Preparation method of CTC capture detection kit based on prostate cancer specific biomarkers

Abstract

This invention discloses a method for preparing a CTC capture detection kit based on prostate cancer-specific biomarkers, relating to the field of circulating tumor cell detection technology. The method includes: Step 1: Based on single-cell transcriptome data of peripheral blood circulating tumor cells from prostate cancer patients, a combination of membrane protein biomarkers stably co-expressed in circulating tumor cells and not expressed in normal peripheral blood nucleated cells is screened. The membrane protein biomarker combination includes at least two of PSMA, STEAP1, and NKX3-1. Step 2: For each member of the membrane protein biomarker combination obtained in Step 1, a corresponding DNA aptamer is obtained through SELEX technology, and the obtained DNA aptamers are modified with a 5' amino group. Step 3: A solid-phase chip with a microporous array structure is provided, and the inner walls of the micropores are passivated with polyethylene glycol. Step 4: The amino-modified DNA aptamers obtained in Step 2 are mixed and then covalently coupled to the passivated inner walls of the micropores obtained in Step 3.

Description

Technical Field

[0001] This invention relates to the field of circulating tumor cell detection technology, and in particular to a method for preparing a CTC capture detection kit based on prostate cancer-specific biomarkers. Background Technology

[0002] Circulating tumor cell (CTC) detection technology refers to a type of liquid biopsy method that isolates, enriches, and identifies exfoliated cells originating from primary or metastatic tumors from peripheral blood and other body fluids of cancer patients. Because CTCs are present in extremely low concentrations in blood, this technology requires a combination of highly sensitive enrichment strategies and specific identification methods to achieve early tumor screening, prognostic assessment, efficacy monitoring, and personalized treatment guidance. Due to its advantages such as being minimally invasive, repeatable, and reflecting tumor heterogeneity, CTC detection has become an important tool in precision oncology.

[0003] In the field of circulating tumor cell detection, mainstream CTC detection platforms rely on EpCAM antibodies for enrichment. However, during the progression of prostate cancer, especially during the epithelial-mesenchymal transition (EMT) or castration resistance stages, CTCs often downregulate or lose EpCAM expression, resulting in reduced capture efficiency and insufficient sensitivity. Furthermore, existing methods do not screen for biomarkers targeting the unique molecular characteristics of prostate cancer CTCs, making it difficult to accurately identify prostate-derived CTCs and easily misclassifying other epithelial-derived exfoliated cells as CTCs. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for preparing a CTC capture detection kit based on prostate cancer-specific biomarkers. This addresses the problem that mainstream CTC detection platforms rely on EpCAM antibodies for enrichment, but during prostate cancer progression, especially during epithelial-mesenchymal transition (EMT) or castration resistance, CTCs often downregulate or lose EpCAM expression, leading to reduced capture efficiency and insufficient sensitivity. Furthermore, existing methods do not screen for biomarkers targeting the unique molecular characteristics of prostate cancer CTCs, making it difficult to accurately identify prostate-derived CTCs and easily misclassifying other epithelial-derived exfoliated cells as CTCs.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a CTC capture detection kit based on prostate cancer-specific biomarkers, comprising: Step 1: Based on single-cell transcriptome data of peripheral blood circulating tumor cells from prostate cancer patients, a combination of membrane protein markers that are stably co-expressed in circulating tumor cells and not expressed in normal peripheral blood nucleated cells is screened. The combination of membrane protein markers includes at least two of PSMA, STEAP1 and NKX3-1. Step 2: For each member of the membrane protein marker combination obtained in Step 1, the corresponding DNA aptamer is obtained by screening using SELEX technology, and the obtained DNA aptamer is modified with 5' amino groups. Step 3: Provide a solid-phase chip with a micropore array structure, and perform polyethylene glycol surface passivation treatment on the inner wall of the micropores; Step 4: After mixing the amino-modified DNA aptamers obtained in Step 2, they are fixed to the passivated micropore inner wall obtained in Step 3 by covalent coupling to form a multivalent synergistic capture layer. Step 5: Pre-place a mixture of lyophilized cell lysis reagent and LAMP amplification primers targeting the TMPRSS2-ERG fusion gene or AR-V7 splice variant at the bottom of the microwell below the multivalent co-capture layer formed in Step 4, and place a water-soluble isolation membrane between the two. Step Six: The microwell array chip constructed in Step Five, the buffer solution for washing unbound cells, and the fluorescent substrate for LAMP amplification signal reading are encapsulated in sealed containers to form the CTC capture detection kit.

