A detection kit and identification method for CTCs based on SCLC molecular typing using microfluidic chips and multiplex immunofluorescence probe technology.

Abstract

This invention discloses a detection kit and identification method for CTCs based on SCLC molecular typing using microfluidic chips and multiplex immunofluorescence probe technology. The kit includes a microfluidic chip, a biotin-labeled trapping agent, a balanced salt solution, a tissue cell fixation solution, a specific hybridization blocking solution, an active nuclear staining solution, and a diluent. It also includes a detection agent composed of key subtype markers of SCLC labeled with different fluorescein groups, SCLC-related marker detection antibodies, and leukocyte auxiliary identification markers, or a detection agent composed of key subtype markers of SCLC labeled with different fluorescein groups and leukocyte auxiliary identification markers. This application enables non-invasive, early, and sensitive detection and analysis of the expression of four immunomarkers involved in CTCs in the peripheral blood of patients: ASCL1, NEUROD1, POU2F3, and YAP1. It is suitable for early tumor screening and can also be used for pan-cancer immunotherapy guidance, immunotherapy efficacy monitoring, and prognostic assessment.

Description

[Technical Field]

[0001] This invention relates to the technical field of molecular subtyping of small cell lung cancer (SCLC), and in particular to the technical field of detection kits and identification methods for CTCs (circulating tumor cells) based on SCLC molecular subtyping using microfluidic chips and multiplex immunofluorescence probe technology. [Background Technology]

[0002] Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with early metastasis and poor prognosis (Byers and Rudin, 2015), accounting for 13-17% of all lung cancer types. Due to its high degree of malignancy and early-stage distant metastasis, SCLC is often diagnosed at an advanced stage, resulting in a very poor prognosis. Chemotherapy and radiotherapy are currently the most traditional treatments for SCLC. In recent years, immunotherapy has made rapid progress. The US FDA has approved atezolizumab or durvalumab in combination with etoposide and platinum-based chemotherapy as first-line treatment for extensive-stage SCLC. Nivolumab ± ipilimumab and pembrolizumab are approved for treating SCLC that has progressed after prior platinum-based chemotherapy and at least one other therapy. Despite the addition of immunotherapy to platinum-based first-line chemotherapy, progression-free survival (PFS) and overall survival (OS) have not been significantly prolonged in SCLC patients (Chung et al., 2020; Paz-Ares et al., 2019). In the 1970s and 80s, the high responsiveness of SCLC to platinum-based chemotherapy regimens, including complete remission, led to the belief that SCLC could soon be cured. Forty years later, due to inherent or most commonly acquired treatment resistance, the 5-year survival rate for SCLC patients remains at 5%. There have also been some advances in targeted therapy for small cell lung cancer, such as temozolomide in combination with the PARP inhibitor olaparib for recurrent small cell lung cancer, while anlotinib has been approved in China for third-line treatment of recurrent small cell lung cancer. PharmaMar's urbinectedin (an RNA polymerase II inhibitor), a seaweed extract derivative, has submitted a marketing application based on phase II study results for second-line treatment of recurrent small cell lung cancer. However, unlike non-small cell lung cancer (NSCLC), where the selection of biomarkers for targeted therapy and immunotherapy has significantly altered traditional treatment paradigms (Zimmermann et al., 2018), clinical targeted therapy studies for small cell lung cancer have primarily focused on unselected populations, with generally disappointing results and no significant progress since the advent of cisplatin and etoposide. The intrinsic driving factors behind the progression of small cell lung cancer from chemotherapeutic sensitivity to chemotherapy resistance and rapid disease progression remain unclear. Therefore, a clearer and more precise definition of the molecular subtypes and subtypes of SCLC to determine the accurate selection and efficacy assessment of targeted and immunotherapies is an urgent problem to be solved.

