A microfluidic nanopillar chip system for rare biological particle capture and release

Abstract

The present application relates to the technical field of in-vitro biological microfluidic analysis, in particular to a microfluidic nanocolumn chip system for rare biological particle capture and release, which comprises a substrate and a microfluidic channel bonded above the substrate, the surface of the substrate is provided with a nanocolumn array, and a comb-shaped herringbone mechanism is integrally formed on the top of the microfluidic channel; the surface of the nanocolumn is modified with a combined polypeptide capture reagent containing p-EpCAM polypeptide and CKAAKN polypeptide through a biotin-avidin system, which is used for specific recognition of circulating tumor cells; the system is also provided with Chymotrypsin polypeptide digestive enzyme, which is used for cutting the polypeptide capture reagent to achieve mild release of the captured cells. Through the synergistic effect of the nanocolumn array, the turbulent synergistic structure and the combined polypeptide capture reagent, efficient capture and active release of rare biological particles are realized, which can be applied to the capture, phenotype analysis and KRAS gene mutation detection of pancreatic cancer circulating tumor cells.

Description

Technical Field

[0001] This invention relates to the field of in vitro biological microfluidic analysis technology, specifically a microfluidic nanopillar chip system for capturing and releasing rare biological particles. Background Technology

[0002] Circulating tumor cells (CTCs), as rare biological particles in peripheral blood, carry complete protein expression characteristics and genomic information of the primary tumor. They are core biomarkers for early tumor screening, prediction of metastasis risk, and monitoring of the efficacy of targeted drugs. Traditional tissue biopsies have inherent technical shortcomings such as invasiveness and inability to perform multiple dynamic sampling and monitoring. Microfluidic chip technology based on peripheral blood CTC separation has become the mainstream research and development direction for liquid biopsies. Continuous industry efforts can realize a microfluidic separation device that integrates rare cell enrichment, in-situ characterization, and downstream molecular detection.

[0003] While existing technologies can achieve basic CTC separation and quantitative statistics, and microfluidic nanopillar structures can reduce cell damage caused by the mechanical shearing of magnetic beads compared to magnetic bead systems, the entire detection process has several unavoidable technical limitations due to multiple technical constraints, including the capture and recognition of molecular types, microchannel fluid enhancement structures, substrate biomodification chemical systems, and cell release mechanisms. It cannot simultaneously complete the entire chain of integrated operation, including efficient capture of rare CTCs, in situ typing of epithelial-mesenchymal transition subtypes, mild activity-preserving release, and single-cell gene mutation sequencing. This makes it difficult to meet the precise molecular detection technology requirements of solid tumors with high incidence of epithelial-mesenchymal transition, such as pancreatic cancer.

[0004] (1) Using only a single EpCAM-type recognition molecule cannot bind to metastatic CTCs that have undergone epithelial-mesenchymal transition, and it is easy to lose high-invasive potential tumor cell subpopulations. The overall capture specificity and capture efficiency have obvious lower limits. (2) The straight microchannel has no active turbulence generation structure. The probability of rare CTCs colliding with the nano-substrate under the laminar blood flow state is extremely low. It is not enough to enrich a small number of tumor cells in the billions of blood cells. (3) The binding stability of captured peptides under the conventional physical adsorption fixation mode is poor. They are easy to fall off prematurely during the sample washing stage. The acid-base dissociation release will destroy the cell membrane integrity and greatly reduce the biological activity of cells after release. It cannot support the amplification of the whole genome of single cells. (4) The existing detection process lacks a multi-antibody co-staining system that can simultaneously distinguish metastatic subtypes. It can only realize the cell number count and cannot predict the risk of tumor metastasis through protein phenotype. (5) After elution, the cells are mixed with a large number of white blood cell impurities. The cell purity is low, making it difficult to stably carry out the amplification and sequencing detection of the pancreatic cancer marker KRAS gene. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic nanopillar chip system for capturing and releasing rare biological particles, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a microfluidic nanopillar chip system for capturing and releasing rare biological particles, comprising a substrate and microfluidic channels bonded to the substrate;

[0007] At least a portion of the surface of the base is provided with a nanopillar array structure; the top of the microfluidic channel is integrally formed with a comb-shaped herringbone structure, which is used to generate turbulence perpendicular to the base surface when fluid flows through the channel;

[0008] The surface of the nanopillars is modified with a biotin-avidin system to form a combined peptide capture reagent, which contains two specific peptides capable of specifically recognizing target rare biological particles.

[0009] The chip system is also equipped with a polypeptide digestive enzyme for shearing and digesting the combined polypeptide capture reagent. The polypeptide digestive enzyme cuts the polypeptide chain, causing the captured rare biological particles to be released from the surface of the nanopillar.

