An integrated platform for circulating tumor cell enrichment, typing analysis and non-destructive recovery and preparation and application thereof

Abstract

The application belongs to the technical field of liquid biopsy and cell sorting, and particularly relates to an integrated platform for circulating tumor cell enrichment, typing analysis and non-destructive recovery as well as preparation and application thereof. The microfluidic chip is obtained by coupling a chip matrix composed of a fishbone structure layer, a capture film and a glass slide and an engineered phage. The upper part of the fishbone structure layer is a fishbone beam repeating structure, and the lower part is a fluid channel. 2+ The CTCs are combined with the engineered phage, chemical information of molecular expression difference of the CTCs is converted into physical position information of spatial distribution rules in the microfluidic chip, the captured cells do not need to be eluted from the chip for complex subsequent single cell sequencing or immunohistochemistry, and typing is realized after capturing.

Description

Technical Field

[0001] This invention belongs to the field of liquid biopsy and cell sorting technology, specifically relating to an integrated platform for the enrichment, typing analysis and non-destructive recovery of circulating tumor cells, as well as its preparation and application. Background Technology

[0002] Circulating tumor cells (CTCs) are cancer cells that detach from the primary solid tumor and enter the bloodstream. The detection and analysis of CTCs are crucial for early cancer diagnosis, prognostic assessment, efficacy monitoring, and personalized treatment. During metastasis, CTCs undergo epithelial-mesenchymal transition (EMT), exhibiting high heterogeneity. CTCs with a mesenchymal phenotype typically demonstrate stronger invasiveness, migration ability, and drug resistance. Therefore, precise phenotypic analysis of CTCs using multiple biomarkers is of vital clinical significance for elucidating tumor metastasis mechanisms and assessing patient prognosis. Achieving precise phenotypic analysis of CTCs with different EMT phenotypes and obtaining more comprehensive information on tumor marker protein expression, such as the epithelial cell adhesion molecule (EpCAM) for epithelial tumors and N-cadherin for mesenchymal tumors, can guide the development of personalized treatment plans.

[0003] Furthermore, compared to other tumor markers such as circulating tumor DNA (ctDNA) and extracellular vesicles (EVs), CTCs, as intact and biologically active tumor cells, are a valuable resource for in-depth downstream analysis, including in vitro culture, drug sensitivity testing, and single-cell sequencing. Therefore, the isolation and enrichment of CTCs in complex matrices should be as gentle and non-destructive as possible, preserving the integrity and viability of the cells to the greatest extent possible.

[0004] To address these needs, various technologies have been developed for CTC enrichment and detection. For example, the CanPatrol detection technology enriches CTCs in blood based on cell size and combines it with multiplex RNA in situ hybridization (FISH) to analyze EMT-related mRNAs of CTCs, enabling simultaneous CTC enrichment and subtype analysis. While this platform expands the analytical dimensions of heterogeneous CTCs, its cell size-based separation process can easily cause physical damage to CTCs, affecting cell viability; it also cannot effectively distinguish leukocytes similar in size to CTCs, failing to guarantee the purity of CTC separation; and FISH-based detection methods struggle to obtain richer protein level information. Besides the aforementioned clinically-oriented technology platforms, flow cytometry and single-cell sequencing are currently the mainstream analytical methods in research scenarios. By labeling cells with antibodies carrying different fluorescent tags, flow cytometry can achieve fine-grained cell segmentation in complex samples. Single-cell sequencing technology is considered the "gold standard" for genome analysis, providing the most comprehensive gene expression information for each cell and revealing the heterogeneity of CTCs. However, flow cytometry and single-cell sequencing technologies cannot simultaneously meet the requirements of efficient enrichment, phenotypic analysis and non-destructive recovery of rare CTCs in complex matrices. Moreover, their complex operation procedures and high costs limit their widespread application in clinical diagnosis.