[0003] From 2019 to 2020, numerous researchers compiled data from cell lines, patient-derived xenografts (PDX), and mouse transgenic models (GEMMs) and conducted comprehensive SCLC genomic studies, leading to the proposal of a molecular subtype classification of heterogeneous SCLC based on mRNA expression profile differentiation program control. Specifically, SCLC subtypes are divided into neuroendocrine (NE) and non-neuroendocrine (non-NE) types based on the prevalent neuroendocrine (NE) phenomenon in SCLC. This includes the previously undiscovered non-NE cluster cell variant of SCLC and supplementary subtypes defined by the transcription factor POU2F3. Furthermore, since many patients still do not belong to these three subtypes, a fourth subtype driven by the transcription factor YAP1 has been proposed, providing a partial solution for this portion of unclassified tumors. Recent consensus recommends classifying SCLC into five subtypes: ASCL1 high expression (SCLC-A), NEUROD1 high expression (SCLC-N), ASCL1 and NEUROD1 co-expression (SCLC-A / N), POU2F3 high expression (SCLC-P), and YAP1 high expression (SCLC-I). Based on the molecular characteristics of SCLC in the Chinese population and correlation analysis of tissue samples and cell line expression profiles, different molecular subtypes of SCLC have been found to have different sensitivities to different drugs. Specifically, SCLC-A (41%) is sensitive to BCL-2 inhibitors or PARP inhibitors combined with immune checkpoint inhibitors; SCLC-N (8%) is sensitive to non-interstitial and non-epithelial Aurora kinase inhibitors; SCLC-A / N (37%); SCLC-P (7%) is sensitive to PARP inhibitors and nucleoside analogs; and SCLC-I (7%) is sensitive to immune checkpoint inhibitors. Current clinical trials are exploring the relationship between different SCLC molecular subtypes and immunobiology. The challenge in the clinical treatment of SCLC is how to select appropriate biomarkers using the optimal strategy to accurately identify the population that will benefit. Precise identification of SCLC molecular subtypes can help provide patients with personalized precision treatment plans.

[0004] Small cell lung cancer (SCLC) is a highly aggressive disease with limited treatment options. Although patients initially respond to treatment, relapse is common. To date, there are no approved targeted therapies or biomarkers to guide treatment for SCLC. Temozolomide has shown some efficacy in relapsed SCLC. Surgery is rarely performed in patients with small cell lung cancer, and the available tissue samples are often insufficient for biomarker analysis. Most SCLC patients (approximately 70%) present with extensive-stage SCLC (ES-SCLC) at diagnosis, while the remaining 30% have limited-stage SCLC (LS-SCLC). The prognosis for small cell lung cancer is poor; the median overall survival (OS) for ES-SCLC patients is 10 months, while for LS-SCLC patients it can reach 4 years. Platinum-based chemotherapy combined with etoposide or irinotecan is the standard first-line treatment for SCLC. Recently, immune checkpoint inhibitors (ICIs) have been approved, alone or in combination with chemotherapy, for the treatment of SCLC. Although most patients initially respond well to chemotherapy (alone or in combination with ICIs), relapse and metastasis often occur rapidly, resulting in a poor prognosis. Topotecan, the only approved second-line drug, has a low response rate and short survival. Unlike non-small cell lung cancer (NSCLC) and other cancer types, small cell lung cancer (SCLC) has few other treatment options, and no targeted therapies are available for advanced-stage patients. The highly aggressive nature of SCLC and the lack of effective and aggressive treatment highlight the urgent need for targeted biomarker analysis and identification, which can aid in the selection of individualized treatment plans and the development of targeted drugs.

[0005] Non-invasive biomarkers in peripheral blood, including circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA), can provide prognostic and / or predictive information for tumors, investigate drug resistance mechanisms, and discover new therapeutic targets. Although ctDNA detection is currently a commonly used method for early auxiliary detection of clinical tumors, a large amount of research in SCLC still focuses on CTCs. Circulating tumor cells (CTCs) are a collective term for tumor cells shed from primary and metastatic tumors and entering the peripheral blood circulation system (one CTC for every 10⁶–10⁷ white blood cells), and are a major cause of cancer development and metastasis. The 2021 version of the CSCO guidelines states that CTCs, as a "liquid biopsy sample" representing the primary tumor, can be used to monitor the condition of cancer patients in real time, dynamically, and non-invasively. Studies have confirmed that SCLC cells have short cell division cycles, rapid proliferation, and are prone to entering the bloodstream and metastasizing to distant sites, while the detection rate of CTCs in the SCLC population is 67–86%. Detecting cytokines (CTCs) helps in accurately determining the clinical stage of the disease, enabling the selection of appropriate clinical protocols, guiding individualized treatment for SCLC patients, monitoring tumor recurrence and metastasis, assessing treatment efficacy, and predicting prognostic survival. It also serves as a means to analyze the molecular mechanisms of drug resistance and address tumor heterogeneity. Previous studies have shown that SCLC patients have a relatively higher number of CTCs compared to NSCLC patients, and patients with extensive-stage SCLC (ES-SCLC) have a relatively higher number of CTCs than those with limited-stage (LS-SCLC). Recent advancements in CTC isolation methods and the possibility of molecular characterization studies of individual CTCs have helped assess the potential role of CTCs as biomarkers for evaluating treatment efficacy and monitoring disease progression in SCLC, in order to investigate tumor heterogeneity and mechanisms of drug resistance. Furthermore, studies using CTC-derived xenograft (CDX) models can provide supplementary information on treatment sensitivity / resistance mechanisms for genomic analysis and CTC counting, allowing for the in vivo investigation of tumor heterogeneity and drug resistance mechanisms within CDX.