[0010] Preferably, the rare biological particles are a suspension of circulating tumor cells; the combined polypeptide capture reagent contains two specific polypeptides, namely p-EpCAM polypeptide and CKAAKN polypeptide.

[0011] Preferably, the diameter of the nanopillars is 20nm-500nm; the width of the microfluidic channel is 1mm-2.5mm, the length is 20mm-45mm, and the total height of the channel is less than or equal to 100μm; the height of the comb-shaped herringbone is 10μm-30μm.

[0012] Preferably, the polypeptide digestive enzyme used in the configuration for cleaving and digesting the combined polypeptide capture reagent is chymotrypsin.

[0013] Preferably, the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system includes the following steps:

[0014] (a) Nanopillar chip modification and biofunctionalization steps: The surface of the nanopillar substrate is cleaned with a mixed solution of concentrated sulfuric acid / hydrogen peroxide, silanized for valence modification, and pretreated with biotin bonding; before use, the chip is rinsed with PBS buffer, and streptavidin solution and combined peptide capture reagent solution are passed through sequentially for incubation. After incubation, the chip is rinsed multiple times with the PBS buffer to stabilize the combined peptide capture reagent on the surface of the nanopillar.

[0015] (b) Cell capture procedure: Venous blood was collected from the patient using ACD anticoagulation blood collection tubes. After discarding a small amount of blood from the first tube to avoid false positives in epithelial cells, the remaining blood was diluted with an equal volume of PBS, centrifuged at gradient density, and the mononuclear cell layer was collected, washed, and resuspended to obtain the test sample. The test sample was then introduced into the chip system at a constant flow rate, and the circulating tumor cells were specifically captured on the surface of the nanopillars by the combined polypeptide capture reagent. After the sample was exhausted, it was rinsed with PBS buffer to remove free leukocyte impurities, and the captured cells were fixed with paraformaldehyde solution.

[0016] (c) Immunostaining and typing steps: After cell fixation, the chip system is incubated with a mixed staining solution containing three 5uM antibodies: anti-pancytokeratin, anti-CD45, and anti-Vimentin. The captured circulating tumor cells are then subjected to immunofluorescence staining to identify epithelial-mesenchymal transition circulating tumor cells with metastatic tendency.

[0017] (d) Cell release step: A release reagent containing polypeptide digestive enzyme is introduced into the chip system. The polypeptide digestive enzyme is brought into full contact with the polypeptide capture reagent at a constant flow rate. The polypeptide capture reagent is sheared and digested, so that the captured circulating tumor cells are detached and released from the surface of the nanopillar.

[0018] (e) Cell collection step: The chip is continuously flushed with the PBS buffer to collect the free circulating tumor cell suspension flowing out of the system.

[0019] Preferably, in the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system, the specific steps of silanization covalent modification and biotin bonding in step (a) are as follows:

[0020] S1. Immerse the cleaned and dried nanopillar chip in a toluene solution (concentration of 1.0%) of freshly prepared 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) and let it stand for 2 hours to complete the aminosilane bonding.

[0021] S2. Remove the bonded nanopillar chip, clean it with anhydrous ethanol, and then dry it with nitrogen.

[0022] S3. Place the dried nanopillar chip from step S2 into a material solution of NHS-PEGn-Biotin (n=1000) (0.05 mg / ml), let it stand for 2 hours to complete biotin modification, wash with anhydrous ethanol, dry with nitrogen and store at 4°C.

[0023] Before use, incubate with streptavidin solution (5.0 μM) for 1 hour; then incubate with the combination peptide solution for 1 hour. After each incubation, rinse thoroughly with PBS buffer.

[0024] Preferably, in the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system, the flow rate of the sample introduced in step (b) is in the range of 0.5 mL / h to 2.0 mL / h.

[0025] Preferably, in the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system, the releasing reagent in step (d) is a 0.5%-2.0% concentration of chymotrypsin aqueous solution; the antibody combination used in step (C) includes three antibodies: anti-pan cytokeratin, anti-CD45, and anti-Vimentin.

[0026] Preferably, the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system further includes a gene detection step after the cell collection step is completed: performing single-cell whole genome amplification on the collected circulating tumor cells, and specifically amplifying and sequencing the pancreatic cancer characteristic KRAS gene to detect KRAS gene mutation subtypes.

[0027] Preferably, the method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system is used to prepare in vitro liquid biopsy products for pancreatic cancer, and the products are used for early diagnosis of pancreatic cancer, prediction of tumor metastasis risk, and guidance for targeted drug therapy for pancreatic cancer.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention uses a combined peptide capture reagent containing p-EpCAM peptide and CKAAKN peptide to replace the single EpCAM recognition molecule, which can simultaneously recognize and capture circulating tumor cells of different phenotypes. This effectively solves the problem that single EpCAM recognition molecules cannot bind to metastatic CTCs that have undergone epithelial-mesenchymal transition and easily lost tumor cell subpopulations, and significantly improves capture specificity and capture efficiency.