[0005] Existing technologies either rely on a single target, thus failing to comprehensively capture heterogeneous CTCs, or suffer cell damage and low purity due to crude enrichment methods, or cannot simultaneously achieve CTC enrichment, phenotypic analysis, and non-destructive recovery. Furthermore, techniques such as flow cytometry and single-cell sequencing typically require a relatively large number of starting cells (>10). 4 More importantly, both techniques struggle to simultaneously achieve CTC enrichment and phenotypic analysis, and the analysis process inevitably results in the loss of rare samples, limiting their application in CTC research. Therefore, there is an urgent need to develop an integrated method capable of efficiently and sensitively enriching CTCs in complex matrices, enabling in-situ labeling, phenotypic analysis, and controlled, mild release, to address the technical challenges in CTC heterogeneity analysis. Summary of the Invention

[0006] The purpose of this invention is to provide an integrated microfluidic chip (hereinafter referred to as the SPARK chip) for the capture, in situ labeling, phenotypic analysis, and non-destructive release of circulating tumor cells (CTCs), and its applications, to solve the problems of fragmented processes, complex operations, significant cell damage, and difficulty in achieving efficient capture and complete information acquisition in complex biological samples in existing technologies. This platform aims to organically integrate the enrichment, labeling, analysis, and release processes of CTCs, enabling the entire process from capture to result output to be completed after a single sample input. It ensures efficient capture, accurate phenotypic analysis, and gentle, non-destructive release of extremely low concentrations of CTCs in complex matrices (such as peripheral blood), thereby maximizing cell viability and the integrity of biological information, and avoiding complex preprocessing and sample loss.

[0007] To address the shortcomings or improvement needs of existing technologies and the heterogeneity of CTCs during EMT, this invention preferably uses EpCAM and N-cadherin as two tumor markers related to EMT, constructing CTC enrichment microfluidic chips EP-SPARK and Ncad-SPARK based on chromatographic separation principles. The affinity interface within these microfluidic chips uses functionalized M13 phage as a backbone, enabling multivalent display of functional peptides and effectively improving the enrichment capacity of the SPARK chip for CTCs, providing strong support for CTC typing analysis. Simultaneously, the fishbone-shaped structure in the chip provides a turbulent effect for the fluid, further enhancing the binding of CTCs to the affinity interface and improving the typing analysis capability of CTCs in complex biological matrices.

[0008] This invention relates to an integrated microfluidic chip for the enrichment, typing analysis, and non-destructive recovery of circulating tumor cells. The chip is a miniaturized processing device with an internal microchannel structure, capable of precisely controlling the flow rate and enriching cells in trace fluid samples (such as human peripheral blood samples).

[0009] The SPARK microfluidic chip includes a chip substrate and engineered bacteriophages coupled to the chip substrate.

[0010] The chip substrate consists of three layers, from top to bottom: a fishbone structure layer, a capture film, and a glass slide.

[0011] The upper part of the fishbone structure layer consists of repeating fishbone beams arranged horizontally from one end of the chip to the other, while the lower part is a fluid channel, forming a complete sheet. Along the direction of the repeating fishbone beams, from the fluid channel inlet at one end of the chip to the fluid channel outlet at the other end, several consecutive fishbone beams constitute a repeating unit, and several consecutive repeating units sequentially constitute the high expression region, the medium expression region, and the low expression region. The capture film corresponding to each expression region is a complete sheet.

[0012] By converting the chemical information of differential expression of CTCs into physical location information of spatial distribution within a microfluidic chip, the captured cells can be eluted from the chip for complex subsequent single-cell sequencing or immunohistochemistry, thus achieving capture-and-type identification.

[0013] The width L1 of the fishbone beam is 80μm, and the interval L2 between two adjacent fishbone beams is 120μm.

[0014] The height of the fishbone beam H1 is 50 μm, the height of the fluid channel H2 is 50 μm, and the total height of the fishbone structure layer is 100 μm. The fluid velocity differs between the fishbone beam and the gaps, thus creating a turbulence effect.

[0015] The surface of the captured film is coated with nickel ion-iminodiacetic acid (Ni 2+ (-IDA), from the fluid channel inlet to the fluid channel outlet, the capture film is divided into three regions, corresponding sequentially to the high expression region, medium expression region, and low expression region in the fishbone structure layer. The capture film thickness H2 is 300 μm.

[0016] Both the fishbone structure layer and the capture film are made of polydimethylsiloxane (PDMS).

[0017] The engineered phage includes phage M13 and a functional polypeptide. The 6His sequence and the 5Gly sequence are expressed on the minor capsid protein pIII and the major capsid protein pVIII of phage M13, respectively. The functional polypeptide has a protease cleavage site and a Sortase A recognition sequence LPETG at its C-terminus. The 5Gly sequence is coupled to the LPETG sequence.

[0018] When the functional peptide is an EP peptide (VRRDAPRFSMQGLDACGGNNCNN) that targets epithelial cell adhesion molecules, the protease cleavage site is the cathepsin B cleavage site GFLG, resulting in the engineered phage EP-H6G5-M13. At this time, the microfluidic chip is the EP-SPARK chip.

