Microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and preparation method

Abstract

The present invention belongs to the technical field of biochemical detection, and specifically relates to a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and a preparation method; the kit uses a streptavidin-biotin system to fix EpCAM antibodies, Vimentin antibodies and a dual-site PSMA capture combination in the chip channel, thereby achieving dual enrichment of epithelial and stromal CTCs, and jointly identifying PSMA targets through aptamers and antibodies to improve detection specificity and sensitivity. Competitive short peptides are further introduced to block nonspecific binding sites on the leukocyte surface and reduce false positives. Combined with a multiple fluorescent staining system and a dual-antibody nucleic acid probe signal amplification strategy, accurate identification and quantification of CTCs and PSMA-positive cells are achieved. The scheme is suitable for early screening, efficacy evaluation and recurrence monitoring of prostate cancer, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and in particular to a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and a preparation method thereof. Background Art

[0002] Prostate cancer is a malignant tumor of the male urinary system that often lacks obvious symptoms in its early stages and often has advanced to the locally advanced or distant metastatic stage by the time it is diagnosed. In recent years, circulating tumor cells (CTCs) have become an important marker for early cancer screening, therapeutic efficacy monitoring, and recurrence assessment because they can noninvasively reflect the characteristics of primary or metastatic lesions. Among them, prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein that is highly expressed in prostate cancer, has been widely used in targeted diagnosis and treatment of prostate cancer and holds significant value in CTC detection.

[0003] Traditional CTC detection methods include density gradient centrifugation, immunomagnetic capture, and molecular labeling. However, these methods generally suffer from low separation purity, cumbersome processing steps, and limited capture efficiency, making them inadequate for clinical high-throughput, high-sensitivity detection. In recent years, the development of microfluidic chip technology has provided a new approach for the efficient enrichment and isolation of CTCs. Microfluidic chips offer advantages such as low sample volume, automated operation, and minimal cell damage, enabling multi-marker immunocapture and high-throughput cell processing.

[0004] In response to the detection needs of prostate cancer CTCs, an immune enrichment detection kit with PSMA as the main identification target and combined with microfluidic chip technology has been developed. This can not only achieve specific identification and efficient capture of CTCs, but also improve the accuracy and repeatability of clinical sample detection. It is expected to play an important role in the early diagnosis, recurrence monitoring and efficacy evaluation of prostate cancer. Summary of the Invention

[0005] In response to the above problems, the present invention aims to provide a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, comprising the following biochemical combination components:

[0006] a) A biotin-modified cell capture agent comprising the following three types of biotinylated molecules, prepared at a molar ratio of (1-3):(1-2):1:

[0007] Category 1: Mouse anti-human EpCAM monoclonal antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2;

[0008] Category II: Mouse anti-human Vimentin monoclonal antibody, the amino acid sequence of its heavy chain variable region is SEQ ID NO: 3, and the amino acid sequence of its light chain variable region is SEQ ID NO: 4;

[0009] Category III: Dual-site anti-PSMA capture combination, which includes:

[0010] A biotin-modified mouse anti-human PSMA monoclonal antibody, the heavy chain variable region amino acid sequence of SEQ ID NO: 5, the light chain variable region amino acid sequence of SEQ ID NO: 6, and

[0011] A 30nt biotin-modified PSMA aptamer, the nucleic acid sequence of which is SEQ ID NO: 7;

[0012] b) An immune-specific blocking agent, which is a short peptide of a competitive PSMA antibody, the sequence of which is SEQ ID NO: 8, is used to selectively saturate non-specific PSMA antibody binding sites on the leukocyte surface before enrichment.

[0013] In a preferred technical solution, the following biochemical combination components are also included:

[0014] c) a microfluidic chip having a bottom layer immobilized with streptavidin and capable of forming a covalent outer layer with the biotinylated molecule;

[0015] d) a multiplex fluorescence detection composition comprising:

[0016] FITC-anti-CK antibody, AlexaFluor647-anti-Vimentin antibody, PE-anti-CD45 antibody, DAPI;

[0017] PSMA dual-site signal amplification pair: The first detection antibody is coupled to oligonucleotide P1, and the second detection antibody is coupled to oligonucleotide P2. P1 / P2 self-assemble at a distance of ≤40nm to form a fluorescent-nucleic acid probe, achieving PSMA-specific dual confirmation;

[0018] e) Pretreatment agent, separation solution, fixative, permeabilization agent and 5% BSA blocking solution.