[0006] Currently, the main CTC detection methods on the market include membrane filtration, immunomagnetic bead technology, and microfluidic chip detection. Membrane filtration can separate CTCs based on cell size differences, but often results in low purity. Immunomagnetic beads primarily utilize the binding of magnetic beads and antibodies, which then capture cells by binding to antigens on the CTC surface; this usually destroys cell viability, preventing further cell culture and other subsequent processes. The number of CTCs detected using the CellSearch system (expressing the epithelial marker EpCAM and cytokeratins CK8 / 18 / 19) has independent prognostic significance in SCLC. However, the evolution of small cell lung cancer pathogenesis from SCLC-A to SCLC-I subtypes involves a transformation of tumor cells from epithelial to mesenchymal (EMT) types. Therefore, CTC detection and identification methods based solely on EPCAM capture cannot accurately classify and identify the five molecular subtypes of SCLC, and thus cannot accurately assist clinicians in providing precise pathological classification and developing personalized treatment strategies for SCLC patients. [Summary of the Invention]

[0007] The purpose of this invention is to solve the problems in the prior art and to propose a detection kit and identification method for CTCs based on SCLC molecular typing using microfluidic chips and multiplex immunofluorescence probe technology. This application employs a combination of key biomarkers for identifying five molecular subtypes of SCLC (SCLC-A subtype: ASCL1-Alexa647 and PanCK488; SCLC-N subtype: NEUROD1-Alexa647 and Myc-488; SCLC-A / N subtype: ASCL1-Alexa647 and NEUROD1-Alexa647; SCLC-P subtype: POU2F3-Alexa647 and AVIL-488; SCLC-I subtype: YAP1-Alexa647 and CSV-FITC or VIM-488), with the addition of the leukocyte identification marker CD45-PE. This allows for the identification and analysis of CTC multiplex immunofluorescent probe combinations enriched by microfluidic chips in five peripheral blood samples (1-2 ml / sample) from the same patient. This enables non-invasive, early, sensitive, and dynamic detection of the SCLC molecular subtype stage in patients. This application targets the characteristic molecular markers of five molecular subtypes of SCLC (A, N, A / N, P, and I) (ASCL1, NEUROD1, POU2F3, and YAP1), and designs a highly efficient microfluidic CTC enrichment chip and corresponding CTC capture reagents combined with multiplex immunofluorescent probes for the identification of the five SCLC molecular subtypes. This approach can utilize existing... The CTC detection system platform is the first in the world to develop reagent kits and methods for identifying and detecting circulating tumor cells (SCLC) that are closely related to clinical treatment. It is used to assist in the molecular subtyping of clinical SCLC, predict clinical efficacy, predict disease recurrence, and screen clinical drugs, thus filling a gap both domestically and internationally.

[0008] To achieve the above objectives, this invention proposes a detection kit for CTCs based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology. The kit includes a microfluidic chip, a biotin-labeled trapping agent, a balanced salt solution, a tissue cell fixation solution, a specific hybridization blocking solution, an active nuclear staining solution, and a diluent. It also includes a detection agent composed of key markers of SCLC subtypes labeled with different fluorescein groups, SCLC-related marker detection antibodies, and leukocyte auxiliary identification markers, or key markers of SCLC subtypes labeled with different fluorescein groups and leukocyte auxiliary identification markers.

[0009] The key markers for the subtypes of SCLC include at least one of ASCL1, NEUROD1, POU2F3, and YAP1;

[0010] The SCLC-related marker detection antibodies include at least one of PanCK, MYC, AVIL, CSV, and VIM.