[0019] In the fishbone structure layer, from the fluid channel inlet to the fluid channel outlet, every 5 fishbone crossbeams constitute a repeat unit. Repeat units 1-13 are high expression regions, repeat units 14-26 are medium expression regions, and repeat units 27-40 are low expression regions. This enables the enrichment, differential expression typing analysis, and non-destructive recovery of the epithelial cell adhesion molecule EpCAM.

[0020] When the functional peptide is an NP peptide (SWTLYTPSGQSK) targeting N-cadherin, the protease cleavage site is the thrombin cleavage site LVPRGS, resulting in the engineered phage NP-H6G5-M13. At this time, the microfluidic chip is the Ncad-SPARK chip.

[0021] In the fishbone structure layer, from the fluid channel inlet to the fluid channel outlet, every 5 fishbone crossbeams constitute a repeat unit. Repeat units 1-12 are high expression regions, repeat units 13-26 are medium expression regions, and repeat units 27-40 are low expression regions; it can achieve enrichment, differential expression typing analysis, and non-destructive recovery of N-cadherin.

[0022] The present invention discloses the preparation of an integrated platform for the enrichment, typing analysis, and non-destructive recovery of circulating tumor cells, comprising the following:

[0023] 1. Fabrication of the chip substrate:

[0024] PDMS and a crosslinking agent were mixed and poured into a fishbone structure layer mold. After curing and molding, the mixture was peeled off from the mold to obtain the fishbone structure layer. Pre-coated Ni... 2+ -IDA's capture film is placed between the fishbone structure layer and the glass slide, and then bonded to form a whole, thus obtaining the chip substrate.

[0025] 2. Preparation of engineered bacteriophages: Two bacteriophages displaying functional peptides, EP-H6G5-M13 and NP-H6G5-M13, were constructed according to the technical solution disclosed in patent CN119916014A. The specific steps are as follows:

[0026] Wild-type M13 bacteriophage was genetically modified to express the 6His and 5Gly sequences on the minor capsid protein pIII and major capsid protein pVIII, respectively. A protease cleavage site (for subsequent cell release) and a Sortase A recognition sequence LPETG were added to the C-terminus of the functional peptide. Subsequently, using a Sortase A-mediated transpeptidation reaction, the LPETG sequence was conjugated to the 5Gly sequence of the pVIII protein to obtain the engineered bacteriophage.

[0027] When the functional peptide is an EP peptide (VRRDAPRFSMQGLDACGGNNCNN) targeting epithelial cell adhesion molecules, the engineered phage EP-H6G5-M13 is obtained by using the cathepsin B restriction site GFLG; when the functional peptide is an NP peptide (SWTLYTPSGQSK) targeting N-cadherin, the engineered phage NP-H6G5-M13 is obtained by using the thrombin restriction site LVPRGS.

[0028] 3. Chip substrate coupling with engineered phage: The engineered phage suspension is introduced into the chip substrate and incubated to complete the coupling, resulting in the SPARK microfluidic chip.

[0029] This invention provides an integrated platform for the enrichment, typing analysis, and non-destructive recovery of circulating tumor cells (CTCs), which can be used to identify CTCs in complex matrix samples, including the enrichment, typing analysis, and controlled release of CTCs. Specifically, it includes the following:

[0030] Before capturing and analyzing CTCs, the SPARK chip was blocked with 3% bovine serum albumin (BSA) at a flow rate of 60 μL / min for 15 minutes to reduce nonspecific adsorption.

[0031] The functional peptides used in the SPARK microfluidic chip were labeled with a fluorescent label (preferably FITC). Cells within the chip were observed under a confocal fluorescence microscope.

[0032] CTCs of the phenotype to be tested are introduced into the chip. The expression level of tumor markers (such as EpCAM and N-cadherin) is determined based on the distribution of CTCs in the capture film. CTCs captured in the high-expression area of ​​the capture film are at the high expression level of the tumor marker, CTCs captured in the medium-expression area of ​​the capture film are at the medium expression level of the tumor marker, and CTCs captured in the low-expression area of ​​the capture film are at the low expression level of the tumor marker, thus achieving capture and typing.

[0033] Specifically, it includes the following:

[0034] 1. CTCs typing analysis:

[0035] The EP-SPARK chip is used to define the differences in EpCAM expression in CTCs. After introducing CTCs of the phenotype to be tested into the chip, the CTCs distributed in the capture film regions of repeat units 1-13, repeat units 14-26, and repeat units 27-40 are defined as high-expression CTCs, medium-expression CTCs, and low-expression CTCs, respectively.

