A cathode photoelectrochemical sensor, a preparation method thereof and application thereof in specific detection of tumor exosomes

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a cathode photoelectrochemical sensor, a preparation method thereof and application thereof in specific detection of tumor exosomes, comprising a heterogeneous photoelectric composite material, a signal amplification probe, an exosome capture probe and exosomes. 88B The heterogeneous photoelectric composite material comprises iodine-doped bismuth oxide bromide of a p-type semiconductor and an n-type semiconductor material; the signal amplification probe comprises a gold-silver composite material and an engineered deoxyribozyme; and the exosome capture probe comprises Fe3O4@NH2-MIL 88B and a biotin-labeled anti-CD81 antibody. The sensor of the application can effectively distinguish between malignant tumors and benign lesions, and can maintain stable and repeatable signal output in a complex biological matrix, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a cathode photoelectrochemical sensor, its preparation method, and its application in the specific detection of tumor exosomes. Background Technology

[0002] Liquid biopsy, with its advantages of being non-invasive, allowing for dynamic monitoring and repeated sampling, has become an important direction for precision oncology diagnosis and treatment. Exosomes, as one of the core biomarkers in liquid biopsy, are stable in origin and carry rich biological information, making them a current research hotspot in this field. Compared to liquid biopsy biomarkers such as circulating tumor DNA (ctDNA), the membrane structure of exosomes protects internal biomolecules from nuclease degradation, resulting in higher detection stability. Furthermore, the exosome membrane surface retains characteristic proteins from the source cells, possessing natural tumor specificity and enabling precise identification of tumor cell origin. Therefore, it has great potential to become a diagnostic biomarker for malignant tumors.

[0003] Currently, methods for detecting exosomes mainly include immunoassay, surface plasmon resonance (LSPR), and surface enhanced Raman scattering (SERS). While these methods have achieved qualitative and quantitative detection of exosomes to some extent, they still have limitations in practical clinical applications. For example, immunoassay lacks sufficient sensitivity, making it difficult to detect low concentrations of exosomes in early-stage tumors. LSPR and SERS methods suffer from significant background interference, affecting the accuracy of results. Furthermore, these techniques are cumbersome and time-consuming, failing to meet the needs of rapid clinical detection. Therefore, developing novel analytical strategies that combine high sensitivity, low background noise, low instrument dependence, and easy integration with biorecognition interfaces is particularly important. Summary of the Invention

[0004] To address the problems of high detection cost, low efficiency, and low sensitivity for cancer in existing exosome detection methods, this invention mainly provides a cathodic photoelectrochemical biosensor for specific detection of tumor exosomes, its preparation method, and its application. The technical solution is as follows:

[0005] A method for fabricating a cathode photoelectrochemical sensor includes a heterogeneous photoelectrochemical composite material, a signal amplification probe, an exosome capture probe, and exosomes; the heterogeneous photoelectrochemical composite material comprises a p-type semiconductor doped with bismuth oxyiodide (I-BiOBr) and an n-type semiconductor material; the signal amplification probe comprises a gold-silver composite material and an engineered deoxyribozyme; the exosome capture probe comprises Fe3O4@NH2-MIL 88B And biotin-labeled anti-CD81 antibody.

[0006] Furthermore, the exosome is disposed between the signal amplification probe and the exosome capture probe; the heterogeneous optoelectronic composite material is attached to the electrode.

[0007] A method for preparing the above-mentioned cathode photoelectrochemical sensor includes the following steps:

[0008] Heterogeneous optoelectronic composite material, signal amplification probe, exosome capture probe and reaction solution of exosome were prepared respectively; the reaction solution of heterogeneous optoelectronic composite material was brought into contact with and combined with the electrode, and after drying, the electrode with heterogeneous optoelectronic composite material was obtained.

[0009] The electrode with the heterogeneous photoelectric composite material was brought into contact with and combined with the reaction solution of the exosome capturing probe. After multiple steps of washing, drying and sealing of non-specific active sites, the electrode with the heterogeneous photoelectric composite material and the exosome capturing probe was obtained.

[0010] Incubate with exosome reaction solution, wash, incubate with signal amplification probe reaction solution, and dry; treat with heme chloride.