[0030] 2. This invention uses a comb-shaped herringbone structure integrally formed at the top of the microfluidic channel to generate turbulence perpendicular to the substrate surface when the fluid flows through it, which increases the probability of collision and contact between rare biological particles and the surface of nanopillars, effectively solving the problem of extremely low cell contact probability caused by the lack of active turbulence generation structure in flat microfluidic channels.

[0031] 3. This invention employs a biotin-avidin system to covalently modify the surface of a combination of polypeptide capture reagents onto the nanopillar surface, and uses chymotrypsin to achieve gentle release of cells through enzymatic cleavage. This effectively solves the problems of poor binding stability of captured molecules and damage to cell membrane integrity caused by acid-base dissociation under conventional physical adsorption and fixation modes, and achieves the purification and collection of captured cells.

[0032] 4. This invention introduces a mixed staining system containing three antibodies—anti-pan cytokeratin, anti-CD45, and anti-Vimentin—into the immunochromatographic step, which can simultaneously distinguish between ordinary CTCs and metastatic CTCs that have undergone epithelial-mesenchymal transition after capture. This effectively solves the problem that existing detection procedures lack a multi-antibody co-staining system that can simultaneously distinguish metastatic subtypes and cannot predict the risk of tumor metastasis through protein phenotype.

[0033] 5. This invention integrates the steps of capture, staining, enzyme release and gene detection, and further conducts single-cell whole genome amplification and specific amplification and sequencing of the pancreatic cancer characteristic KRAS gene after cell collection. It realizes the whole-chain integrated operation from rare cell enrichment and in situ characterization to downstream molecular detection, which can meet the precise molecular detection needs of solid tumors with high incidence of epithelial-mesenchymal transformation such as pancreatic cancer. Attached Figure Description

[0034] Figure 1 This is a flowchart illustrating the steps of the microfluidic nanopillar chip system of the present invention for capturing and releasing rare biological particles.

[0035] Figure 2 This is a flowchart of the nanopillar chip modification and biofunctionalization method of the present invention;

[0036] Figure 3 This is a schematic diagram illustrating the three stages of CTC capture and release established in this invention;

[0037] Figure 4 This is a schematic diagram of the modification and biofunctionalization of the nanopillar chip of the present invention;

[0038] Figure 5 This is a graph showing the results of the optimized CTC capture conditions for pancreatic cancer in Example 1 of the present invention;

[0039] Figure 6 The figure shows the results of the optimized release conditions of pancreatic cancer CTCs after capture in Example 1 of the present invention.

[0040] Figure 7 This is a diagram showing the results of capturing and immunofluorescence staining of circulating tumor cells in a clinical patient in Example 2 of the present invention;

[0041] Figure 8 This is a sequencing result diagram of KRAS gene mutation detection in CTC of a clinical pancreatic cancer patient in Example 2 of the present invention. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0043] Please see Figure 1-8 This invention provides a microfluidic nanopillar chip system for capturing and releasing rare biological particles, comprising a substrate and a microfluidic channel bonded to the substrate; at least a portion of the substrate surface is provided with a nanopillar array structure; the top of the microfluidic channel is integrally formed with a comb-like herringbone structure, the herringbone structure being used to generate turbulence perpendicular to the substrate surface when fluid flows through the channel; the surface of the nanopillars is modified with a biotin-avidin system to form a combined peptide capture reagent, the combined peptide capture reagent containing two specific peptides capable of specifically recognizing target rare biological particles; the chip system is also equipped with a peptide digestive enzyme for shearing and digesting the combined peptide capture reagent, the peptide digestive enzyme cleaving the peptide chain, thereby releasing the captured rare biological particles from the surface of the nanopillars.

[0044] Furthermore, the rare biological particles are a suspension of circulating tumor cells; the combined polypeptide capture reagent contains two specific polypeptides, namely p-EpCAM polypeptide and CKAAKN polypeptide.

[0045] Furthermore, the diameter of the nanopillars is 20nm-500nm; the width of the microfluidic channels is 1mm-2.5mm, the length is 20mm-45mm, and the total height of the channels is less than or equal to 100μm; the height of the comb-shaped herringbone structure is 10μm-30μm.

[0046] Furthermore, the peptide digesting enzyme used to prepare the combined peptide capture reagent for shearing and digestion is chymotrypsin.