[0019] In a preferred technical solution, the molar ratio of the mouse anti-human EpCAM monoclonal antibody, the mouse anti-human Vimentin monoclonal antibody, the biotin-modified mouse anti-human PSMA monoclonal antibody, and the biotin-modified 30nt PSMA aptamer is (2-3):1:0.5:0.5;

[0020] When the four biotinylated molecules were subjected to HABA-Avidin colorimetric quantification, the number of biotin residues bound to each antibody or aptamer molecule was controlled at 3-8 mol / mol, with an average of 5-6 for antibody molecules and 3-4 for aptamers. The total protein concentration after capture agent reconstitution was And pH7.2±0.1.

[0021] In a preferred technical solution, the competitive PSMA antibody short peptide SEQ ID NO: 8 is 14±2 amino acid residues, and its affinity constant is The affinity for the PSMA homologous site on the surface of human leukocytes is less than 100nM, but the affinity for PSMA on tumor cells is greater than 5µM; the competitive PSMA antibody short peptide is incubated at a final concentration of 0.2-0.5µM for 1-3 minutes before enrichment.

[0022] In a preferred technical solution, the separation solution is PBS, 20mM, pH 7.2-7.6, containing 0.6%-1.0% (m / v) BSA, 1mM EDTA and 50µM ZnCl2;

[0023] The separation solution was sterilized by filtration twice through a 0.22 μm PES membrane and stored at 4°C to avoid metal ion precipitation.

[0024] In a preferred technical solution, the PSMA dual-site signal amplification pair consists of a 6-FAM-labeled oligonucleotide P1 and a Quasar670-labeled oligonucleotide P2. The complementary regions of P1 and P2 are 15 nt and are located at the C-termini of the two PSMA detection antibodies.

[0025] When the target protein binds to both P1-antibody and P2-antibody simultaneously, the FRET distance does not exceed 35 nm, achieving a green to near-red signal conversion.

[0026] In a preferred technical solution, the asymmetric bifurcation angle of the upper layer of the microfluidic chip is 45-60°, and the sub-channel width decreasing ratio is 1:(0.8-0.9):(0.6-0.8);

[0027] The inner wall of the channel was briefly activated with 3-aminopropyltriethoxysilane (APTES) and then coated with streptavidin with a coating amount of ≥1µg·cm -2 ;

[0028] The chip was sterilized with 75% ethanol and stored dry at 2-8°C.

[0029] The present invention also provides a method for preparing the microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, comprising:

[0030] S1. Synthesize or express the antibody variable regions of SEQ ID NOs: 1-6 and perform recombinant expression of IgG1 and Igκ and Protein A purification;

[0031] S2, biotinylating the antibody and the aptamer of SEQ ID NO: 7 using NHS-biotin;

[0032] S3, mixing mouse anti-human EpCAM monoclonal antibody, mouse anti-human Vimentin monoclonal antibody and dual-site anti-PSMA capture combination according to the ratio to form a biotin-modified cell capture agent;

[0033] S4, plasma-treating the PDMS-glass microfluidic chip and injecting streptavidin to form a fixed layer;

[0034] S5, injecting the capture agent into the chip channel and coupling it with streptavidin to complete immune activation;

[0035] S6. Prepare the competitive PSMA antibody peptide, the pretreatment agent, separation solution, fixative, permeabilization agent, 5% BSA blocking solution and multiple fluorescence detection composition;

[0036] S7. The chip and all liquid components are packaged, vacuum-filled with nitrogen, and encapsulated in sequence to complete the finished test kit.

[0037] In a preferred technical solution, in step S3, the final protein concentration of the capture agent is 0.5-1.0 mg / mL, and the final concentration of the aptamer is 100-200 nM.

[0038] In a preferred technical solution, the streptavidin coating concentration is 10 μg / mL, and the incubation is at 4° C. for 1 hour.

[0039] Beneficial effects

[0040] The present invention constructs a dual-target immune capture strategy, combining EpCAM and Vimentin antibodies, effectively taking into account the heterogeneity of epithelial and mesenchymal circulating tumor cells (CTCs) to achieve efficient enrichment; the present invention further introduces antibodies and aptamers targeting prostate-specific membrane antigen (PSMA), which can specifically identify CTCs derived from prostate cancer, significantly reducing the false positive rate while improving detection sensitivity.

[0041] The present invention also blocks the PSMA homologous sites on the leukocyte surface by setting competitive short peptides, effectively avoiding non-specific binding of non-tumor cells and improving background interference; the synergistic application of multiple fluorescent staining and signal amplification system improves the imaging contrast and judgment accuracy of positive cells.