[0011] Furthermore, the concentration of the heterogeneous photoelectric composite material reaction solution is 1.5~2.5 mg / mL; the concentration of the exosome capturing probe reaction solution is 0.5~1.5 mg / mL; and the concentration of the exosome reaction solution is 10 mg / mL. 4 ~10 6 particles / mL.

[0012] Furthermore, the preparation of the heterogeneous optoelectronic composite material includes the following steps:

[0013] S1, dissolve bromide and iodide salts in water to prepare a mixed solution; prepare an acetic acid solution, add bismuth salt and mix well, add the mixed solution dropwise, and stir the reaction until the precipitate no longer increases; separate and collect the product, wash thoroughly and dry to obtain iodine-doped bismuth oxybromide;

[0014] S2, tetrakis(4-carboxyphenyl)pyrene was dispersed in dimethylformamide, then methanol was quickly added, mixed evenly, and allowed to stand to stabilize the system; the product was separated and collected, washed thoroughly, and freeze-dried to obtain HOF-101;

[0015] S3, add HOF-101 to phosphate buffer solution, then add iodine-doped bismuth oxybromide, mix well and stir vigorously for 6-10 hours; separate and collect the product, wash thoroughly and dry to obtain the final product.

[0016] Furthermore, the concentration of the acetic acid solution is 0.5~0.8 g / mL; the molar ratio of the bismuth salt to the iodine salt is 9~10:1; the molar ratio of the bromide salt to the iodine salt is 18~20:1; the mass ratio of the tetra(4-carboxyphenyl)pyrene to methanol is 1~1.5:1; and the mass ratio of HOF-101 to iodinated bismuth oxybromide is 1:2~2.5.

[0017] Furthermore, the preparation of the signal amplification probe includes the following steps:

[0018] a. Prepare a tetrachloroauric acid solution and a sodium borohydride solution, and cool the sodium borohydride solution; prepare a micelle solution by dissolving hexadecyltrimethylammonium bromide in water;

[0019] b. Take the micelle solution, add tetrachloroauric acid solution, and then quickly add sodium borohydride solution to nucleate and obtain a gold seed nanoparticle suspension;

[0020] c. Prepare hexadecyltrimethylammonium bromide solution A and L-ascorbic acid solution; mix hexadecyltrimethylammonium bromide solution A and L-ascorbic acid solution and add tetrachloroauric acid solution to obtain growth solution;

[0021] d. Prepare hexadecyltrimethylammonium bromide solution B. Mix the gold seed nanoparticle suspension with the growth solution and let it stand for 8-16 hours to obtain a gold precursor suspension. Mix L-ascorbic acid solution, sodium hydroxide solution, gold nanoparticle solution and hexadecyltrimethylammonium bromide solution B evenly, add silver nitrate, and stir at low speed for 8-16 hours.

[0022] Further, the concentration of the micelle solution is 70-80 mmol / L; the concentration of the tetrachloroauric acid solution is 15-25 mmol / L; the concentration of the sodium borohydride solution is 8-12 mmol / L; the sodium borohydride solution is cooled to below 5°C; the concentration of the L-ascorbic acid solution is 35-40 mmol / L; the concentration of hexadecyltrimethylammonium bromide solution A is 40-45 mmol / L; and the concentration of hexadecyltrimethylammonium bromide solution B is 15-20 mmol / L.

[0023] The mass ratio of tetrachloroauric acid to sodium borohydride in step b is 2.5~3.5:1; the mass ratio of tetrachloroauric acid to L-ascorbic acid in step c is 1:10~15; the mass ratio of tetrachloroauric acid to gold seed nanoparticles in the growth solution in step d is 25~35:1; the mass ratio of L-ascorbic acid to sodium hydroxide in step d is 4~5:1; the mass ratio of sodium hydroxide to gold seed nanoparticles in step d is 90~110:1; and the mass ratio of silver nitrate to gold seed nanoparticles in step d is 20~25:1.

[0024] Furthermore, the preparation of the exosome capture probe includes the following steps:

[0025] Take Fe3O4@NH2-MIL 88BAfter thorough activation, the product was reacted with streptomycin solution at room temperature with shaking for 3-5 hours to obtain the modified streptomycin precursor. The modified streptomycin precursor was then incubated with biotin-labeled anti-CD81 antibody for 40-80 minutes, and the product was collected and thoroughly washed. The Fe3O4@NH2-MIL... 88B The mass ratio of the modified streptomycin precursor to the biotin-labeled anti-CD81 antibody is 3-4:1; the mass ratio of the modified streptomycin precursor to the biotin-labeled anti-CD81 antibody is 1:1-1.5.