[0036] Similarly, the Ncad-SPARK chip is used to define the differences in N-cadherin expression among CTCs. After introducing CTCs of the phenotype to be tested into the chip, the CTCs distributed in the capture film regions of repeat units 1-12, repeat units 13-26, and repeat units 27-40 of the Ncad-SPARK chip are respectively CTCs with high N-cadherin expression levels, CTCs with medium N-cadherin expression levels, and CTCs with low N-cadherin expression levels.

[0037] 2. Controlled release of CTCs:

[0038] The fishbone layer of the chip was carefully peeled off, and the capture films of the three regions were removed. The EP-SPARK microfluidic chip was immersed in cathepsin B (0.4 U / mL) solution to release circulating tumor cells (CTCs), and the Ncad-SPARK microfluidic chip was immersed in thrombin (0.2 U / mL) solution to release CTCs. The release efficiency of circulating tumor cells (%) was calculated as: (number of released cells / number of captured cells) × 100%. The released CTCs were collected, and cell viability was analyzed using an apoptosis reagent. Simultaneously, the released CTCs were placed in culture medium for further culture, and the state of the cultured cells was observed under a microscope.

[0039] The key technical points and beneficial effects of this invention are as follows:

[0040] First, this invention provides a non-destructive, cell-preserving in-situ identification method for CTCs. The core lies in employing a live-cell compatible labeling system containing recognition ligands with high affinity for tumor markers (EpCAM, N-cadherin) on the surface of CTCs. This fluorescent labeling reaction is carried out under mild conditions, eliminating the need for cell fixation and membrane disruption, thereby achieving high signal-to-noise ratio sensitive identification and labeling of CTCs in complex biological matrices while ensuring complete preservation of their activity.

[0041] Secondly, this invention simultaneously achieves efficient capture and in-situ phenotypic analysis of CTCs. The key lies in the fact that the EP-SPARK and Ncad-SPARK chips allow CTCs with different phenotypes to be captured in different regions within the chip due to differences in the expression of tumor markers on their surfaces. This transforms the molecular expression differences (chemical information) of tumor cells into spatial distribution patterns (physical location information) within the microfluidic chip, eliminating the need for complex subsequent single-cell sequencing or immunohistochemistry after eluting cells from the chip, thus achieving capture-and-type analysis. This avoids the cell loss caused by elution and metastasis in traditional phenotypic analysis methods that rely on a "enrichment-then-analysis" process.

[0042] Third, this method designs specific cell release switches (such as the cathepsin B recognition sequence GFLG and the thrombin recognition sequence LVPRGS) in the sequences of CTCs-bound peptides. After CTCs are captured, the connection between the peptide and the chip can be precisely cut by adding cathepsin B or thrombin. This release strategy is mild and maintains the activity and intact biological information of CTCs, which can be used for high-value analysis and research such as downstream single-cell sequencing and cell culture. Attached Figure Description

[0043] Figure 1 This is a picture of the SPARK microfluidic chip.

[0044] Figure 2 This is a schematic diagram of the fishbone structure layers;

[0045] Figure 3 This is a magnified view of a portion of the fishbone structure layer;

[0046] Figure 4 This is a simulation diagram of the fluid velocity in the channel of the SPARK microfluidic chip;

[0047] Figure 5 This is a graph showing the fluorescence identification performance of the Light-up strategy of the SPARK microfluidic chip for CTC subtypes and non-CTCs (such as leukocytes, MCF-10A, etc.); where (a) is a fluorescence microscopy image of cells in the chip, (b) is the fluorescence intensity statistics of different cell lines, and (c) is the performance evaluation of the Light-up strategy for CTC fluorescent labeling.

[0048] Figure 6 The graph shows the release efficiency of CTCs by the SPARK microfluidic chip; where (a) is the release efficiency of CTCs in the EP-SPARK chip by Cathepsin B, and (b) is the release efficiency of CTCs in the Ncad-SPARK chip by Thrombin.

[0049] Figure 7 This is a graph showing the cell viability assessment results after CTCs were released from the SPARK chip;

[0050] Figure 8 It is the SPARK microfluidic chip and CellSearch ® Comparison chart of CTC detection rates;

[0051] Figure 9 The image shows the sorting effect of CTCs; where (a) is the fluorescence intensity of the model cells as measured by flow cytometry, and (b) is the location of the model cells captured in the EP-SPARK chip. Detailed Implementation

[0052] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. It should be noted that the embodiments described in this invention are only for further explanation and illustration, and not for limiting their application scope. Based on this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention.