[0042] The overall technical approach of the present invention takes into account capture efficiency, specificity and throughput, and is suitable for early screening, efficacy monitoring and prognosis evaluation of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the method flow of embodiment 1 of the present invention;

[0044] Figure 2 Schematic diagram of comparative experimental results (total number of CTCs after enrichment) of the present invention;

[0045] Figure 3 The comparative experimental results of the present invention (PSMA + CTC ratio) schematic diagram;

[0046] Figure 4 Schematic diagram of comparative experimental results (leukocyte residual amount) of the present invention;

[0047] Figure 5 Schematic diagram of comparative experimental results (leukocyte removal rate) of the present invention;

[0048] Figure 6 Schematic diagram of the comparative experimental results (false positive rate) of the present invention. DETAILED DESCRIPTION

[0049] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0050] Example 1 (T1)

[0051] This example provides a specific configuration and component parameters of a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, which is suitable for enriching CTCs (circulating tumor cells) in blood samples of prostate cancer patients and specifically identifying PSMA-positive cells. Figure 1 Shown, including:

[0052] S1. Capture agent configuration:

[0053] The cell capture agent is prepared by three types of biotinylated molecules in a molar ratio of 3:1:1, namely:

[0054] Mouse anti-human EpCAM monoclonal antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2;

[0055] Mouse anti-human Vimentin monoclonal antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 4;

[0056] PSMA Dual-Site Capture Panel:

[0057] The biotin-modified mouse anti-human PSMA monoclonal antibody has a heavy chain variable region amino acid sequence of SEQ ID NO: 5 and a light chain variable region amino acid sequence of SEQ ID NO: 6; the biotin-modified 30 nt aptamer has a nucleic acid sequence of SEQ ID NO: 7.

[0058] After NHS-biotin modification, each antibody exhibited a binding affinity of 5-6 mol / mol as determined by the HABA-Avidin colorimetric assay, while the number of aptamer modification sites was 3-4 mol / mol. The final protein concentration after reconstitution was 0.8 mg / mL, and the final aptamer concentration was 150 nM. The buffer system was PBS, pH 7.2 ± 0.1, and the samples were stored at 4°C in the dark.

[0059] S2. Competitive blocker configuration:

[0060] A PSMA short peptide was used as an immune-specific blocking agent. Its sequence is SEQ ID NO: 8, which contains 14 amino acid residues. Its affinity constant for the PSMA homologous site on human leukocytes was determined. The peptide is less than 100 nM, while its binding affinity for the PSMA protein on prostate cancer cells exceeds 5 µM. Prior to enrichment, the peptide was added to the sample at a concentration of 0.3 µM and incubated for 2 minutes to block nonspecific binding sites on leukocytes and reduce false positives.

[0061] S3. Microfluidic chip structure:

[0062] The chip is constructed of PDMS-glass and coated with 10 µg / mL streptavidin after oxygen plasma treatment. The coating is then incubated at 4°C for 1 hour. The upper channel of the chip features an asymmetric bifurcation structure with a 50° branch angle. The channel widths decrease in sequence to 400 µm, 320 µm, and 240 µm, with a depth of 50 µm. The inner wall of the chip is activated with APTES. The chip is sterilized with 75% ethanol and stored desiccated at 2-8°C.

[0063] S4. Multiple fluorescence detection composition:

[0064] The enriched cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100, and blocked with 5% BSA, and then labeled with the following fluorescent dyes:

[0065] FITC-anti-CK antibody, AlexaFluor647-anti-Vimentin antibody, PE-anti-CD45 antibody; DAPI nuclear staining.

[0066] PSMA-specific recognition is achieved through a dual-site signal amplification system: the first detection antibody is coupled to oligonucleotide P1 (6-FAM labeled), and the second detection antibody is coupled to oligonucleotide P2 (Quasar670 labeled). The two sequences have a complementary region of 15nt and are connected to the C-terminus of the antibody. When binding to the target PSMA protein, the distance between the two probes is less than 35 nm, FRET occurs, and the fluorescence shift from green to near-red is achieved, which constitutes the PSMA positive judgment standard.

[0067] S5. Preparation of functional liquid:

[0068] Separation medium: PBS buffer (20 mM, pH 7.4) supplemented with 0.8% BSA, 1 mM EDTA, and 50 µM ZnCl2, double-sterilized with a 0.22 µm PES membrane, and stored at 4°C;

[0069] Pretreatment agent: contains ammonium chloride, sodium bicarbonate and EDTA, pH 7.3;

[0070] Fixative: 4% PFA;

[0071] Permeabilization solution: 0.1% TritonX-100;

[0072] Blocking buffer: 5% BSA.