[0007] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, wherein the membrane protein biomarker combination consists of PSMA and STEAP1.

[0008] Furthermore, the co-expression rate of PSMA and STEAP1 on the surface of circulating tumor cells in prostate cancer was verified by single-cell flow cytometry to be no less than 85%, and the expression rate in nucleated cells of peripheral blood of patients with benign prostatic hyperplasia was less than 2%.

[0009] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, wherein the membrane protein biomarker combination consists of PSMA, STEAP1 and NKX3-1.

[0010] Furthermore, the combination of the three biomarkers maintained stable co-expression in CTCs from castration-resistant prostate cancer patients, overcoming the problem of expression loss during epithelial-mesenchymal transition (EMT) when PSA or EpCAM is used alone, thus increasing the CTC capture sensitivity to over 92% in advanced patient samples.

[0011] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, wherein: the covalent coupling method adopts an activation system of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to fix the amino-modified DNA aptamer to the inner wall of the micropore.

[0012] Furthermore, the coupling reaction is carried out in a MES buffer system with pH 5.0–6.0, the reaction temperature is controlled at 4°C, and the reaction time is 2 hours, in order to maximize the preservation of the three-dimensional conformation of the aptamer and its binding activity with the target, and to avoid inactivation caused by high temperature or strong acid and alkaline conditions.

[0013] In a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer-specific biomarkers according to the present invention, the immobilization density of each DNA aptamer in the multivalent synergistic capture layer on the inner wall of the micropore is 5 × 10⁻⁶. 9 Up to 2×10 12 One molecule per square centimeter.

[0014] Furthermore, when the fixed density is less than 5×10 9 When the concentration of CTC molecules per square centimeter is 2 × 10⁻⁶, the capture efficiency of CTC decreases significantly; when it exceeds 2 × 10⁻⁶, the capture efficiency of CTC decreases significantly. 12 At a density of 1 molecule per square centimeter, the aptamer conformation is restricted due to steric hindrance, which actually reduces binding affinity; the optimal density range is 1 × 10⁻⁶. 11 Up to 5×10 11 One molecule per square centimeter.

[0015] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific markers of the present invention, the water-soluble isolation membrane is made of polyvinyl alcohol and has a thickness of 10 to 50 micrometers.

[0016] Furthermore, the dissolution time of the polyvinyl alcohol isolation membrane is controlled within 30–90 seconds after the addition of the trigger buffer to ensure that the lysis reagent is accurately released after the washing step of CTC is completed, avoiding premature contact that could lead to nonspecific cell lysis or primer degradation.

[0017] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, wherein: the LAMP amplification primer mixture contains four primers, F3, B3, FIP and BIP, designed for the breakpoint region of the TMPRSS2-ERG fusion gene.

[0018] Furthermore, the FIP primers contain a linker sequence of TMPRSS2 exon1 and ERG exon4, and the BIP primers contain reverse complementary sequences of TMPRSS2 exon1 and ERG exon4, ensuring that circular amplification can only be initiated when the fusion gene is present, and there is no cross-amplification of wild-type TMPRSS2 or ERG genes.

[0019] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, the LAMP amplification primer mixture contains four primers, F3, B3, FIP and BIP, designed for the skipping exon boundary region of AR-V7 splicing variant.

[0020] Furthermore, the primers are designed to span the splicing site of AR gene exon3 and cryptic exon CE3. The FIP primers contain downstream exon3 sequences and upstream CE3 sequences, thereby specifically recognizing AR-V7 transcripts without amplifying full-length AR mRNA, making them suitable for drug resistance monitoring in castration-resistant prostate cancer.

[0021] In a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers of the present invention, the fluorescent substrate is hydroxynaphthol blue or calcein-manganese ion complex.

[0022] Furthermore, the hydroxynaphthol blue changes from purple to blue during LAMP amplification due to the precipitation of magnesium pyrophosphate, which is visually perceptible; the calcein-manganese ion complex emits green fluorescence in the presence of the amplification product, making it suitable for reading with a portable fluorescence detector; neither substrate requires opening the cap, preventing aerosol contamination.

[0023] As a preferred embodiment of the preparation method of the CTC capture detection kit based on prostate cancer specific markers of the present invention, wherein: in the prepared kit, the inner wall of the microwell is fixed with no less than two types of DNA aptamers, and the LAMP amplification primer mixture and fluorescent substrate are configured to generate a detectable fluorescent signal only when the captured cells simultaneously bind to at least two different DNA aptamers and release the TMPRSS2-ERG fusion gene or the AR-V7 splice variant target sequence.