[0026] Application of the above-mentioned cathode photoelectrochemical sensor in the prevention, diagnosis or treatment of tumors.

[0027] By adopting the above scheme, the method of the present invention has the following advantages:

[0028] 1. This invention constructs a cathode photoelectrochemical sensor based on a sandwich structure, which achieves highly sensitive and specific analysis of tumor exosomes. It has high sensitivity and good linear response to different concentrations of exosomes. The photoelectric signal is stable and repeatable in complex biological samples. In clinical serum sample testing, it can effectively distinguish between malignant tumor groups, benign lesion groups and healthy groups, and has potential application value for precise tumor identification and early diagnosis.

[0029] 2. This invention uses I-BiOBr@HOF-101 heterojunctions as photoelectric response units, achieving significant enhancement of photoelectric signals through I-doping and p–n heterojunction structure formation. To improve the specificity of tumor diagnosis, a Biotin-CD81 aptamer is modified onto Fe3O4@NH2-MIL88B magnetic beads. Utilizing its specific interaction with the highly expressed CD81 protein on exosomes, tumor exosomes are recognized and captured.

[0030] 3. This invention effectively promotes the separation of photogenerated electron-hole pairs through the I-BiOBr@HOF-101 heterojunction, and the I-BiOBr conduction band reduces dissolved oxygen to generate O2. - This further triggers the Au@AgNPs disproportionation reaction and Engineered DNAzyme catalytic cycle, resulting in a significant amplification of the cathode photocurrent signal. The sensor exhibits high sensitivity and specificity, good storage stability over 4 weeks, and no significant signal attenuation after 10 light cycles, demonstrating excellent photoelectric stability.

[0031] 3. This invention developed the Au@Ag@E-DNAzyme complex as a signal amplification probe. Utilizing the specific recognition of aptamers and the dual activities of peroxidase-like and superoxide dismutase-like enzymes, a sandwich recognition structure is formed, thereby constructing an exosome photoelectrochemical analysis after PSA / CD81 dual-target co-recognition, which improves the diagnostic accuracy of malignant tumors. Attached Figure Description

[0032] Figure 1 Transient photocurrent response spectra of HOF-101, I-BiOBr and HOF-101@I-BiOBr composite modified electrodes under intermittent visible light irradiation;

[0033] Figure 2 Photocurrent response curves of different modified electrode sensors in oxygen-unsaturated PBS electrolyte; a) I-BiOBr@HOF-101; b) I-BiOBr@HOF-101 / TDN; c) I-BiOBr@HOF-101 / EngineeredDNAzyme; d) I-BiOBr@HOF-101 / Au@Ag@E-DNAzyme;

[0034] Figure 3 This invention provides a comparison of the specific responses of the sensor to exosomes from different cell sources and mixed samples.

[0035] Figure 4 Signal stability test of the sensor of the present invention during a 4-week storage period;

[0036] Figure 5 The photoelectric stability test of the sensor of the present invention under 10 consecutive light-on-off cycles;

[0037] Figure 6 The photocurrent response distribution of serum samples from the healthy group (n=20), the benign prostatic hyperplasia group (n=30), and the cancer group (n=35) is shown; (A) photocurrent histogram of each sample; (B) comparison of photocurrent intensity box plots of the three groups.

[0038] Figure 7 Heatmaps of exosome concentrations in individuals from cohorts of cancer, benign prostatic hyperplasia, and healthy individuals, with cancer samples labeled according to Gleason scores. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: (1) First, 4.85 g of Bi(NO3)3·5H2O was added to a mixed solution of 20 mL glacial acetic acid and 30 mL ultrapure water; then, another 50 mL solution containing 1.95 g NaBr and 0.17 g KI was added dropwise to the above mixed solution; after stirring continuously for 180 min, a yellow precipitate was obtained, washed with ultrapure water and dried overnight at 70 °C; 10 mg of the precursor H4TBAPy was dispersed in 1 mL of anhydrous DMF solution. Subsequently, 8 mL of methanol was rapidly introduced into the solution and stirred for 10 min; after standing for 30 min, the resulting mixture was centrifuged at 12000 g to collect the precipitate, and then thoroughly washed multiple times with ultrapure water; finally, the precipitate was freeze-dried to obtain an n-type semiconductor material (HOF-101).