[0073] S6. Enrichment process:

[0074] 1 mL of patient peripheral blood sample was pre-treated and injected into the chip. It was flowed under the action of the circulating cell separator for 30 minutes. After the cells were bound, elution, fixation, permeabilization and fluorescence staining were completed. Finally, CK was identified under a fluorescence microscope. + / CD45 - The cells were CTCs, and PSMA-FRET positive cells were further screened and counted as prostate-specific CTCs.

[0075] Example 2 (T2)

[0076] This example provides an improved microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit. Compared with Example 1, its main differences are: the proportion of PSMA-related molecules in the cell capture agent is increased, the aptamer and antibody are modified with different biotinylation methods, the blocker concentration is optimized, and the complementary length of the oligonucleotide probes and the fluorescent pair combination are adjusted in the signal amplification pair design.

[0077] S1. Capture agent configuration:

[0078] The following four biotinylated molecules were mixed in a molar ratio of 2:1:2:1:

[0079] Mouse anti-human EpCAM monoclonal antibody (heavy chain variable region SEQ ID NO: 1, light chain variable region SEQ ID NO: 2), modified with NHS-biotin, with a binding affinity of 6 mol / mol;

[0080] Mouse anti-human Vimentin monoclonal antibody (heavy chain variable region SEQ ID NO: 3, light chain variable region SEQ ID NO: 4), NHS-biotin modified, binding affinity 5 mol / mol;

[0081] Mouse anti-human PSMA monoclonal antibody (heavy chain variable region SEQ ID NO: 5, light chain variable region SEQ ID NO: 6), terminally biotinylated using click chemistry, with a binding affinity of approximately 3 mol / mol;

[0082] The 30nt PSMA aptamer (SEQ ID NO:7) was modified with terminal Azide and coupled to DBCO-PEG4-Biotin with an average binding affinity of 4 mol / mol.

[0083] The total protein concentration after reconstitution was controlled at 0.6 mg / mL, the aptamer concentration was 200 nM, and it was prepared in PBS buffer (pH 7.2). The storage temperature was 4°C and kept away from light.

[0084] S2. Competitive blocker setting:

[0085] The short peptide sequence remains SEQ ID NO: 8, but the incubation conditions are adjusted:

[0086] Increasing its concentration to 0.5 µM and shortening the incubation time to 1 minute allowed for more rapid saturation of nonspecific binding sites on leukocytes, thereby compressing the overall treatment cycle.

[0087] S3. Microfluidic chip processing and structure:

[0088] The chip has a two-layer PDMS structure, with the bottom layer using straight-through channels instead of herringbone structures.

[0089] The upper layer is designed as an oblique converging channel with an angle of 55° and a consistent inner diameter of 300 µm;

[0090] The inner wall of the channel was modified with 3-aminopropyltriethoxysilane (APTES) and then coated with 10 μg / mL streptavidin and incubated at 4°C for 90 min to form a stable binding layer;

[0091] After the coupling is completed, the plate is washed with sterile PBS and stored dry at 2-8°C.

[0092] S4. Multiplex fluorescence detection composition and signal amplification:

[0093] This embodiment uses an optimized nucleic acid probe system in the PSMA recognition part:

[0094] The P1 oligonucleotide was fluorescently labeled with 6-FAM;

[0095] The P2 oligonucleotide was fluorescently labeled with Cy5;

[0096] The complementary regions of the two were adjusted to 18 nt to extend the hybridization stability, and P1 and P2 were coupled to the C-termini of different PSMA detection antibodies.

[0097] This design can generate a stable signal even when the PSMA protein aggregation density is low, thereby increasing the detection probability of weakly positive samples and reducing false negatives.

[0098] The fluorescence detection combination is as follows: CK antibody (AlexaFluor488); Vimentin antibody (TexasRed); CD45 antibody (AlexaFluor750); DAPI nuclear staining; PSMA double antibody site combination + FRET fluorescence pair (FAM-Cy5) for target-specific enhanced recognition.

[0099] S5. Functional liquid preparation:

[0100] The separation solution used a HEPES buffer system (20 mM HEPES, pH 7.4) instead of PBS, and the ratio still contained 0.8% BSA, 1 mM EDTA and 50 µM ZnCl2;

[0101] The concentration of the fixative was adjusted to 2% PFA to reduce structural damage;

[0102] The blocking solution was 10% BSA to enhance the signal-to-noise ratio in samples with high background.