[0024] Furthermore, the dual confirmation mechanism effectively eliminates interference from circulating epithelial cells that are positive for a single marker but not of prostate cancer origin. In clinical validation, it reduced the false positive rate from 35% in traditional PSA screening to less than 5%, significantly improving the positive predictive value of early screening.

[0025] The beneficial effects of this invention are as follows: By integrating a single-cell transcriptome-guided multi-marker combination (PSMA / STEAP1 / NKX3-1), a highly stable DNA aptamer recognition element, a microwell array multivalent synergistic capture structure, and an integrated capture-lysis-in situ LAMP amplification design, it achieves highly sensitive and specific detection of circulating tumor cells in prostate cancer. The method not only overcomes the shortcomings of high false positive rates in traditional PSA screening and missed detections during EMT in EpCAM-dependent CTC technology, but also improves the ability to differentiate between benign and malignant lesions through a dual confirmation mechanism of dual-marker binding and specific gene amplification. In clinical validation, the false positive rate has been reduced to below 5%, providing a reliable, mass-producible, and grassroots-suitable molecular diagnostic tool for early prostate cancer screening, risk stratification, and drug resistance monitoring. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a flowchart of the preparation method of the CTC capture detection kit based on prostate cancer specific biomarkers in Example 1. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] Example 1, please refer to Figure 1 This is the first embodiment of the present invention, which provides a specific preparation method for a CTC capture detection kit based on a combination of PSMA and STEAP1 dual markers, including: A polymethyl methacrylate (PMMA) microporous array chip with injection molding is provided, the surface of which has micropores with a diameter of 80 micrometers and a depth of 100 micrometers. The chip is subjected to oxygen plasma treatment for 5 minutes, and then immersed in 2% (v / v) polyethylene glycol silane ethanol solution and reacted at 60°C for 2 hours to complete the passivation of the inner wall of the micropores. DNA aptamers targeting the extracellular domain of PSMA (sequence as SEQ ID NO:1) and DNA aptamers targeting STEAP1 (sequence as SEQ ID NO:2) were obtained using SELEX technology, and amino groups were introduced at the 5' end of both aptamers. The two aptamers were dissolved in MES buffer at pH 5.5 at a 1:1 molar ratio, and activated with EDC and NHS for 30 minutes. They were then injected into a passivated microchip and incubated at 4°C for 12 hours to allow the aptamers to covalently couple to the inner wall of the micropores, forming a multivalent co-capture layer. At the bottom of the microwell, a lyophilized cell lysis buffer (containing 0.5% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA) and a TMPRSS2-ERG fusion gene-specific LAMP primer mixture (F3 / B3 / FIP / BIP) were pre-placed in the form of a micro-spotting device, and a 30-micrometer-thick layer of polyvinyl alcohol aqueous solution was sprayed on top of it. After drying, a water-soluble isolation membrane was formed. The microporous chip constructed as described above is placed in an aluminum foil bag, and phosphate washing buffer (pH 7.4) and hydroxynaphthol blue fluorescent substrate solution are encapsulated. The bag is then vacuum sealed to obtain the CTC capture detection kit of the present invention.

[0032] In summary, scanning electron microscopy and fluorescence labeling confirmed that the aptamers were uniformly distributed on the inner wall of the micropores, with a fixation density of approximately 1.2 × 10⁻⁶. 11 molecule / cm 2 It possesses good capture activity.

[0033] Example 2, the second embodiment of the present invention, provides a specific preparation method for a CTC capture detection kit based on a combination of three biomarkers: PSMA, STEAP1, and NKX3-1, including: The same PMMA microporous array chip as in Example 1 was provided, and after activation by oxygen plasma, the surface was passivated with polyethylene glycol silane. DNA aptamers targeting PSMA, STEAP1, and NKX3-1 were obtained using SELEX technology, with sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and amino groups were modified at the 5' end of each aptamer. The three aptamers were mixed in a 1:1:1 molar ratio and dissolved in MES buffer at pH 5.5. EDC and NHS were added to a final concentration of 2 mM and 5 mM respectively. After activation at room temperature for 30 minutes, the mixture was injected into a passivated microporous chip and coupled at 4 °C for 12 hours to form a trivalent synergistic capture layer. A lyophilized mixture of AR-V7-specific LAMP primers (F3 / B3 / FIP / BIP, sequence as shown in SEQ ID NO:8–11) and cell lysis buffer were pre-placed at the bottom of the microwells, and a 40-micrometer-thick polyvinyl alcohol solution was sprayed on top and dried to form a film as a water-soluble isolation layer. The constructed chip, washing buffer, and calcein-manganese ion fluorescent substrate were encapsulated in light-proof aluminum foil bags and vacuum-sealed to obtain a CTC capture detection kit suitable for the detection of castration-resistant prostate cancer.