[0053] Example 1

[0054] Construct the EP-SPARK microfluidic chip.

[0055] (1) Fabrication of a microfluidic chip substrate with a fishbone shape. The chip consists of three layers, from top to bottom: a fishbone structure layer, a Ni 2+ -IDA-coated capture film, glass slide, physical example Figure 1 As shown.

[0056] Fishbone structure layers such as Figure 2 As shown in the enlarged view, Figure 3 As shown, the width L1 of the fishbone beam is 80 μm, and the width L2 between two adjacent fishbone beams is 120 μm. The height H1 of the fishbone beam is 50 μm, the height H2 of the fluid channel is 50 μm, and the total height of the fishbone structure layer is 100 μm. Every 5 fishbone beams constitute a repeating unit, for a total of 40 repeating units.

[0057] Based on the chip structure and size, a fishbone structure layer mold is made. PDMS prepolymer and crosslinking agent are mixed in a mass ratio of 10:0.7 and poured into the mold. After vacuuming to remove air bubbles, the mixture is placed at 80°C for complete curing. The fishbone structure layer is obtained after the mold is peeled off.

[0058] Holes are drilled at the fluid channel inlet and outlet for oxygen plasma treatment, allowing for the connection of conduits to facilitate fluid flow; (This is in contrast to Ni...) 2+ -IDA-coated capture films and glass slides are bonded together to obtain a fishbone-shaped microfluidic chip substrate. The microfluidic chip substrate is then cleaned with ethanol for 30 minutes, and then dried at 60°C for later use.

[0059] (2) Preparation of engineered bacteriophages.

[0060] (2.1) Phage modification. The engineered phage H6G5-M13 was prepared according to the technical solution disclosed in patent application CN119916014A. The N-terminus of the major capsid protein pVIII expresses the 5Gly fragment, and the minor capsid protein pIII expresses the 6His fragment.

[0061] The specific preparation steps are as follows: Gene fragments encoding 5Gly and 6His were inserted into the N-terminus of the major capsid protein pVIII and the minor capsid protein pIII of the M13KE phage vector to construct a genetically modified phage vector. This phage vector was then transformed into *E. coli* XL1-Blue competent cells and cultured. Sequencing and screening yielded H6G5-M13 phage particles that perfectly matched the theoretical sequence.

[0062] (2.2) Coupling of functional peptides. A cathepsin B restriction site GFLG and a Sortase A recognition sequence LPETG were added to the C-terminus of the EP peptide. The functional peptide LPETG sequence was coupled to the 5Gly of the pVIII protein of H6G5-M13 phage particles using a Sortase A-mediated transpeptidation reaction.

[0063] In 500 μL of transpeptidase A reaction buffer (Tris 50 mM, NaCl 150 mM, CaCl2 10 mM, pH 7.5), phage particles H6G5-M13 and the peptide EP with a C-terminus increased by cathepsin B cleavage site GFLG were added to the reaction at an optimized molar ratio of pVIII:EP = 1:10. The reaction was carried out at 37 °C for 1 h with transpeptidase A (20 μM). After the reaction, the sample was passed through a polyethylene glycol 8000 / sodium chloride solution (PEG). 8000 Excess components were removed by sedimentation or ultrafiltration centrifugation with NaCl to obtain the engineered phage EP-H6G5-M13.

[0064] (3) Engineered phages are coupled to the chip substrate.

[0065] The above-mentioned engineered bacteriophage (1×10) 12 PFU / mL (50 μL) was introduced into a fishbone-shaped microfluidic chip substrate at a flow rate of 0.2 mL / h and incubated for 3 h to obtain a phage-functionalized EP-SPARK microfluidic chip. PBS buffer containing 3% BSA was then introduced into the chip for blocking to reduce non-specific adsorption. The simulated flow rate of the fluid in the chip channels is shown below. Figure 4 As shown.

[0066] Example 2

[0067] Construct the Ncad-SPARK microfluidic chip.

[0068] The difference from Example 1 is that, in step (2) of preparing the engineered phage, a thrombin restriction site LVPRGS and a Sortase A recognition sequence LPETG were added to the C-terminus of the NP polypeptide, and coupled with the H6G5-M13 phage particles to obtain the NP-H6G5-M13 engineered phage. The prepared engineered phage was then introduced into a microfluidic chip substrate with a fishbone shape and incubated for 3 hours to obtain a phage-functionalized Ncad-SPARK microfluidic chip. Other methods were the same as in Example 1.