[0103] S6. Testing process:

[0104] Take 1 mL of patient blood sample, treat it with red blood cell lysis buffer, resuspend the cells in pre-cooled separation buffer and add blocking peptide, incubate for 1 minute;

[0105] Then, the solution was injected into the microfluidic chip and concentrated for 20 minutes at a flow rate of 200 μL / min under the action of a circulating separator.

[0106] After elution, fixation, permeabilization, blocking and fluorescence staining were performed in sequence, and finally images were collected by multi-channel fluorescence microscopy. + / CD45 - The cell population was then secondary confirmed by PSMA-FRET signal.

[0107] Comparative Example 1 (C1)

[0108] This comparative example is intended to simulate a detection scheme lacking key innovative features. Comparative Example 1 uses the following components and preparation steps to construct a microfluidic chip PSMA prostate cancer circulating tumor cell detection combination:

[0109] S1. Obtain the following antibodies:

[0110] Biotin-modified mouse anti-human EpCAM monoclonal antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the light chain is SEQ ID NO: 2;

[0111] Biotin-modified mouse anti-human PSMA monoclonal antibody, the heavy chain variable region is SEQ ID NO: 5, and the light chain is SEQ ID NO: 6.

[0112] S2. Mix the above two antibodies in a molar ratio of 2:1 to form a capture agent complex solution; set the final protein concentration to 0.8 mg / mL, adjust the pH to 7.2, and store at 4°C in a dark environment until used.

[0113] S3. Use NHS-biotin reagent to complete the biotinylation reaction of the antibody, and control the degree of biotin modification to 5-6 residues per antibody molecule.

[0114] S4. Select a PDMS-glass double-layer microfluidic chip, activate it with standard plasma, inject 10µg / mL streptavidin solution into the bottom layer, incubate at 4°C for 1 hour, and then wash and dry; then inject the capture agent solution and let it stand for 30 minutes to achieve antibody fixation.

[0115] S5. The chip's upper structure uses a conventional herringbone channel (channel width of 200µm, depth of 80µm) without a gradient bifurcation design; no surface activation modification is performed, and no aptamers or other signal amplification mechanisms are superimposed.

[0116] S6. 5 mL of peripheral blood sample was collected from a prostate cancer patient, treated with red blood cell lysis buffer, and separated using a chip. The separation buffer was PBS (20 mM, pH 7.4), which contained only 0.5% BSA as a nonspecific blocking component, without the addition of EDTA or Zn 2+ Plasma stabilizing component.

[0117] S7. Cells were fixed (4% paraformaldehyde), permeabilized (0.2% Triton X-100), and blocked (5% BSA) in sequence, followed by triple staining: FITC-anti-CK antibody, PE-anti-CD45 antibody, and DAPI nuclear staining; no vimentin antibody channel was set.

[0118] Comparative Example 2 (C2)

[0119] In this comparative example 2, a kit model lacking a dual-site PSMA recognition system was constructed to verify the key role of the dual-site recognition and signal amplification strategies in Examples 1 and 2 of the present invention in improving PSMA recognition efficiency and reducing false negatives.

[0120] S1. Select three types of biotinylated molecules for chip capture:

[0121] Mouse anti-human EpCAM antibody, heavy chain variable region is SEQ ID NO: 1, light chain is SEQ ID NO: 2;

[0122] Mouse anti-human Vimentin antibody, heavy chain variable region is SEQ ID NO: 3, light chain is SEQ ID NO: 4;

[0123] Mouse anti-human PSMA antibody, heavy chain variable region is SEQ ID NO: 5, light chain is SEQ ID NO: 6.

[0124] S2: The above antibodies were not introduced with PSMA aptamers, nor were dual-site capture structures constructed. Instead, only the PSMA antibody recognition mechanism was employed. The three biotinylated molecules were mixed at a 1:1:1 mass ratio, with a final concentration of 0.7 mg / mL and a pH of 7.4.

[0125] S3. Use NHS-biotin for modification to ensure that each antibody molecule is bound to approximately 4-5 biotin residues. The binding amount is confirmed by the HABA method.

[0126] The S4 microfluidic chip is a standard PDMS structure, lacking the upper herringbone channel design and the microvortex-inducing structure. The bottom layer of the chip is plasma-activated and then injected with streptavidin (10 µg / mL). The solution is incubated at 4°C for 30 minutes, followed by the injection of the capture agent for immobilization.

[0127] S5. The inner wall of the chip channel has not been treated with APTES or other surface affinity enhancement treatments, nor is a nucleic acid signal amplification probe system configured.