[0034] Example 3 is the third embodiment of the present invention. This embodiment provides a specific preparation method for optimizing aptamer fixation density and controlling coupling process, including: Three sets of microporous chips were prepared, all of which underwent the same passivation treatment; Group 1: The aminated PSMA / STEAP1 aptamer mixture was diluted to a low concentration (10 nM), and the fixed density was measured to be 3 × 10⁻⁶ after coupling. 9 molecule / cm 2 ; Group 2: Using a medium concentration (100 nM), a fixed density of 1.5 × 10⁻⁶ was obtained. 11 molecule / cm 2 ; Group 3: Using high concentration (1 μM), the fixation density reached 3 × 10⁻⁶. 12 molecule / cm 2 ; All coupling reactions were carried out in a pH 5.5 MES buffer system at 4°C for 12 hours. Subsequently, TMPRSS2-ERG LAMP primer freeze-dried blocks and 30-micron polyvinyl alcohol isolation membranes were uniformly pre-placed at the bottom of the microwells of each group of chips, and the kit assembly was completed. The density differences were quantitatively confirmed by fluorescently labeled aptamers and retained for subsequent capture efficiency testing to verify the process feasibility of the density range.

[0035] Example 4, the fourth embodiment of the present invention, provides a specific method for fabricating an integrated and closed signal readout system for a LAMP amplification module, including: At the bottom of the microwell of the multivalent capture chip obtained in Example 1, 1 μL of a premix containing LAMP primers (F3 / B3 / FIP / BIP), Bst DNA polymerase, dNTPs and betaine was precisely added using a non-contact piezoelectric spotting instrument. The spotted chip was placed in a freeze dryer and freeze-dried at -50℃ and 10 Pa for 4 hours to form a stable solid reagent layer. Subsequently, a 2% polyvinyl alcohol aqueous solution was sprayed on top of the freeze-dried layer. The spray flow rate and platform movement speed were controlled to ensure that the film thickness was uniform at 35 micrometers. The film was then dried at 60°C for 10 minutes to form a water-soluble isolation film. Finally, the chip and the independently packaged trigger buffer (containing Tris-HCl, MgSO4 and hydroxynaphthol blue) are put into a dual-chamber sealed detection cartridge to achieve an integrated closed detection structure of "sample in - result out" and avoid contamination by amplification products.

[0036] Example 5, the fifth embodiment of the present invention, provides a standard operating procedure for the reagent kit of the present invention in clinical samples, including: Collect 2 mL of peripheral blood from a suspected prostate cancer patient and add it to an anticoagulant tube containing EDTA; Whole blood samples were directly injected into the micro-hole chip inlet of the reagent kit prepared in Example 1 and left to stand at 37°C for 30 minutes to allow CTCs to fully bind to the multivalent aptamers. The chip was rinsed with 10 mL of PBS buffer at a flow rate of 0.5 mL / min to remove unbound cells. Inject 100 μL of trigger buffer to dissolve the polyvinyl alcohol isolation membrane and release the lyophilized lysis-amplification reagent; Transfer the chip to a 63°C constant temperature metal bath and incubate for 45 minutes to start LAMP amplification; The color change inside the micropore was observed using a portable blue light excitation device: if it changed from purple to blue, it was interpreted as TMPRSS2-ERG positive. Combined with the results of dual aptamer capture, it was finally determined to be CTC positive of prostate cancer origin. The entire process requires no nucleic acid extraction, opening of the lid, or complex instruments, and the operation time is less than 90 minutes.

[0037] Example 6, the sixth embodiment of the present invention, provides a performance verification experiment on the integrated detection system of PSMA / STEAP1 dual-marker aptamer microchip and in-situ LAMP in the detection of prostate cancer CTCs, including: To systematically evaluate the detection performance of the kit of this invention in a real clinical setting, 80 subjects who visited the urology department of a tertiary hospital between January 2025 and December 2025 were selected as the experimental subjects. These included 40 patients with pathologically confirmed prostate cancer (20 with localized carcinoma and 20 with castration-resistant prostate cancer (CRPC), 20 patients with benign prostatic hyperplasia (BPH), 10 patients with prostatitis, and 10 healthy volunteers. All subjects signed informed consent forms, and 2 mL of peripheral venous blood was collected according to standardized procedures and anticoagulated with EDTA. The experimental group was detected using the PSMA / STEAP1 dual-marker CTC capture detection kit prepared in Example 1 of this invention; the control group was detected in parallel using Comparative Example 1 (CellSearch system) and Comparative Example 2 (serum PSA chemiluminescence method), respectively. All operations were strictly performed according to their respective instructions or standard procedures.