[0041] (2) Weigh 3 mg of HOF-101 and add it to 10 mL of PBS and stir for 30 min. Then add 7 mg of I-BiOBr to the above solution and stir vigorously for 8 h to mix thoroughly. After mixing, centrifuge at 8000 g, add ultrapure water and ethanol for washing, and finally dry overnight at 60 °C.

[0042] (3) Dissolve CTAB in 7 mL of ultrapure water at 30 °C to form a micelle solution of 75 mmol / L, and then add 87.5 μL of 20 mmol / L HAuCl4 solution. Nucleation was initiated by rapidly injecting 0.6 mL of a 10 mmol / L NaBH4 solution pre-cooled to 4 °C, forming a brown Au seed nanoparticle suspension. The seed solution was then gently stirred at 30 °C for 2–5 h to remove excess reducing agent. During the growth phase, a growth solution was prepared by mixing 43 mmol / L CTAB with 90 μL HAuCl4 and 1.39 mL of AA (38.8 mmol / L). Next, 0.54 mL of the freshly prepared Au seed nanoparticle solution was added, and the mixture was incubated overnight at 30 °C to complete the growth of AuNPs. Then, 4.64 mL of 38.8 mM AA, 180 µL of 1M NaOH, and 1.8 mL of Au seed nanoparticle solution were sequentially added to 38 mL of 18.9 mM CTAB solution, ensuring complete mixing before adding 0.45 mL of 20 mM AgNO3. The mixture was then incubated at 40 °C. Stir at low speed overnight at ℃; when the color turns yellow, it indicates that the surface of the Au core has been covered by the Ag shell, and Au@AgNPs are obtained;

[0043] The lyophilized DNA strand powder was dissolved in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to prepare a 100 μM stock solution; then quantified by UV-Vis spectrometry and stored at -20 °C. Different framework nucleic acids were prepared by mixing equimolar oligonucleotides in Tris-HCl buffer (pH 8.0); the mixture was heated at 95 °C for 10 min, then rapidly cooled to 4 °C and held for 15 min; the folded DNA nanostructures were used to prepare Engineered DNAzyme; Au@AgNPs and TDN solution (400 nM) were mixed at a 2:1 volume ratio and incubated with shaking on a vertical suspension for 16 h. After incubation, the mixture was centrifuged at 8000 rpm, washed twice with Tris-HCl, and incubated at 37 °C for 30 min with Hemin (200 nM) to obtain Au@Ag@E-DNAzyme.

[0044] (4) Iron solution was prepared by mixing FeCl3·6H2O (1.12 M), FeCl2·4H2O (0.64 M) and HCl (0.4 M), and Fe3O4 NPs were prepared by NH4OH (2 M); 2-aminoterephthalic acid (NH2-BDC) (10 mM) and FeCl3·6H2O (20 mM) were dispersed in 60 mL DMF, and then 0.5 g of dried magnetic Fe3O4 NPs were added to the solution and ultrasonically dispersed for 15 min; then placed in a polytetrafluoroethylene autoclave and heated to 110 °C for 36 h; after the reaction was complete, the autoclave was cooled to room temperature; the magnetic absorption product was collected and washed three times with ultrapure water and ethanol to remove impurities; finally, it was vacuum dried at 40 °C to obtain amino-functionalized magnetic Fe3O4-metal-organic framework nanorods (Fe3O4@NH2-MIL88B).

[0045] (5) Weigh 1.5 mg of Fe3O4@NH2-MIL88B into 7.5 mL of PBS buffer (pH 6.0), then add 142.5 mg of EDC and 165 mg of NHS, disperse by sonication, place on a vertical suspension apparatus, and activate by shaking at room temperature in the dark for 30 min; after activation, magnetically remove the solid and wash twice with pH 6.0 PBS, then react with 400 μL (1 mg / mL) streptavidin solution (SA) at room temperature for 4 h with shaking; after magnetic separation, remove the supernatant, wash twice with pH 7.0 PBS, and resuspend the obtained solid in 1.5 mL of PBS to obtain Fe3O4@NH2-MIL88B@SA with a concentration of 1 mg / mL, and store at 4 ℃ for later use; take 100 μL of Fe3O4@NH2-MIL88B@SA and 100 μL of Biotin-CD81 aptamer and incubate at room temperature with shaking for 1 h. h, after magnetic washing, the Fe3O4@NH2-MIL88B@Apt capture probe is prepared;