[0011] Preferably, the microfluidic chip has several diversion channels with flow segments arranged in a double-row herringbone structure.

[0012] Furthermore, the microfluidic chip is equipped with eight grooved microfluidic array units with a double-row herringbone structure, which are staggered and connected in series at a specific angle to form a diversion channel.

[0013] Preferably, the silane modifier, bifunctional crosslinking agent, and streptavidin are sequentially coupled within the split-flow lane.

[0014] As a preferred method, the fabrication of the microfluidic chip is as follows: ① Fabrication of the microfluidic channel PDMS substrate: Photoresist is uniformly coated on a silicon wafer after plasma cleaning, and after heating and drying, exposure, heating and development are performed sequentially to obtain a photoresist nanoarray. Then, PDMS adhesive is cast based on the photoresist nanoarray, holes are drilled and the adhesive is cut, and plasma cleaning is performed. After bonding, a PDMS substrate is obtained; ② Fabrication of a functionalized probe-loaded glass slide: The surface of the glass slide after oxygen plasma treatment is silanized with 3-aminopropyltriethoxy, and then a bifunctional amine-thiol crosslinking agent and streptavidin (SA) are sequentially combined; ③ Bonding: After oxygen plasma treatment, the microfluidic channel PDMS substrate and the functionalized probe-loaded functionalized glass slide are bonded to the non-channel edge regions around the perimeter.

[0015] Preferably, the biotin-labeled scavengers are classified into SCLC-A subtype scavengers, SCLC-N subtype scavengers, SCLC-A / N subtype scavengers, SCLC-P subtype scavengers, and SCLC-I subtype scavengers according to SCLC molecular typing.

[0016] The SCLC-A subtype capture agent is the biotinylated epithelial tumor marker EpCAM;

[0017] The SCLC-N subtype capture agent is either biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin CSV, or biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin VIM.

[0018] The SCLC-A / N subtype capture agent is either biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin CSV, or biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin VIM.

[0019] The SCLC-P subtype capture agent is the biotinylated epithelial tumor marker EpCAM and the characteristic marker of small cell lung cancer P subtype P, POU2F3.

[0020] The SCLC-I subtype capture agent is the biotinylated mesenchymal tumor marker vimentin CSV and the tumor marker receptor tyrosine kinase AXL, which is highly expressed in small cell lung cancer subtype I.

[0021] Preferably, the balanced salt solution is PBS buffer; the tissue cell fixative is PFA fixative; the specific hybridization blocking solution is FC receptor blocking solution; the active nuclear staining solution is Hoechst 33342 DNA fluorescent staining solution; and the diluent is 1×ADB antibody dilution buffer.

[0022] Preferably, the detection reagents are classified according to the type of analyte and SCLC molecular typing into SCLC-A subtype fluorescent probe detection reagents, SCLC-N subtype fluorescent probe detection reagents, SCLC-A / N subtype fluorescent probe detection reagents, SCLC-P subtype fluorescent probe detection reagents, SCLC-I subtype fluorescent probe detection reagents, and leukocyte fluorescent probe detection reagents;

[0023] The SCLC-A subtype fluorescent probe detection reagent consists of ASCL1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled PanCK.

[0024] The SCLC-N subtype fluorescent probe detection reagent consists of NEUROD1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled MYC.

[0025] The SCLC-A / N subtype fluorescent probe detection group consists of ASCL1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, NEUROD1 primary antibody, and Alexa647 fluorescently labeled IgG secondary antibody.

[0026] The SCLC-P subtype fluorescent probe detection reagent consists of POU2F3 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled AVIL.

[0027] The SCLC-I subtype fluorescent probe detection reagent is either YAP1 primary antibody, Alexa647 fluorescein-labeled IgG secondary antibody, and FITC fluorescein-labeled CSV, or YAP1 primary antibody, Alexa647 fluorescein-labeled IgG secondary antibody, and 488 fluorescein-labeled VIM.

[0028] The leukocyte fluorescent probe detection agent is CD45-PE.

[0029] The above five groups of SCLC molecular subtype marker probes can be used to simultaneously identify multiple immunofluorescence probe combinations in five peripheral blood samples (1-2 ml / sample) divided equally from the same patient.