[0069] Example 3

[0070] CTCs classification analysis using EP-SPARK chip.

[0071] Five different cell lines, namely Hoechst-prestained MCF-7, TGF-β1-induced EMT MCF-7, MDA-MB-231, leukocytes, and MCF-10A, were resuspended in 1 mL of PBS buffer and injected into the EP-SPARK chip at a flow rate of 1 mL / h. After incubation at room temperature in the dark for 30 min, uncaptured cells were rinsed with PBS buffer.

[0072] The capture membrane was peeled off, and the EP-SPARK chip was immersed in Cathepsin B (0.4 U / mL) solution to release circulating tumor cells (CTCs). The release efficiency (%) of circulating tumor cells was calculated as: (number of released cells / number of captured cells) × 100%. The released CTCs were collected, and cell viability was analyzed using an apoptosis reagent; the cell viability reached 98.7%.

[0073] MCF-7 cells, representing high EpCAM expression patterns, were denoted as E-CTCs; TGF-β1-induced EMT MCF-7 cells, representing moderate EpCAM expression patterns, were denoted as i-CTCs; and MDA-MB-231 cells, representing low EpCAM expression patterns, were denoted as M-CTCs. The results showed that repeat units 1-13 specifically captured cells with high EpCAM expression, repeat units 14-26 specifically captured cells with moderate EpCAM expression, and repeat units 27-40 specifically captured cells with low EpCAM expression.

[0074] Example 4

[0075] Classification analysis of CTCs using Ncad-SPARK chips.

[0076] The difference from Example 3 is that five different cell lines—Hoechst-prestained MCF-7, TGF-β1-induced EMTMCF-7, MDA-MB-231, leukocytes, and MCF-10A—were resuspended in 1 mL of PBS buffer and injected into the Ncad-SPARK chip at a flow rate of 1 mL / h. After incubation at room temperature in the dark for 30 min, uncaptured cells were rinsed with PBS buffer. The capture membrane was peeled off, and the Ncad-SPARK chip was immersed in Thrombin (0.2 U / mL) solution to release CTCs. Other methods were the same as in Example 3.

[0077] The results showed that repeat units 1-12 could specifically capture cells with high N-cadherin expression, repeat units 13-26 could specifically capture cells with medium N-cadherin expression, and repeat units 27-40 could specifically capture cells with low N-cadherin expression.

[0078] Example 5

[0079] The EP-SPARK chip is used to identify CTCs in human peripheral blood samples, and to capture, controllably release, and genotype CTCs.

[0080] (1) CTCs in peripheral blood samples of the identified individuals:

[0081] A 1 mL sample of human peripheral blood was fed into the EP-SPARK chip at a constant rate (1.5 mL / h). As the peripheral blood sample flowed through the chip, circulating tumor cells (CTCs) in the peripheral blood specifically bound to the engineered phage EP-H6G5-M13 coupled to the capture membrane within the chip, thus being specifically captured. As the engineered phage EP-H6G5-M13 accumulated on the surface of the captured CTCs, it formed localized fluorescent hotspots. Normal cells and other non-target cells, which did not bind to the engineered phage EP-H6G5-M13, were not captured and were expelled from the chip with the fluid. The distribution of these fluorescent hotspots could be observed using a fluorescence microscope, thus enabling the specific identification of CTCs.

[0082] (2) Controllable release of CTCs: After capture, carefully peel off the fishbone structure layer of the SPARK chip, remove the capture membrane, and immerse it in cathepsin B solution (0.4 U / mL, 500 μL) to release the captured CTCs of different phenotypes. Release efficiency (%) = (number of captured cells - number of residual cells) / number of captured cells × 100%.

[0083] Comparative Example 1

[0084] As an engineered phage control, the difference from Example 1 is that a microfluidic chip (EP-Chip) monovalently modified with EP peptides having only C-terminal restriction sites was prepared. The EP peptides were introduced into the fishbone-shaped microfluidic chip substrate prepared in Example 1, incubated at room temperature for 3 hours, and then washed with PBS buffer to obtain the EP-Chip monovalently modified with EP peptides. Equal volumes of resuspensions of five different Hoechst-prestained cell lines were introduced into this monovalent EP-Chip chip, and the captured cells and their distribution locations were counted. The results showed that the multivalent chip EP-SPARK had a significantly higher cell capture efficiency than the monovalent chip, and its sorting effect was also superior.