[0046] (6) The bare SPCE electrode was activated in 0.1 M PBS buffer (pH 7.4) at +1.7 V for 3 min; then, 10 μL of I-BiOBr@HOF-101 dispersion (2.0 mg / mL) was added to the working electrode area and dried under an infrared lamp for 30 min; subsequently, 10 μL of Fe3O4@NH2-MIL88B@Apt probe (1.0 mg / mL) was added to the electrode immobilized with I-BiOBr@HOF-101, dried under an infrared lamp for 30 min, and then the electrode was gently rinsed twice with 0.1 M PBST to remove unbound probes, and dried for another 10 min; next, 10 μL of 1% BSA buffer was introduced onto the electrode surface to block non-specific active sites on the electrode, and dried under an infrared lamp for 20 min; after washing twice with PBST, 10 μL of exosome solutions (Exo) of different concentrations were introduced into the working electrode and incubated at room temperature for 45 min. After washing twice with PBST to remove unbound exosomes, 10 μL of Au@Ag@E-DNAzyme complex was added and incubated for 30 min. The mixture was then washed twice with PBST, dried under infrared light for 10 min, and then 10 μL of Hemin solution was added and incubated for another 30 min. After drying, the mixture was washed to obtain the cathodic photoelectrochemical sensor (I-BiOBr@HOF-101 / Fe3O4@NH2-MIL88B@Apt / Exo / Au@Ag@E-DNAzyme).

[0047] Example Sample Testing:

[0048] 1. Evaluation of the photocurrent response of I-BiOBr@HOF-101: The photocurrent response of I-BiOBr@HOF-101 was examined by scanning the it photocurrent curves of several modified electrodes. Electrodes were prepared by dropping 2 mg / mL of each of the prepared I-BiOBr, HOF-101, and I-BiOBr@HOF-101 photoelectric materials onto SPCE electrodes. For I-BiOBr, oxygen-saturated PBS (pH 7.5) was used as the electrolyte; for HOF-101, 0.1 MAA was used; and for I-BiOBr@HOF-101, oxygen-saturated PBS (pH 7.5) was used. The it photocurrent curves were measured at -0.5 V (with illumination turned on every 10 s), and the photocurrent magnitudes of different electrodes were compared.

[0049] The results are as follows Figure 1 As shown, after 0-10 s and 20 s, the light source was turned off, and the system was in a dark state. The current values ​​of all three materials were close to 0 μA, indicating that the dark current of the electrodes was extremely low and there was almost no background signal interference when there was no light excitation. After 10 s of illumination, the photocurrent rose rapidly. After 20 s of light source being turned off, the current quickly dropped back to the initial level, indicating that all three photoelectric materials have good reversible photoresponse characteristics. Comparing the current responses of the three groups of materials, it can be seen that the photocurrent intensity of I-BiOBr@HOF-101 is much higher than that of the two single components, about 6 times that of HOF-101 and 3 times that of I-BiOBr. This comparison result fully demonstrates that the combination of I-BiOBr and HOF-101 can effectively promote the separation of photogenerated electron-hole pairs, reduce carrier recombination losses, improve photoelectric conversion efficiency, and ultimately achieve significant photocurrent signal enhancement.

[0050] 2. In oxygen-unsaturated PBS electrolyte, photocurrent responses of four modified electrodes—I-BiOBr@HOF-101, I-BiOBr@HOF-101 / TDN, I-BiOBr@HOF-101 / DNAzyme, and I-BiOBr@HOF-101 / Au@Ag@E-DNAzyme—were detected and compared using IT technology at a voltage of -0.5 V. The concentration ratio of I-BiOBr to HOF-101 was 7:3, the TDN concentration was 2 μM, and the volume ratio of Au@AgNPs to Engineered DNAzyme was 2:1.