[0038] The specific experimental procedure is as follows: Whole blood samples were directly injected into the microchip inlet of this invention and incubated at 37°C for 30 minutes to complete CTC capture; then, 10 mL of PBS was used to wash away background cells at a flow rate of 0.5 mL / min; 100 μL of trigger buffer was injected to dissolve the polyvinyl alcohol isolation membrane and release the lyophilized lysis-LAMP reagent; after incubation at 63°C for 45 minutes, the color change was observed using a portable blue light lamp (purple → blue was considered positive). 7.5 mL of whole blood was processed using the CellSearch system according to standard procedures, enriched with magnetic beads, and then counted using an automated fluorescence scanning system for CK+ / CD45- / DAPI+ cells; PSA detection was performed using the Roche Cobase801 platform, with a cutoff value set at 4 ng / mL. All results were interpreted blinded by two independent physicians and validated using histopathological or long-term follow-up results as the gold standard. In addition, single-cell qRT-PCR was performed on the CTCs captured by this invention to confirm the expression of the TMPRSS2-ERG fusion gene to verify molecular specificity.

[0039] Table 1: Performance comparison of the kit of the present invention with existing technologies in the detection of CTCs in prostate cancer (n=80) ; Data Analysis: The data listed in Table 1 clearly demonstrate the significant advantages of this invention in the detection of circulating tumor cells (CTCs) in prostate cancer. Firstly, in terms of sensitivity, this invention achieves 92.5%, significantly higher than the 67.5% of the CellSearch system and the aptamer capture method without molecular validation (85.0%). This improvement stems from the synergistic recognition strategy of dual biomarkers PSMA and STEAP1—both are prostate cancer-specific membrane proteins that remain stably expressed during epithelial-mesenchymal transition (EMT), effectively avoiding the inherent defect of traditional EpCAM-dependent methods that miss CTCs during EMT. Although serum PSA detection is the fastest (30 minutes), it is not suitable for direct CTC detection and therefore cannot provide sensitivity data, serving only as an indirect reference.

[0040] Secondly, in terms of specificity, this invention achieves a high accuracy of 95.0%, far superior to serum PSA (65.0%) and the aptamer method without LAMP verification (77.5%). Particularly noteworthy is the false positive rate of only 5.0% in benign prostatic hyperplasia (BPH) samples, compared to 35.0% for PSA detection and 20.0% for aptamer-only capture. This clearly demonstrates that relying solely on surface marker capture is insufficient to distinguish malignant CTCs from benign exfoliated epithelial cells; while this invention, by integrating an in situ LAMP amplification module of the TMPRSS2-ERG fusion gene within microwells, achieves a dual verification mechanism of "phenotypic capture + genotypic confirmation," fundamentally improving interpretation accuracy.

[0041] Furthermore, in terms of testing efficiency, this invention can complete the entire process from whole blood sample introduction to result interpretation in just 85 minutes, far faster than the 240 minutes required by CellSearch, and requires only 2 mL of blood (compared to 7.5 mL for CellSearch), greatly reducing the burden of blood collection for patients and making it more suitable for primary care or bedside settings. Although PSA testing is faster, it lacks cellular-level evidence and cannot be used for dynamic monitoring of CTCs or drug resistance assessment.

[0042] Comparative Example 1 (EpCAM antibody-dependent CTC capture): The commercially available CellSearch system kit was used, with the surface of the capture magnetic beads coated with anti-EpCAM monoclonal antibody. Follow the instructions: Collect 7.5 mL of the patient's whole blood, enrich it with immunomagnetic beads, stain it with CK / CD45 / DAPI three-color fluorescence, and then identify CTCs by an automated scanning system; This method relies solely on EpCAM expression, which makes it prone to missing CTCs in the epithelial-mesenchymal transition (EMT) state, and it cannot distinguish prostate cancer from other epithelial tumors.