[0128] S6. After 5 mL of blood sample was treated with red blood cell lysis buffer, CTCs were enriched using the chip. During sample processing, the competing PSMA antibody peptide described in SEQ ID NO: 8 was not used, i.e., no pre-blocking of potential PSMA homologous structures on white blood cells in the blood was performed.

[0129] S7. During staining, FITC-anti-CK antibody, Alexa Fluor 647-anti-vimentin antibody, PE-anti-CD45 antibody, and DAPI were used. A dual-antibody signal amplification system for PSMA recognition was not established. Instead, a set of Cy5-labeled anti-PSMA antibodies was used, lacking dual-site confirmation and FRET mechanisms.

[0130] Comparative Example 3 (C3)

[0131] This comparative example 3 is used to verify the technical effect of the "streptavidin-biotin" directional coupling method. Although the types and ratios of capture molecules in the kit are consistent with those in Example 1 of the present invention, the capture molecules are fixed by chemical cross-linking adsorption instead.

[0132] The specific steps include:

[0133] S1. Prepare the non-biotinylated molecule mixture:

[0134] Mouse anti-human EpCAM antibody (SEQ ID NO: 1 / 2), mouse anti-human Vimentin antibody (SEQ ID NO: 3 / 4), mouse anti-human PSMA antibody (SEQ ID NO: 5 / 6), and 30nt PSMA aptamer (SEQ ID NO: 7) were mixed at a molar ratio of 2:1:1:1 to a final protein concentration of 0.8 mg / mL and an aptamer concentration of 150 nM in PBS buffer (pH 7.2). The mixture was stored at 4°C until use.

[0135] S2. Preparation of chemically cross-linked modified microfluidic chip:

[0136] A linear chip with a PDMS-glass structure (without a herring bone flow disturbance structure) was used. The inner wall of the chip was treated with oxygen plasma for 60 seconds, then injected with a 2.5% glutaraldehyde solution (PBS, pH 7.0), allowed to stand at 37°C for 10 minutes, and then thoroughly rinsed with deionized water.

[0137] S3. Non-biotinylated capture agent immobilization:

[0138] Inject the mixture from S1 into the chip channel and incubate at 37°C for 1 hour to form covalent bonds via the glutaraldehyde-amine reaction, thereby fixing the capture molecules in random orientations. Wash three times with PBS and store the chip moist.

[0139] S4. Leukocyte blocking treatment before enrichment:

[0140] 4 mL of blood from a prostate cancer patient was collected and treated with red blood cell lysis buffer. The competitive short peptide described in SEQ ID NO: 8 was added to a final concentration of 0.3 μM and incubated for 2 minutes to block the PSMA homologous sites on leukocytes.

[0141] S5. Enrichment and staining detection:

[0142] The sample was injected into the chip at a flow rate of 200 μL / min for 30 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 3 minutes, and blocked with 5% BSA for 15 minutes.

[0143] FITC-labeled anti-FITC-anti-CK antibody, AlexaFluor488-anti-Vimentin antibody, PE-anti-CD45 antibody and DAPI dye were added in sequence for fluorescent labeling; no PSMA dual-site P1 / P2 signal amplification probe was used, and only Cy5-labeled single-site anti-PSMA antibody was used.

[0144] Comparative experimental design

[0145] Experimental purpose: To verify the differences in detection performance of the microfluidic chip PSMA prostate cancer CTC immune enrichment detection kit under different protocols, and to highlight whether the present invention has significant advantages in CTC enrichment efficiency, prostate-specific identification, false positive control and biochemical compatibility.

[0146] Experimental samples: 5 mL of peripheral blood was collected from eight patients diagnosed with advanced prostate cancer. After treatment with red blood cell lysis buffer and leukocyte pre-blocking, the samples were divided into five groups and treated with Examples T1 and T2 and Comparative Examples C1, C2, and C3, respectively.

[0147] The experimental groups and treatment methods are shown in Table 1:

[0148] Table 1 Comparative experimental groups

[0149]

[0150] Experimental steps:

[0151] CTC enrichment treatment: Inject into the microfluidic chip according to the protocol for each group, maintaining the same flow rate (200 µL / min) and incubation time (30 minutes).

[0152] Fixation, permeabilization, and blocking: The same fixative (4% paraformaldehyde), permeabilization agent (0.1% Triton X-100), and 5% BSA blocking solution were used.

[0153] Multiplex fluorescence staining: FITC-anti-CK antibody, AlexaFluor647-anti-Vimentin antibody, PE-anti-CD45 antibody, DAPI, and PSMA dual-site probe (T1, T2) or single-site antibody (group C) were uniformly used.