[0051] The results are as follows Figure 2As shown, the photocurrent response of different modified electrodes differed significantly in oxygen-unsaturated PBS electrolyte. I-BiOBr@HOF-101, under photoexcitation, generated photogenerated electrons, resulting in a current response of approximately 3.1 μA in the it curve (a). The photocurrent decreased slightly after the introduction of TDN (b), indicating that the presence of TDN increased the interfacial charge transport resistance to some extent. When Engineered DNAzyme was further introduced onto the electrode, the photocurrent increased again (c), demonstrating that the catalytic function of Engineered DNAzyme improved the reaction kinetics at the sensor interface. Further modification with Au@Ag@E-DNAzyme (d) significantly increased the photocurrent to 12 μA compared to Engineered DNAzyme. These results clearly demonstrate that the composite of Au@AgNPs on Engineered DNAzyme not only provides an excellent electron transport channel, but its SOD-like activity can also improve the utilization efficiency of photogenerated carriers by regulating the oxygen molecule level in the system, thereby achieving effective amplification of the photoelectric signal.

[0052] 3. Detection Specificity of the Cathodophotochemical Sensor: A549, T24, SW480, HeLa, and HGC cells were selected as control cells. After the above cells were cultured, exosomes were extracted according to the steps in Part 2, 2.3.2. The exosome concentrations of control cells and LNCaP cells were diluted to 10⁷ particles / mL, and the control cells and LNCaP cells were mixed in a certain proportion to form a mixed cell. Then, electrodes were fabricated, and photoelectric response detection was performed. By comparing the current response, the specificity of this method for tumor exosome detection was explored. The experiment was repeated three times.

[0053] The results are as follows Figure 3 As shown, only exosomes derived from LNCaP could generate a significantly enhanced photocurrent signal on the sensor, while the response signals of other cell-derived exosomes were all low (P<0.01). Furthermore, the response signal of mixed exosomes was not significantly different from that of LNCaP exosomes alone. These results fully demonstrate that the cathodic photoelectrochemical biosensor we constructed has excellent specific recognition ability for tumor-derived exosomes. Simultaneously, it further confirms that the sensor can still respond stably and efficiently to target exosomes in complex sample matrices. This advantage can be attributed to the joint recognition of tumor exosomes by the CD81 aptamer in the capture probe and the PSA aptamer in the signal amplification probe, as well as the enhancement effect of the signal amplification probe on the photocurrent.

[0054] 4. Storage stability of the cathode photoelectrochemical sensor: 10 7The sensor was prepared using exosomes with a particle / mL ratio, following the steps in Example 1. It was then placed in a 4 °C refrigerator and stored for 0, 1, 2, 3, and 4 weeks, respectively. The photoelectric it curve was then detected, and the storage stability of the sensor was examined by comparing the photocurrent response.

[0055] The results are as follows Figure 4 As shown, the photocurrent response of the sensor remained largely stable during the 4-week storage period, exhibiting only slight fluctuations, and the signal attenuation rate was less than 5%, indicating that the biosensor we fabricated possesses high stability. This is mainly attributed to the excellent chemical stability and interfacial compatibility of the I-BiOBr@HOF-101 heterojunction, which provides a stable loading substrate for the functional layer on the electrode surface; simultaneously, the specific binding of the Au@Ag@E-DNAzyme probe to exosomes further ensures the stability of the recognition interface.

[0056] 5. Signal stability of the cathode photoelectrochemical sensor: 10 1 particles / mL and 10 7 The sensor was prepared using exosomes with a particle / mL density according to step 2.2 in Part 3. After preparation, the photoelectric response was detected by turning on the xenon lamp light source at 10-second intervals for a total duration of 200-second. The signal stability of the sensor was examined by comparing the photocurrent at each interval.

[0057] The results are as follows Figure 5 As shown, during multiple light source on-off cycles, the sensor did not exhibit significant attenuation or drift in the photocurrent signal of exosomes. This was true even at high concentrations (10...). 7 (particles / mL) or low concentration (10) 1 The photocurrent response of exosomes (particles / mL) showed good reproducibility, which fully demonstrates that the photogenerated electron transport process on the electrode surface is stable and has high reproducibility.