[0085] Comparative Example 2

[0086] As a comparison of sorting technologies, flow cytometry was used to sort E-CTCs, i-CTCs, and M-CTCs. The specific steps were as follows: Preparation of three cell suspensions (1×10⁻⁶) of E-CTCs, i-CTCs, and M-CTCs. 6 CoraLite was added to the cell suspensions of the three model cells mentioned above (cells / mL). ®Plus 488-labeled anti-EpCAM antibody (Proteintech) ® (Catalog No.: 21050-1-AP), incubated at 4℃ in the dark for 2 hours. After incubation, cells were collected by centrifugation and washed three times with PBS buffer. The cells were then resuspended in 1 mL of PBS buffer. The cell resuspended solution was then passed through a flow cytometer (BD Accuri™ C6 Plus) for detection. Phenotypic analysis was performed based on the fluorescence signals collected by the FLTC channel, and the phenotypic analysis results of the flow cytometry were compared with the EP-SPARK sorting results. Figure 9 (a) and Figure 9 As shown in (b), the sorting results of the three model cells by EP-SPARK were consistent with those of flow cytometry, and the separation degree (1.228) was better than that of flow cytometry (0.866).

[0087] The following results can be obtained from the above embodiments and comparative examples:

[0088] 1. Improved capture performance for heterogeneous CTCs. The SPARK chip can effectively capture 91% of CTCs, which is 3.1 times higher than the 22% capture efficiency of monovalent modified microfluidic chips.

[0089] 2. During capture, the flexibility of the bacteriophage's bend causes ligands to accumulate at the cell surface contact area, forming localized fluorescent hotspots, such as... Figure 5 As shown in (a); this allows for clear differentiation of CTCs in complex matrices, such as Figure 5 As shown in (b), the sensitivity reaches 97%, the specificity reaches 95%, and the accuracy is improved by 1.4 times compared with the classic immunofluorescence staining method. Figure 5 As shown in (c).

[0090] 3. EP-SPARK enables efficient capture and phenotypic analysis of CTCs with different phenotypes. In the 40 repeat units of the EP-SPARK chip, the distribution peak of EpCAM-highly expressed CTCs (E-CTCs) was located in unit 7 ± 0.6, EpCAM-medium expressed (i-CTCs) was mainly concentrated in unit 18 ± 0.6, and EpCAM-low expressed (M-CTCs) was mainly concentrated in unit 34 ± 1. This result is consistent with the flow cytometry analysis results. Not only is the separation significantly better than that of flow cytometry, but the minimum analytical quantity (50 cells) is also much lower than the analytical requirement of flow cytometry (10... 4 (cells).

[0091] 4. The method described in this invention achieves mild and controllable release of CTCs with different phenotypes, Cathpsin B. Figure 6 (a) and Thrombin Figure 6(b) The protease cleavage release strategy achieved a release efficiency of >90% for CTCs, with a gentle and non-destructive release process that maintained 98.7% cell viability, such as... Figure 7 As shown, this provides a reliable guarantee for downstream cell analysis.

[0092] 5. In clinical sample analysis, the SPARK chip achieved better performance than CellSearch in 4 cancer patients. ® A higher detection rate of circulating tumor cells was observed, and no false positive results were observed in two healthy volunteers. Figure 8 As shown, this demonstrates the robustness of the SPARK chip platform and its practical clinical application value.

Claims

1. An integrated platform for the enrichment, typing analysis, and non-destructive recovery of circulating tumor cells, the integrated platform being denoted as a SPARK chip, characterized in that, It includes a chip substrate and engineered bacteriophages coupled to the chip substrate; the chip substrate has three layers, from top to bottom: a fishbone structure layer, a capture film, and a glass slide; The upper part of the fishbone structure layer is a repeating fishbone beam structure, arranged horizontally from one end of the chip to the other, and the lower part is a fluid channel in sheet shape. Along the arrangement direction of the repeating fishbone beam structure, from the fluid channel inlet at one end of the chip to the fluid channel outlet at the other end of the chip, several consecutive fishbone beams constitute a repeating unit, and several consecutive repeating units sequentially constitute a high expression region, a medium expression region, and a low expression region.

2. The integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, The SPARK chip transforms the chemical information of differential molecular expression in circulating tumor cells into physical location information of spatial distribution patterns within the chip, eliminating the need to wash captured cells from the chip for complex subsequent single-cell sequencing or immunohistochemistry, thus achieving immediate typing upon capture.

