following steps: Step 1: Add L-cysteine ​​and thiourea in a molar ratio of 4:6 to ultrapure water and mix well. Transfer the mixture to a polytetrafluoroethylene-lined autoclave and heat at 200°C to obtain an N,S-CQDs solution. Freeze-dry the solution to form pure N,S-CQDs solid. Step 2: MoS2 / N,S-CQDs / Bi2S3 nanocomposite material was constructed by a one-step hydrothermal solvent method. Sodium molybdate dihydrate was dissolved in ultrapure water and stirred at 40°C. An aqueous solution of N,S-CQDs was added and stirring continued. Finally, bismuth nitrate pentahydrate was added and stirred to ensure complete reaction, resulting in a homogeneous mixed solution of sodium molybdate dihydrate and N,S-CQDs. The obtained homogeneous mixed solution was transferred to a polytetrafluoroethylene-lined autoclave and heated to 200°C. After cooling to room temperature, centrifugation was performed to obtain a black precipitate, which was washed and dried to obtain the photoactive composite material, labeled as MoS2 / N,S-CQDs / Bi2S3. Step 3: Rinse the FTO glass electrode sequentially with acetone, ethanol, and ultrapure water. Coat the FTO glass electrode evenly with a MoS2 / N,S-CQDs / Bi2S3 suspension. After drying, obtain an electrode labeled FTO / MoS2 / N,S-CQDs / Bi2S3. Then, sequentially add mercaptoacetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and other pre-treatments to the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode. Prostate antibodies and bovine serum albumin were reacted to block non-specific sites, resulting in an electrode labeled FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA. The FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode was co-incubated with PSA solution, and the incubated electrode was washed with ultrapure water before photocurrent measurement experiments were performed.

2. The method for preparing the immunosensor for prostate antigen detection according to claim 1, characterized in that, A 5–10 μmol / L solution of mercaptoacetic acid was added dropwise to the FTO / MoS2 / N,S-CQDs / Bi2S3 electrode and allowed to stand for 30–60 min. The electrode was then washed with a PBS buffer solution with a pH of 7.2–7.

4. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide solution were then sequentially modified onto the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA electrode for 30–60 min at concentrations of 10–20 μmol / L and 1–5 μmol / L, respectively, to activate the -COOH group of mercaptoacetic acid. Excess mercaptoacetic acid was then washed away to obtain the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode.

3. The method for preparing the immunosensor for prostate antigen detection according to claim 2, characterized in that, A prostate antibody solution with a mass concentration of 5–10 μg / mL was used to modify the surface of the FTO / MoS2 / N,S-CQDs / Bi2S3 / (EDC / NHS) electrode described in claim 2, and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA electrode. Subsequently, bovine serum albumin with a mass concentration of 10–20 ng / mL was dropped onto the modified electrode surface and reacted at room temperature for 1–3 h to block nonspecific sites, and labeled as FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode.

4. The method for preparing the immunosensor for prostate antigen detection according to claim 3, characterized in that, The PSA solution was incubated with the FTO / MoS2 / N,S-CQDs / Bi2S3 / TGA / (EDC / NHS) / Anti-PSA / BSA electrode described in claim 3 at room temperature for 1-3 hours. The electrode was then rinsed with ultrapure water and kept in a refrigerator at 4°C for subsequent photocurrent measurement experiments.

5. An integrated portable detector for prostate antigen detection, prepared using the immunosensor described in any one of claims 1-4, characterized in that, The testing instrument includes a microcontroller, a socket, a resistor array, the immunosensor used for prostate antigen detection, a transistor, a crystal oscillator, a capacitor, a universal board, and an LCD display, and includes the following steps: Step 1: The display, detector, light source, power supply and electrolytic cell are integrated into one system. All microprocessors are fixed to the circuit board by soldering and placed in the sealed housing of the photoelectrochemical workstation. Step 2: The sensor is reset by the controller connected to the microcontroller. After receiving the reset signal, the immune sensor for prostate antigen detection will send back a presence pulse. After the reset process is completed, the controller will pull the bus of the data sheet high and receive the presence pulse. Step 3: After receiving the pulse, the controller sends an operation command to the immunosensor used for prostate antigen detection, reads the current intensity data, and inputs it into the microcontroller. In the microcontroller, the relationship table between current magnitude and concentration is stored in advance. The controller searches for the PSA concentration value corresponding to the input current intensity value in the relationship table, inputs it into the sensor chip, and displays it on the LCD screen.

6. The integrated portable detector according to claim 5, characterized in that, The sealed casing of the photoelectrochemical workstation is made of environmentally friendly materials using 3D printing technology.

7. The integrated portable detector according to claim 5, characterized in that, A three-electrode system was used: the prepared photoelectrochemical immunosensor was used as the working electrode, the platinum wire electrode was used as the control electrode, and the Ag / AgCl electrode was used as the reference electrode; Ascorbic acid is added to PBS buffer solution as an electron donor; the specific application method includes the following steps: Step 1: Centrifuge the blood and urine samples, filter and dilute the supernatant for later use; Step 2: The diluted sample is combined with the MoS2 / N,S-CQDs / Bi2S3 composite material and attached to the working electrode of the sensor. The sample is placed in a PBS buffer solution containing ascorbic acid and irradiated with a xenon lamp to generate photocurrent. Step 3: Obtain the PSA concentration using the conversion table between photocurrent and PSA concentration, and then display the final PSA concentration on the detector's screen.

8. The integrated portable detector according to claim 7, characterized in that, Blood and urine samples were centrifuged at 8000–12000 rpm for 10–20 min.

9. The integrated portable detector according to claim 7, characterized in that, The filtration system uses a nylon filter with a pore size of 0.45μm.

10. The integrated portable detector according to claim 7, characterized in that, The excitation source was a 500W xenon lamp, and the applied potential was 0V throughout the experiment.

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