[0047] Furthermore, the method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system includes the following steps, detailed in reference [reference needed]. Figure 1 :

[0048] (a) Nanopillar chip modification and biofunctionalization steps: The nanopillar substrate surface was cleaned with a mixed solution of concentrated sulfuric acid / hydrogen peroxide, silanized for valence modification, and pretreated with biotin bonding; before use, the chip was rinsed with PBS buffer, and streptavidin solution and combined peptide capture reagent solution were passed through sequentially for incubation. After incubation, the chip was rinsed multiple times with PBS buffer to stabilize the combined peptide capture reagent on the nanopillar surface.

[0049] (b) Cell capture procedure: Venous blood was collected from the patient using ACD anticoagulation blood collection tubes. After discarding a small amount of blood from the first tube to avoid false positives in epithelial cells, the remaining blood was diluted with an equal volume of PBS, centrifuged at gradient density, and the mononuclear cell layer was collected, washed, and resuspended to obtain the test sample. The test sample was then introduced into the chip system at a constant flow rate. Circulating tumor cells were specifically captured on the surface of the nanopillars by the combined polypeptide capture reagent. After the sample was exhausted, PBS buffer was introduced to wash away free leukocyte impurities, and paraformaldehyde solution was introduced to fix the captured cells.

[0050] (c) Immunostaining and typing steps: After cell fixation, the chip system is incubated with a mixed staining solution containing three 5uM antibodies: anti-pancytokeratin, anti-CD45, and anti-Vimentin. The captured circulating tumor cells are then subjected to immunofluorescence staining to identify epithelial-mesenchymal transition circulating tumor cells with metastatic tendency.

[0051] (d) Cell release step: A release reagent containing peptide digestive enzyme is introduced into the chip system. The peptide digestive enzyme is brought into full contact with the peptide capture reagent at a constant flow rate. The peptide capture reagent is sheared and digested, so that the captured circulating tumor cells are detached and released from the surface of the nanopillar.

[0052] (e) Cell collection procedure: PBS buffer is continuously circulated to rinse the chip and collect the free circulating tumor cell suspension flowing out of the system.

[0053] Furthermore, regarding the method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system, the specific operational steps of silanization covalent modification and biotin bonding in step (a) are as follows, please refer to [link / reference needed]. Figure 2 :

[0054] S1. Immerse the cleaned and dried nanopillar chip in a toluene solution (concentration of 1.0%) of freshly prepared 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) and let it stand for 2 hours to complete the aminosilane bonding.

[0055] S2. Remove the bonded nanopillar chip, clean it with anhydrous ethanol, and then dry it with nitrogen.

[0056] S3. Place the dried nanopillar chip from step S2 into a material solution of NHS-PEGn-Biotin (n=1000) (0.05 mg / ml), let it stand for 2 hours to complete biotin modification, wash with anhydrous ethanol, dry with nitrogen and store at 4°C.

[0057] Before use, incubate with streptavidin solution (5.0 μM) for 1 hour; then incubate with the combination peptide solution for 1 hour. After each incubation, rinse thoroughly with PBS buffer.

[0058] Furthermore, in the method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system, the flow rate of the sample introduced in step (b) is in the range of 0.5 mL / h to 2.0 mL / h.

[0059] Furthermore, in the method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system, the releasing reagent in step (d) is a 0.5%-2.0% concentration of Chymotrypsin aqueous solution; the antibody combination used in step (C) includes three antibodies: anti-pancytokeratin, anti-CD45, and anti-Vimentin.

[0060] Furthermore, the method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system includes a gene detection step after the cell collection step: single-cell whole genome amplification is performed on the collected circulating tumor cells, and the pancreatic cancer characteristic KRAS gene is specifically amplified and sequenced to detect KRAS gene mutation subtypes.

[0061] Furthermore, a method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system is developed. This microfluidic nanopillar chip system is used to prepare in vitro liquid biopsy products for pancreatic cancer. These products are used for early diagnosis of pancreatic cancer, prediction of tumor metastasis risk, and guidance for targeted drug therapy in pancreatic cancer.

[0062] Example 1

[0063] 1. Fabrication of nanopillar chips

[0064] First, a nanopillar array structure with a diameter of 20nm-500nm is etched on the substrate surface using standard semiconductor photolithography. After etching, the nanopillar chip modification and biofunctionalization steps were followed: the nanopillar surface was further cleaned with a concentrated sulfuric acid / hydrogen peroxide mixture (70:30) and washed with deionized water, followed by further cleaning with anhydrous ethanol, and then dried under N2 gas in an ultra-clean environment; a 1.0% toluene solution of 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) was freshly prepared, and the cleaned and dried nanopillar-modified chip was immersed in it and left to stand for 2 hours; the APTES-bonded chip was removed, cleaned with anhydrous ethanol, and then dried under N2 gas; 200 ml of a freshly prepared NHS-PEGn-Biotin (n=1000) material solution (0.05 mg / ml) was placed in it, and left to stand for another 2 hours. Finally, after cleaning with anhydrous ethanol, drying under N2 gas, and storing in a dry environment at 4°C for later use.