3. The integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, The width of the fishbone beam is 80 μm, the interval between two adjacent fishbone beams is 120 μm, the height of the fishbone beam is 50 μm, the height of the fluid channel is 50 μm, and the total height of the fishbone structure layer is 100 μm.

4. The integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, The surface of the capture film is coated with Ni 2+ -IDA, from the fluid channel inlet to the fluid channel outlet, the capture film is divided into three regions, which correspond to the high expression region, medium expression region and low expression region in the fishbone structure layer in sequence; The thickness of the capture film is 300 μm; both the fishbone structure layer and the capture film are made of polydimethylsiloxane.

5. The integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, The engineered phage includes phage M13 and a functional polypeptide. The 6His sequence and the 5Gly sequence are expressed on the minor capsid protein pIII and the major capsid protein pVIII of phage M13, respectively. The functional polypeptide has a protease cleavage site and a Sortase A recognition sequence LPETG at its C-terminus. The 5Gly sequence is coupled to the LPETG sequence.

6. The integrated platform for circulating tumor cell enrichment, typing analysis, and non-destructive recovery according to claim 5, characterized in that, When the functional polypeptide is an EP polypeptide that targets epithelial cell adhesion molecules, the protease cleavage site is the cathepsin B cleavage site GFLG, resulting in the engineered bacteriophage EP-H6G5-M13 and the EP-SPARK chip. When the functional polypeptide is an NP polypeptide targeting N-cadherin, the protease cleavage site is the thrombin cleavage site LVPRGS, resulting in engineered phage NP-H6G5-M13 and an Ncad-SPARK chip.

7. The integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 6, characterized in that, The EP-SPARK chip features a fishbone structure layer where, from the fluid channel inlet to the fluid channel outlet, every five fishbone crossbeams form a repeating unit. Repeating units 1-13 are high-expression regions, repeating units 14-26 are medium-expression regions, and repeating units 27-40 are low-expression regions. This allows for the enrichment, differential expression typing analysis, and non-destructive recovery of the epithelial cell adhesion molecule EpCAM. The Ncad-SPARK chip has a fishbone structure layer in which every 5 fishbone crossbeams form a repeating unit from the fluid channel inlet to the fluid channel outlet. Repeating units 1-12 are high expression regions, repeating units 13-26 are medium expression regions, and repeating units 27-40 are low expression regions. It can achieve enrichment, differential expression genotyping analysis, and non-destructive recovery of N-cadherin.

8. The method for preparing an integrated platform for the enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, Includes the following: (1) Preparation of chip substrate: Polydimethylsiloxane and crosslinking agent were mixed and poured into a fishbone structure layer mold, and solidified to obtain a fishbone structure layer; Ni-coated substrate was then prepared. 2+ -IDA's capture film is placed between the fishbone structure layer and the glass slide, and bonded together to form a whole, thus obtaining the chip substrate; (2) Preparation of engineered phages: Wild-type M13 phages were genetically modified so that the 6His sequence and 5Gly sequence were expressed on the minor capsid protein pIII and major capsid protein pVIII of phage M13, respectively. A protease cleavage site and a Sortase A recognition sequence LPETG were added to the C-terminus of the functional peptides, respectively. Using a Sortase A-mediated transpeptidation reaction, the functional peptide LPETG sequence was coupled to the 5Gly of the phage pVIII protein to obtain engineered phages; (3) Coupled engineering phage to chip substrate: The engineered phage suspension is introduced into the chip substrate and incubated to complete the coupling, thus obtaining the SPARK chip.

9. The application of the integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 1, characterized in that, Circulating tumor cells (CTCs) of the phenotype to be tested are introduced into the SPARK chip. The expression level of tumor markers is determined based on the distribution of CTCs in the capture membrane. CTCs captured in the high-expression area of ​​the capture membrane are considered to have a high level of tumor marker expression, CTCs captured in the medium-expression area of ​​the capture membrane are considered to have a medium level of tumor marker expression, and CTCs captured in the low-expression area of ​​the capture membrane are considered to have a low level of tumor marker expression, thus achieving capture-based typing.

10. The application of the integrated platform for enrichment, typing analysis, and non-destructive recovery of circulating tumor cells according to claim 9, characterized in that, The SPARK chip was first blocked with bovine serum albumin, and then circulating tumor cell phenotype testing was performed; the tumor markers included epithelial cell adhesion molecule EpCAM and N-cadherin.

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