[0058] 6. Detection of exosomes in the serum of patients with PCa (prostate cancer), patients with benign prostatic hyperplasia (BPH), and healthy adults: Collect whole blood into a blood collection tube without anticoagulant. Centrifuge at 3000-4000 g for 15-20 min at 4 °C. Carefully aspirate the supernatant, which is the serum. Remove cell debris and apoptotic bodies. Transfer the serum obtained in the previous step to an ultracentrifuge tube and centrifuge at 10000-20000 g for 30-60 min at 4 °C. Collect the supernatant after centrifugation. Then, enrich exosomes by ultracentrifugation at 100000-120000 g for 70-120 min at 4 °C. After centrifugation, discard the supernatant, retain the bottom precipitate, resuspend the precipitate with a large amount of pre-cooled PBS, gently mix by pipetting, and centrifuge again at 100000-120000 g for 70-120 min at 4 °C. After completion, all supernatant was discarded, and the samples were resuspended in a small amount of PBS, aliquoted, and stored at -80°C. Eighty-five serum samples were collected, including 35 PCa patients, 30 BPH patients, and 20 healthy individuals.

[0059] The results are as follows Figure 6 As shown, under optimized experimental conditions, the photocurrent response values ​​of all samples were recorded and analyzed. Figure 6 As shown, the photocurrent signal in the PCa group was significantly higher than that in the BPH and Health groups (P < 0.0001). The increased exosome concentration in the PCa group was due to the high expression of PSA and CD81, which allowed the Au@Ag@E-DNAzyme signal amplification probe to be specifically immobilized on the electrode surface and complete the catalytic reaction. There was no statistically significant difference between the BPH and Health groups, and the exosome level in the BPH group was slightly higher than that in the healthy group. This may be because the PSA level in this group was relatively higher, prompting exosomes to bind to the electrode. After the Au@Ag@E-DNAzyme signal probe was added, slight non-specific adsorption may also occur. These results indicate that the sensor can effectively distinguish between malignant and non-malignant prostate disease states and has good clinical diagnostic potential.

[0060] 7. Signal visualization and cluster analysis: Comparative analysis of the differences in photocurrent response of photoelectrochemical sensors in different clinical groups.

[0061] The results are as follows Figure 7 As shown, the photocurrent intensity of PCa patients was significantly higher than that of the Health control and BPH patients, and the photocurrent level increased with the increase of PCa Gleason score (GS 9-10>GS 8>GS 6-7); the photocurrent response of the Health control was slightly lower than that of the BPH patients, which further intuitively demonstrated the distribution differences among the different groups.

[0062] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A cathode photoelectrochemical sensor, characterized in that, The invention comprises a heterogeneous optoelectronic composite material, a signal amplification probe, an exosome capture probe, and exosomes; the heterogeneous optoelectronic composite material includes a p-type semiconductor doped with bismuth oxy iodide and an n-type semiconductor material; the signal amplification probe includes a gold-silver composite material and an engineered deoxyribozyme; the exosome capture probe includes Fe3O4@NH2-MIL. 88B And biotin-labeled anti-CD81 antibody.

2. The cathode photoelectrochemical sensor according to claim 1, characterized in that, The exosomes are positioned between the signal amplification probe and the exosome capture probe; the heterogeneous optoelectronic composite material is attached to the electrode.

3. A method for preparing the cathode photoelectrochemical sensor according to claim 1, characterized in that, Includes the following steps: Heterogeneous optoelectronic composite material, signal amplification probe, exosome capture probe and reaction solution of exosome were prepared respectively; the reaction solution of heterogeneous optoelectronic composite material was brought into contact with and combined with the electrode, and after drying, the electrode with heterogeneous optoelectronic composite material was obtained. The electrode with the heterogeneous photoelectric composite material was brought into contact with and combined with the reaction solution of the exosome capturing probe. After multiple steps of washing, drying and sealing of non-specific active sites, the electrode with the heterogeneous photoelectric composite material and the exosome capturing probe was obtained. Incubate with exosome reaction solution, wash, incubate with signal amplification probe reaction solution, and dry; treat with heme chloride.

4. The method for preparing the cathode photoelectrochemical sensor according to claim 3, characterized in that, The concentration of the heterogeneous optoelectronic composite material reaction solution is 1.5~2.5 mg / mL; the concentration of the exosome capturing probe reaction solution is 0.5~1.5 mg / mL; the concentration of the exosome reaction solution is 10 mg / mL. 4 ~10 6 particles / mL.