[0065] 2. Fabrication of microfluidic chips

[0066] The microfluidic channel system was fabricated using standard semiconductor photolithography techniques. The specific steps included: designing and printing photomasks for both the microfluidic channel structure and the herringbone structure; then spin-coating SU-8 photoresist to a thickness of 70 micrometers onto a silicon wafer; imagering the channel structure region using the fluid channel photomask to obtain the channel structure; subsequently, spin-coating a 100-micrometer-thick layer of SU-8 photoresist onto the same silicon substrate. After drying, using the herringbone structure photomask, the channel region was precisely aligned, irradiated with ultraviolet light, and developed to obtain the herringbone structure built onto the channel structure; after cleaning and drying the microfluidic channel photoresist film, trichloro(1H,1H,2H,2H-perfluorooctyl)silane was vapor-deposited under reduced pressure and stored in a dust-free environment; finally, Dow Corning Sylgard 184 was used, with a 10:1 ratio of 184 silicone elastomer base to Sylgard 184 silicone elastomer curing agent. The overall thickness of the PDMS microfluidic channel system was 4 mm-5 mm. After curing, the chips are cut to the size suitable for nanostructure chips, and holes are drilled, cleaned, and sterilized at the inlet and outlet positions before use.

[0067] In this embodiment, before testing biological samples, the chip surface was cleaned with phosphate-buffered saline (PBS), and then 200 μL of streptavidin solution (5.0 μM) was added and incubated for 1 hour. After cleaning with PBS, it was incubated with 200 μL of PBS containing p-EpCAM and CKAAKN peptides for another hour. After cleaning, it was used for CTC capture. For details, please refer to [link to relevant documentation]. Figure 2 .

[0068] 3. System optimization and testing using pancreatic cancer cells as an analogy sample

[0069] (1) Preparation of simulated samples: From the culture dish of human pancreatic cancer cell line BxPC3 with a confluency of about 95%, a cell suspension with a density of 105 cells / mL was prepared by digestion with trypsin, and 20 μL of cell solution was diluted to prepare a sample for use. 180 μL of suspension containing 2×10^6 white blood cells was added (all cells were diluted and prepared with buffered phosphate solution (PBS)) to prepare 200 μL of analog sample;

[0070] (2) Chip Assembly and Cell Capture: The biofunctionalized nanopillar chip was assembled with the PDMS microfluidic channel layer. After connecting the inlet and outlet fluid delivery tubes, 200 μL of analog sample was injected into the microfluidic chip system at an optimal flow rate of 1.0 mL / h, allowing the analog sample to flow through the nanopillar base. Circulating tumor cells were specifically recognized by the combinatorial peptide capture reagent and captured on the nanopillar surface. After the sample flow was complete, 200 μL of PBS buffer was continuously injected for washing to remove unbound free leukocytes and other impurities. Subsequently, 200 μL of 2%-4% paraformaldehyde (PFA) solution was injected and flowed through the chip assembly at a flow rate not exceeding 1.0 mL / h to fix the captured cells.

[0071] (3) Determination of capture efficiency: After cell fixation, the chip assembly was disassembled, the nanopillar substrate slide was removed, and after washing with buffered phosphate solution (PBS), 200 μL of a mixed solution containing 5 μM anti-pan cytokeratin antibody (PanCK) and 5 μM CD45 antibody (anti-CD45) was applied to the nanopillar region. The solution was incubated at 4°C for 24 hours for immunofluorescence staining. After washing, secondary antibody staining was performed according to standard methods. The CK antibody was labeled with FITC, and the CD45 antibody with TRITC. The secondary antibody staining time was 30 minutes at room temperature (25°C). After washing with buffered phosphate solution (PBS), the surface solution residue was removed, and the sample was sealed with 80 μL of fluorescent protective solution containing Hoechst nuclear staining reagent. After covering with a 0.17 mm coverslip and sealing, fluorescence imaging and counting were performed using a fluorescence microscope. The number of CK+ / CD45- / DAPI+ cells in the imaging results was taken as the captured cancer cells. The capture efficiency was calculated by comparing it with the initial number of cancer cells (2000 cells).