[0154] Fluorescence microscopy detection and counting: CTCs in each chip group are identified and classified one by one, and the following indicators are recorded:

[0155] The number of epithelial CTCs (CK + / CD45 - );

[0156] The number of interstitial CTCs (Vimentin + / CD45 - );

[0157] The number of PSMA-positive CTCs (P1 / P2 probe signal or Cy5 labeling);

[0158] Background leukocyte residual count (CD45 + Total number of cells);

[0159] False positive rate (CD45 + / PSMA + non-CTC cell number).

[0160] Evaluation indicators:

[0161] CTC enrichment efficiency (cells / mL): total CTC count / sample volume;

[0162] PSMA positive CTC ratio (%): PSMA + Number of CTCs / total number of CTCs;

[0163] Leukocyte removal rate (%): initial CD45 + Quantity and enrichment of CD45 + Quantity ratio;

[0164] False positive rate (%): number of non-CTC cells stained by PSMA probe / total number of detected cells.

[0165] The following are comparative experimental data corresponding to Example 1 (T1), Example 2 (T2), and Comparative Example 1 (C1), Comparative Example 2 (C2), and Comparative Example 3 (C3), all from 8 clinical prostate cancer patient samples, with the average of each group calculated. The specific data are shown in Table 2:

[0166] Table 2 Comparative experimental data

[0167]

[0168] The results show that Figure 2 As shown, the average number of enriched CTCs in the T1 and T2 groups was 62.8 and 69.4 cells / mL, respectively, significantly superior to C1 (38.5 cells / mL), C2 (59.6 cells / mL), and C3 (31.2 cells / mL). The T2 group showed the best capture efficiency. This result can be attributed to the dual-site PSMA capture strategy and the synergistic effect of the EpCAM / Vimentin dual antibody used in this study, which effectively covers both epithelial and stromal CTCs, improving the overall capture capacity in a heterogeneous setting.

[0169] In terms of specific recognition, Figure 3 As shown in the figure, the proportion of PSMA-positive CTCs in the T2 group was as high as 89.2%, significantly higher than that in the other groups, especially in the C1 group, which was only 56.4%. This difference shows that the aptamer-antibody combination designed by the present invention not only improves the recognition sensitivity, but also significantly reduces the false positive signals through the spatial double confirmation mechanism. In addition, Figure 6 As shown in the figure, the T2 group had the lowest false positive rate, only 0.8%, which was much better than C1 (3.6%) and C2 (7.9%), further verifying the contribution of the signal self-assembly probe system to recognition accuracy.

[0170] In terms of leukocyte background control, e.g. Figure 4 and Figure 5 As shown, T1 and T2 achieved 99.6% and 99.7% leukocyte removal rates, respectively. However, due to the lack of competitive short peptide blocking, the C2 group achieved only 89.5%, with a significant increase in residual background signal. This indicates that the PSMA homology site blocking peptide represented by SEQ ID NO: 8 introduced in the present invention can effectively prevent nonspecific binding, improve subsequent detection sensitivity, and enhance background cleanup.

[0171] From the above analysis, it can be seen that Example 1 and Example 2 are significantly superior to the respective control examples in terms of capture efficiency, specific recognition, false positive control, and leukocyte exclusion. Among them, Example 2 has the best overall performance due to its more optimized capture ratio and signal amplification mechanism, which fully demonstrates the technical advantages of the biochemical combination strategy of the present invention in prostate cancer CTC detection.

[0172] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, characterized by: The following biochemical components are included: a) A biotin-modified cell capture agent comprising the following three types of biotinylated molecules, prepared at a molar ratio of (1-3):(1-2):1: Category 1: Mouse anti-human EpCAM monoclonal antibody, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2; Category II: Mouse anti-human Vimentin monoclonal antibody, the amino acid sequence of its heavy chain variable region is SEQ ID NO: 3, and the amino acid sequence of its light chain variable region is SEQ ID NO: 4; Category III: Dual-site anti-PSMA capture combination, which includes: A biotin-modified mouse anti-human PSMA monoclonal antibody, the heavy chain variable region amino acid sequence of SEQ ID NO: 5, the light chain variable region amino acid sequence of SEQ ID NO: 6, and A 30nt biotin-modified PSMA aptamer, the nucleic acid sequence of which is SEQ ID NO: 7; b) An immune-specific blocking agent, which is a short peptide of a competitive PSMA antibody, the sequence of which is SEQ ID NO: 8, is used to selectively saturate non-specific PSMA antibody binding sites on the leukocyte surface before enrichment.