5. The method for preparing the cathode photoelectrochemical sensor according to claim 1, characterized in that, The preparation of the heterogeneous optoelectronic composite material includes the following steps: S1, dissolve bromide and iodide salts in water to prepare a mixed solution; prepare an acetic acid solution, add bismuth salt and mix well, add the mixed solution dropwise, and stir the reaction until the precipitate no longer increases; separate and collect the product, wash thoroughly and dry to obtain iodine-doped bismuth oxybromide; S2, tetrakis(4-carboxyphenyl)pyrene was dispersed in dimethylformamide, then methanol was quickly added, mixed evenly, and allowed to stand to stabilize the system; the product was separated and collected, washed thoroughly, and freeze-dried to obtain HOF-101; S3, add HOF-101 to phosphate buffer solution, then add iodine-doped bismuth oxybromide, mix well and stir vigorously for 6-10 hours; separate and collect the product, wash thoroughly and dry to obtain the final product.

6. The method for preparing the cathode photoelectrochemical sensor according to claim 5, characterized in that, The concentration of the acetic acid solution is 0.5~0.8 g / mL; the molar ratio of the bismuth salt to the iodine salt is 9~10:1; the molar ratio of the bromide salt to the iodine salt is 18~20:1; the mass ratio of the tetrakis(4-carboxyphenyl)pyrene to methanol is 1~1.5:1; and the mass ratio of HOF-101 to iodinated bismuth oxybromide is 1:2~2.

5.

7. The method for preparing the cathode photoelectrochemical sensor according to claim 1, characterized in that, The preparation of the signal amplification probe includes the following steps: a. Prepare a tetrachloroauric acid solution and a sodium borohydride solution, and cool the sodium borohydride solution; prepare a micelle solution by dissolving hexadecyltrimethylammonium bromide in water; b. Take the micelle solution, add tetrachloroauric acid solution, and then quickly add sodium borohydride solution to nucleate and obtain a gold seed nanoparticle suspension; c. Prepare hexadecyltrimethylammonium bromide solution A and L-ascorbic acid solution; mix hexadecyltrimethylammonium bromide solution A and L-ascorbic acid solution and add tetrachloroauric acid solution to obtain growth solution; d. Prepare hexadecyltrimethylammonium bromide solution B. Take the gold seed nanoparticle suspension and the growth solution, mix them and let stand for 8-16 h to obtain the gold nanoparticle solution. Take L-ascorbic acid solution, sodium hydroxide solution, gold nanoparticle solution and hexadecyltrimethylammonium bromide solution B, mix them evenly, add silver nitrate, and stir at low speed for 8-16 h.

8. The method for preparing the cathode photoelectrochemical sensor according to claim 7, characterized in that, The concentration of the micelle solution is 70-80 mmol / L; the concentration of the tetrachloroauric acid solution is 15-25 mmol / L; the concentration of the sodium borohydride solution is 8-12 mmol / L; the sodium borohydride solution is cooled to below 5°C; the concentration of the L-ascorbic acid solution is 35-40 mmol / L; the concentration of hexadecyltrimethylammonium bromide solution A is 40-45 mmol / L; and the concentration of hexadecyltrimethylammonium bromide solution B is 15-20 mmol / L. The mass ratio of tetrachloroauric acid to sodium borohydride in step b is 2.5~3.5:1; the mass ratio of tetrachloroauric acid to L-ascorbic acid in step c is 1:10~15; the mass ratio of tetrachloroauric acid added to the growth solution in step d to the gold seed nanoparticles in step d is 25~35:1; the mass ratio of L-ascorbic acid to sodium hydroxide in step d is 4~5:1; the mass ratio of sodium hydroxide to the gold seed nanoparticles in step d is 90~110:1; and the mass ratio of silver nitrate to the gold seed nanoparticles in step d is 20~25:

1.

9. The method for preparing the cathode photoelectrochemical sensor according to claim 1, characterized in that, The preparation of the exosome capture probe includes the following steps: Take Fe3O4@NH2-MIL 88B After thorough activation, the product was reacted with streptomycin solution at room temperature with shaking for 3-5 hours to obtain the modified streptomycin precursor. The modified streptomycin precursor was then incubated with biotin-labeled anti-CD81 antibody for 40-80 minutes, and the product was collected and thoroughly washed. The Fe3O4@NH2-MIL... 88B The mass ratio of the modified streptomycin precursor to the biotin-labeled anti-CD81 antibody is 3-4:1; the mass ratio of the modified streptomycin precursor to the biotin-labeled anti-CD81 antibody is 1:1-1.

5.

10. The application of the cathode photoelectrochemical sensor according to claim 1 or 2 in the prevention, diagnosis or treatment of cancer.

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