[0072] (4) Experiment on optimization of capture conditions: refer to the following for details. Figure 5 ;

[0073] a. Comparison of capture efficiency between combinatorial peptides and single peptides: Capture experiments were conducted on BxPC3 cell analog samples using nanopillar chips modified with single p-EpCAM peptides, single CKAAKN peptides, and a combinatorial peptide mixture of p-EpCAM and CKAAKN, respectively, following the method described above. The results showed that the capture efficiency of the combinatorial peptides was significantly higher than that of any single peptide.

[0074] b. Determination of optimal flow rate: Using BxPC3 cell analog samples as the target, capture experiments were conducted at flow rates of 0.5 mL / h, 0.8 mL / h, 1.0 mL / h, 1.2 mL / h, 1.5 mL / h, and 2.0 mL / h, respectively. The results showed that the capture efficiency was highest at a flow rate of 1.0 mL / h.

[0075] c. Capture effect on different pancreatic cancer cell lines: Analog samples were prepared using three pancreatic cancer cell lines, BxPC3, Panc-1, and AsPC-1, respectively. Capture experiments were conducted under the optimal conditions described above, with healthy human leukocytes used as a negative control. The results showed that all three types of pancreatic cancer cells could be effectively captured, with a capture efficiency significantly higher than that against the leukocyte background, indicating that the system has good capture ability for multiple pancreatic cancer cell lines.

[0076] d. Comparison of different substrate structures and modification conditions: Four different chips were prepared using planar substrates (without nanopillars) and nanopillar substrates, with no peptide modification and p-EpCAM+CKAAKN combined peptide modification as the conditions. Capture experiments on three types of pancreatic cancer cells were conducted using the methods described above. The results showed that the chip with nanopillar substrate + combined peptide modification had the highest capture efficiency, significantly better than the planar substrate or modified chip.

[0077] (5) Optimization of CTC release conditions after capture in pancreatic cancer, please refer to the following for details. Figure 6 ;

[0078] a. Screening of release reagents: After preparing BxPC3 cell analog samples and completing CTC capture, nine different peptide cleavage / digestion reagents were prepared. The CTC release effect was tested using the same 200 μL solution (0.25% concentration) and the same rinsing rate of 1.0 mL / h. The effluent was collected, and the cells remaining on the nanopillar substrate were subjected to immunofluorescence staining. The release efficiency was calculated using the following formula: Release efficiency = Number of released cells / (Number of released cells + Number of remaining cells) x 100%. The results showed that chymotrypsin and trypsin exhibited superior release effects compared to the other seven reagents.

[0079] b. Optimization of enzyme concentration and release conditions: Hydroxymotrypsin and trypsin aqueous solutions with concentrations of 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, and 2.0% were prepared and introduced into the pre-captured cell array system at a flow rate of 1.0 mL / h. The release efficiency was calculated according to method (a) above. The results showed that the release efficiency of both chymotrypsin and trypsin increased with increasing concentration. Further AO / EB dual-fluorescence staining was used to determine the cell viability after release of different concentrations of chymotrypsin. The optimal balance between release efficiency and cell viability was observed when the chymotrypsin concentration was 0.5%–1.0%. At a concentration of 1.0%, the optimal balance between release efficiency and cell viability was achieved, while cell viability decreased significantly above 1.5%.

[0080] c. Comparison of cell activity and purity before and after release

[0081] AO / EB staining was used to stain the samples after capture (on-chip).

[0082] Furthermore, cell viability was measured by comparing cells collected after capture (on-chip) and after release (off-chip). Simultaneously, the purity change of CTCs relative to leukocyte impurities in the samples before and after release was compared using fluorescence staining and counting. The results showed no significant difference in cell viability between the released and captured cells, indicating that the enzymatic release method has minimal impact on cell viability. Meanwhile, the purity of the CTC samples collected after release was significantly higher than that after capture, indicating that enzymatic release effectively removes residual leukocytes and other impurities while releasing CTCs, achieving further purification of the CTCs.

[0083] Example 2

[0084] (1) For the capture and phenotypic analysis of CTCs in clinical samples, please refer to [reference needed]. Figure 7

[0085] a. Collection and Pretreatment of Clinical Samples: Blood collection for clinical samples requires a BD Vacutainer Glass ACDSolution A tube (8.5 ml) to avoid EDTA anticoagulation damage to cell surface antigens in biological samples. The first 2 ml of blood drawn is discarded to avoid false positives caused by epithelial cells sloughed off during puncture. Taking 4 mL of venous blood as an example, preliminary purification is performed by gradient density centrifugation: 4 mL of venous blood is diluted with an equal volume of 4 mL of PBS buffer, and then slowly added to the upper layer of a 15 mL centrifuge tube containing 4 mL of gradient density centrifugation buffer. Centrifuge at 400 xg for 30 minutes. After centrifugation, remove the upper serum layer and collect approximately 200 μL of peripheral blood mononuclear cell (PBMC) layer. Wash with PBS buffer, centrifuge at 400 xg for 5 minutes, remove the supernatant, wash with 2 mL of PBS buffer, and then bring the volume to 200 μL for later use.