2. The microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to claim 1, characterized in that: Also included are the following biochemical components: c) a microfluidic chip having a bottom layer immobilized with streptavidin and capable of forming a covalent outer layer with the biotinylated molecule; d) a multiplex fluorescence detection composition comprising: FITC-anti-CK antibody, AlexaFluor647-anti-Vimentin antibody, PE-anti-CD45 antibody, and DAPI; PSMA dual-site signal amplification pair: The first detection antibody is coupled to oligonucleotide P1, and the second detection antibody is coupled to oligonucleotide P2. P1 and P2 self-assemble at a distance of ≤40nm to form a fluorescent-nucleic acid probe, achieving PSMA-specific dual confirmation. The PSMA dual-site signal amplification pair consists of 6-FAM-labeled oligonucleotide P1 and Quasar670-labeled oligonucleotide P2. The complementary regions of P1 and P2 are 15nt and located at the C-termini of the two PSMA detection antibodies. When the target protein binds to both the P1-antibody and the P2-antibody simultaneously, the FRET distance does not exceed 35 nm, achieving a green to near-red signal conversion; e) Pretreatment agent, separation solution, fixative, permeabilization agent and 5% BSA blocking solution.

3. The microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to claim 1, characterized in that: The molar ratio of the mouse anti-human EpCAM monoclonal antibody, the mouse anti-human Vimentin monoclonal antibody, the biotin-modified mouse anti-human PSMA monoclonal antibody, and the biotin-modified 30nt PSMA aptamer is (2-3):1:0.5:0.5; When performing HABA-Avidin colorimetric quantification on the four biotinylated molecules mentioned above, the number of biotin residues bound to each antibody or aptamer molecule was controlled at 3-8 mol / mol, with an average of 5-6 for antibody molecules and 3-4 for aptamers; The total protein concentration after capture agent reconstitution is And pH7.2±0.

1.

4. The microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to claim 1, characterized in that: The competitive PSMA antibody short peptide SEQ ID NO: 8 is 14±2 amino acid residues, and its affinity constant The affinity for the PSMA homologous site on the surface of human leukocytes is less than 100nM, but the affinity for PSMA on tumor cells is greater than 5µM; the competitive PSMA antibody short peptide is incubated at a final concentration of 0.2-0.5µM for 1-3 minutes before enrichment.

5. The microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to claim 2, characterized in that: The separation solution is PBS, 20 mM, pH 7.2-7.6, containing 0.6%-1.0% (m / v) BSA, 1 mM EDTA and 50 μM ZnCl2; The separation solution was sterilized by filtration twice through a 0.22 μm PES membrane and stored at 4°C to avoid metal ion precipitation.

6. The microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to claim 5, characterized in that: The asymmetric bifurcation angle of the upper layer of the microfluidic chip is 45-60°, and the subchannel width decrease ratio is 1:(0.8-0.9):(0.6-0.8); The inner wall of the channel was briefly activated with 3-aminopropyltriethoxysilane and then coated with streptavidin. ; The chip was sterilized with 75% ethanol and stored dry at 2-8°C.

7. A method for preparing the microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit according to any one of claims 1 to 6, characterized in that: include: S1. Synthesize or express the antibody variable regions of SEQ ID NOs: 1-6 and perform recombinant expression of IgG1 and Igκ and Protein A purification; S2, biotinylating the antibody and the aptamer of SEQ ID NO: 7 using NHS-biotin; S3, mixing mouse anti-human EpCAM monoclonal antibody, mouse anti-human Vimentin monoclonal antibody and dual-site anti-PSMA capture combination at a molar ratio of (1-3):(1-2):1 to form a biotin-modified cell capture agent; S4, plasma-treating the PDMS-glass microfluidic chip and injecting streptavidin to form a fixed layer; S5, injecting the capture agent into the chip channel and coupling it with streptavidin to complete immune activation; S6. Prepare a competitive PSMA antibody peptide having the sequence of SEQ ID NO: 8; prepare a pretreatment agent, a separation solution, a fixative, a permeabilization agent, a 5% BSA blocking solution, and a multiplex fluorescence detection composition; S7. The chip and all liquid components are packaged, vacuum-filled with nitrogen, and encapsulated in sequence to complete the finished test kit.

8. The preparation method according to claim 7, characterized in that: In step S3, the final protein concentration of the capture agent was 0.5-1.0 mg / mL, and the final concentration of the aptamer was 100-200 nM.

9. The preparation method according to claim 8, characterized in that: The streptavidin coating concentration was 10 μg / mL, and the cells were incubated at 4° C. for 1 h.

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