[0043] Comparative Example 2 (Detection of a Single PSA Marker): Serum samples were collected from the same patients as in Example 5, and the total PSA concentration was detected by chemiluminescence immunoassay. A positive result was determined using 4 ng / mL as the cutoff value; This method cannot locate CTCs and often produces false positives in patients with benign prostatic hyperplasia and prostatitis, lacking direct evidence of tumor cells.

[0044] Comparative Example 3 (Ampamibole capture without molecular verification): Microporous chips were prepared according to Example 1, but the LAMP primers and isolation membrane in step five were omitted, and only the PSMA / STEAP1 dual aptamer trapping layer was retained. The presence of CTCs was confirmed by immunofluorescence staining (anti-CK antibody) after capture; The lack of confirmation by TMPRSS2-ERG or AR-V7 molecules makes it impossible to rule out interference from benign circulating epithelial cells, leading to false positive signals in some BPH samples.

[0045] In summary, this invention achieves highly sensitive and specific detection of circulating tumor cells (CTCs) in prostate cancer by integrating a single-cell transcriptome-guided multi-marker combination, a highly stable DNA aptamer recognition element, a microwell array multivalent synergistic capture structure, and an integrated capture-lysis-in situ LAMP amplification design. The method not only overcomes the shortcomings of traditional PSA screening (high false positive rate) and EpCAM-dependent CTC technology (missed detection during EMT), but also enhances the ability to differentiate between benign and malignant lesions through a dual-marker binding + specific gene amplification dual confirmation mechanism. In clinical validation, the false positive rate has been reduced to below 5%, providing a reliable, mass-producible, and grassroots-suitable molecular diagnostic tool for early prostate cancer screening, risk stratification, and drug resistance monitoring.

[0046] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a CTC capture detection kit based on prostate cancer-specific biomarkers, characterized in that: include: Step 1: Based on single-cell transcriptome data of circulating tumor cells in peripheral blood of prostate cancer patients, a combination of membrane protein markers that are stably co-expressed in circulating tumor cells and not expressed in normal peripheral blood nucleated cells was screened. The combination of membrane protein markers includes PSMA, STEAP1 and NKX3-1. Step 2: For each member of the membrane protein marker combination obtained in Step 1, the corresponding DNA aptamer is obtained by screening using SELEX technology, and the obtained DNA aptamer is modified with 5' amino groups. Step 3: Provide a solid-phase chip with a micropore array structure, and perform polyethylene glycol surface passivation treatment on the inner wall of the micropores; Step 4: After mixing the amino-modified DNA aptamers obtained in Step 2, they are fixed to the passivated micropore inner wall obtained in Step 3 by covalent coupling to form a multivalent synergistic capture layer. Step 5: A mixture of lyophilized cell lysis reagent and LAMP amplification primers targeting the TMPRSS2-ERG fusion gene or AR-V7 splice variant is pre-placed at the bottom of the micropores below the multivalent synergistic capture layer formed in Step 4, and a water-soluble isolation membrane made of polyvinyl alcohol with a thickness of 10 to 50 micrometers is placed on top of it. Step Six: The microwell array chip constructed in Step Five, the buffer solution for washing unbound cells, and the fluorescent substrate for LAMP amplification signal reading are encapsulated in sealed containers to form the CTC capture detection kit.

2. The method for preparing the CTC capture detection kit based on prostate cancer-specific biomarkers as described in claim 1, characterized in that: The membrane protein biomarker combination consists of PSMA, STEAP1, and NKX3-1.

3. The method for preparing the CTC capture detection kit based on prostate cancer-specific biomarkers as described in claim 1, characterized in that: The fixation density of each DNA aptamer on the inner wall of the micropore in the multivalent synergistic capture layer is 5 × 10⁻⁶. 9 Up to 2×10 12 One molecule per square centimeter.

4. The method for preparing the CTC capture detection kit based on prostate cancer-specific biomarkers as described in claim 1, characterized in that: The LAMP amplification primer mixture contains four primers, F3, B3, FIP, and BIP, designed for the breakpoint region of the TMPRSS2-ERG fusion gene.

5. The method for preparing the CTC capture detection kit based on prostate cancer-specific biomarkers as described in claim 1, characterized in that: The LAMP amplification primer mixture contains four primers, F3, B3, FIP, and BIP, designed for the skip exon boundary region of the AR-V7 splicing variant.

6. The method for preparing the CTC capture detection kit based on prostate cancer-specific biomarkers as described in claim 1, characterized in that: The fluorescent substrate is hydroxynaphthol blue or calcein-manganese ion complex.

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