[0086] b. CTC Capture and Triple Immunofluorescence Staining for Typing: 200 μL of pretreated clinical sample was introduced into a biofunctionalized microfluidic nanopillar chip system under the optimal conditions (1.0 mL / h flow rate) determined in the first embodiment, allowing CTCs to be specifically captured on the nanopillar surface by a combination of peptide capture reagents. After the sample was exhausted, it was rinsed with PBS buffer to remove free leukocytes and other impurities, and then the captured cells were fixed with 2%–4% paraformaldehyde solution. After cell fixation, 200 μL of a mixed antibody solution containing 5 μM anti-pan cytokeratin (PanCK, FITC-labeled, green fluorescence), 5 μM anti-CD45 (TRITC-labeled, red fluorescence), and 5 μM anti-Vimentin (Vimentin, using another label compatible with the aforementioned fluorescent labeling) was placed over the nanopillar region and incubated at 4°C for 24 hours. Subsequently, the sample was washed three times with PBS and stained with the corresponding secondary antibody for 30 minutes at room temperature. After PBS washing, the slide was mounted with a fluorescent protective solution containing Hoechst nuclear staining reagent for fluorescence microscopy imaging.

[0087] c. Phenotypic Analysis Results: In fluorescence imaging, PanCK+ / CD45- / DAPI+ cells were identified as circulating tumor cells (CTCs). Based on this, Vimentin+ / PanCK+ / CD45- / DAPI+ cells were further identified as a subtype of CTCs with a metastatic tendency exhibiting the epithelial-mesenchymal transition (EMT) phenotype. The results showed that the system could stably capture CTCs from peripheral blood samples of pancreatic cancer patients. Based on counting, it could simultaneously detect CTC subpopulations expressing the EMT marker Vimentin, identifying tumor cells with high metastatic potential and providing protein phenotypic evidence for clinical prediction of tumor metastasis risk.

[0088] (2) For details on the capture, release, and gene mutation detection of CTCs in clinical samples, please refer to [reference needed]. Figure 8

[0089] a. CTC Release and Collection from Clinical Samples: Following the method described in Part I of this embodiment, peripheral blood samples from 7 clinical samples (6 pancreatic cancer patients and 1 healthy control) were pretreated and CTCs were captured. After capture, under the optimal conditions determined in Part II of this embodiment, 1.0% Chymotrypsin aqueous solution (1.0 mL / h flow rate) was passed into the chip system for enzymatic release, and the CTC suspension flowing out of the system was collected. After neutralization and washing with PBS buffer, the CTC cell pellet was obtained by centrifugation.

[0090] b. Single-cell whole-genome amplification and KRAS gene sequencing: The collected CTC cell pellet was subjected to single-cell whole-genome amplification using a WGA (whole-cell genome amplification) kit, following the kit instructions to obtain sufficient genomic DNA for subsequent PCR amplification. Specific primers were designed targeting the 12th and 13th codon regions of the KRAS gene (GenBank accession number: NM_004985.5), a characteristic gene of pancreatic cancer, for PCR amplification of the WGA product. After purification, the PCR product was sequenced using the Sanger dideoxy chain termination method, and the sequencing results were compared with the KRAS wild-type sequence.

[0091] c. Comparative experimental results: Sequencing results showed that among the 6 pancreatic cancer patient samples, 5 had KRAS gene mutation subtypes (including common mutation types such as G12D and G12V), and 1 was KRAS wild-type, while no KRAS gene mutation was detected in the healthy control samples.

[0092] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A microfluidic nanopillar chip system for capturing and releasing rare biological particles, characterized in that, Includes a substrate and microfluidic channels bonded to the substrate; At least a portion of the surface of the base is provided with a nanopillar array structure; the top of the microfluidic channel is integrally formed with a comb-shaped herringbone structure, which is used to generate turbulence perpendicular to the base surface when fluid flows through the channel; The surface of the nanopillars is modified with a biotin-avidin system to form a combined peptide capture reagent, which contains two specific peptides capable of specifically recognizing target rare biological particles. The chip system is also equipped with a polypeptide digestive enzyme for shearing and digesting the combined polypeptide capture reagent. The polypeptide digestive enzyme cuts the polypeptide chain, causing the captured rare biological particles to be released from the surface of the nanopillar.

2. The microfluidic nanopillar chip system for capturing and releasing rare biological particles according to claim 1, characterized in that, The rare biological particles are a suspension of circulating tumor cells; the combined polypeptide capture reagent contains two specific polypeptides, namely p-EpCAM polypeptide and CKAAKN polypeptide.

3. The microfluidic nanopillar chip system for capturing and releasing rare biological particles according to claim 1, characterized in that, The diameter of the nanopillars is 20nm-500nm; the width of the microfluidic channel is 1mm-2.5mm, the length is 20mm-45mm, and the total height of the channel is less than or equal to 100μm; the height of the comb-shaped herringbone is 10μm-30μm.

4. The microfluidic nanopillar chip system for capturing and releasing rare biological particles according to claim 1, characterized in that, The peptide digesting enzyme used in the configuration for cleaving and digesting the combined peptide capture reagent is chymotrypsin.

5. A method for capturing and releasing rare biological particles using the microfluidic nanopillar chip system according to any one of claims 1-4, characterized in that, Includes the following steps: (a) Nanopillar chip modification and biofunctionalization steps: The surface of the nanopillar substrate is cleaned with a mixed solution of concentrated sulfuric acid / hydrogen peroxide, silanized for valence modification, and pretreated with biotin bonding; Before use, the chip was rinsed with PBS buffer, and then streptavidin solution and combined peptide capture reagent solution were passed through it for incubation. After incubation, the chip was rinsed multiple times with PBS buffer to stabilize the combined peptide capture reagent on the surface of the nanopillar. (b) Cell capture procedure: Venous blood was collected from the patient using an ACD anticoagulation blood collection tube. After discarding a small amount of blood from the first tube, the remaining blood was diluted with PBS, centrifuged using a gradient density method, and the mononuclear cell layer was collected, washed, and resuspended to obtain the test sample. The test sample was then introduced into the chip system at a constant flow rate. The circulating tumor cells were specifically captured on the surface of the nanopillars by the combined polypeptide capture reagent. After the sample was exhausted, it was rinsed with PBS buffer to remove free leukocyte impurities, and the captured cells were fixed with paraformaldehyde solution. (c) Immunostaining and typing steps: After cell fixation, mixed antibody staining solution is introduced into the chip system to perform immunofluorescence staining on the captured circulating tumor cells to identify epithelial-mesenchymal transition circulating tumor cells with metastatic tendency; (d) Cell release step: A release reagent containing polypeptide digestive enzyme is introduced into the chip system. The polypeptide digestive enzyme is brought into full contact with the polypeptide capture reagent at a constant flow rate. The polypeptide capture reagent is sheared and digested, so that the captured circulating tumor cells are detached and released from the surface of the nanopillar. (e) Cell collection step: The chip is continuously flushed with the PBS buffer solution to collect the free circulating tumor cell suspension flowing out of the system.

6. The method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system according to claim 5, characterized in that, The specific steps for silanization covalent modification and biotin bonding in step (a) are as follows: S1. Immerse the cleaned and dried nanopillar chip in a toluene solution (concentration of 1.0%) of freshly prepared 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) and let it stand for 2 hours to complete the aminosilane bonding. S2. Remove the bonded nanopillar chip, clean it with anhydrous ethanol, and then dry it with nitrogen. S3. Place the dried nanopillar chip from step S2 into a material solution of NHS-PEGn-Biotin (n=1000) (0.05mg / ml), let it stand for 2 hours to complete the biotin modification, wash it with anhydrous ethanol, dry it with nitrogen gas, and then store it at 4℃. Before use, incubate with streptavidin solution (5.0 μM) for 1 hour. Incubate with the combined peptide solution for 1 hour, and rinse thoroughly with PBS buffer after each incubation.

7. The method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system according to claim 5, characterized in that, In step (b), the flow rate of the sample introduced is in the range of 0.5 mL / h to 2.0 mL / h.

8. The method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system according to claim 5, characterized in that, In step (d), the release reagent is a 0.5%-2.0% aqueous solution of chymotrypsin; the antibody combination used in step (C) includes three antibodies: anti-pan cytokeratin, anti-CD45, and anti-Vimentin.

9. The method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system according to claim 5, characterized in that, After the cell collection step is completed, the gene detection step is also included: single-cell whole genome amplification is performed on the collected circulating tumor cells, and the pancreatic cancer characteristic KRAS gene is specifically amplified and sequenced to detect KRAS gene mutation subtypes.

10. The method for capturing and releasing rare biological particles using a microfluidic nanopillar chip system according to claim 5, characterized in that, The microfluidic nanopillar chip system is used to prepare in vitro liquid biopsy products for pancreatic cancer. These products are used for early diagnosis of pancreatic cancer, prediction of tumor metastasis risk, and guidance on targeted drug therapy for pancreatic cancer.

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