[0030] The biotin-labeled trapping and detection reagent combinations for the one-time enrichment, separation, and identification of CTCs for five molecular subtypes of SCLC are shown in Table 1 below:

[0031]

[0032]

[0033] Table 1. Biotin-labeled trapping agent combinations and detection kits for the single-use CTC enrichment, separation, and identification of five molecular subtypes of SCLC.

[0034] In this invention, the multiplex immunoassay probes are each labeled with a different emission wavelength of fluorescein to distinguish different molecular subtypes of SCLC: ASCL1, NEUROD1, POU2F3, and YAP1 are labeled with primary antibodies and corresponding Alexa Fluor 647 red fluorescein-labeled IgG secondary antibodies; PanCK, MYC, AVIL, and CSV (or VIM) are labeled with FITC (isothiocyanate) or 488 green fluorescein; and CD45 is labeled with PE (phycoerythrin) orange fluorescein. The five molecular subtypes of CTC cells identified by this invention using a combination of multiplex immunofluorescence probes with different fluorescein labels can be completely distinguished under a fluorescence microscope using different filters.

[0035] The identification method for CTCs based on SCLC molecular typing using a microfluidic chip and multiplex immunofluorescence probe technology includes the following steps:

[0036] a) Coating and blocking of biotin-labeled trapping agents in microfluidic chips: The biotin-labeled trapping agent was diluted with a balanced salt solution and injected into the split lane through the chip inlet and incubated. After washing the microfluidic chip with a balanced salt solution, tissue cell fixation solution was injected and fixed. After washing the microfluidic chip with a balanced salt solution, a specific hybridization blocking solution diluted with a diluent was injected and incubated.

[0037] b) Isolation of peripheral blood mononuclear cells (PBMCs) from SCLC patients: Blood was drawn from SCLC patients and PBMCs were isolated from SCLC patients by density gradient centrifugation using human peripheral blood lymphocyte separation medium.

[0038] c) Enrichment and capture of CTC cells in a microfluidic chip: Peripheral blood mononuclear cells (PBMCs) from SCLC patients were injected into a microfluidic chip to enrich and capture CTC cells;

[0039] d) Multiplex fluorescent immunoassay probe hybridization of CTC cells: The reagents in the detection kit, except for the Alexa647 fluorescently labeled IgG secondary antibody, were diluted and injected into the microfluidic chip and incubated. After washing the microfluidic chip with balanced salt solution, the Alexa647 fluorescently labeled IgG secondary antibody diluted with diluent was added and incubated.

[0040] e) Viable nuclear staining of CTC cells: After cleaning the microfluidic chip with balanced salt solution, viable nuclear staining solution diluted with diluent was injected and incubated.

[0041] f) Scanning and interpretation of CTC cells: CTC cells were scanned, identified and analyzed using a four-color channel automated fluorescence scanning system.

[0042] Preferably, in step a), the biotin-labeled capture agent is diluted 50 to 100 times and incubated at room temperature for 0.5 to 2 hours; the tissue cell fixative is fixed for 5 to 15 minutes at room temperature; and the specific hybridization blocking solution is diluted 200 to 400 times and incubated at room temperature for 10 to 30 minutes.

[0043] Preferably, in step d), the detection reagent is diluted 50 to 200 times and incubated at room temperature for 0.5 to 1.5 hours.

[0044] Preferably, in step e), the incubation time of the active nuclear stain solution at room temperature is 5 to 15 minutes.

[0045] The beneficial effects of this invention are:

[0046] 1) This invention designs and develops a microfluidic matrix CTC enrichment chip based on a grooved herringbone double-row herringbone structure. This geometric design effectively enhances the probability of contact between CTC surface antigens and the capture agent. Furthermore, by adjusting the microfluidic flow rate and the direction and magnitude of shear force, efficient and specific CTC enrichment is achieved while maintaining a small amount of patient blood sample (0.2-1 ml). This not only greatly improves the capture efficiency of CTC cells in body fluids such as peripheral blood, cerebrospinal fluid, pleural and peritoneal cerebrospinal fluid, and urine, but also maintains the integrity of the enriched CTC cell morphology. At the same time, it reduces the retention of white blood cells (WBCs), greatly improving the purity of CTC enrichment by this microfluidic chip.

[0047] 2) This invention achieves CTC immunoenrichment based on the streptavidin and biotin cascade signal amplification reaction by sequentially silanizing, applying a bifunctional protein crosslinking agent, modifying with streptavidin (SA) coupling, and coating with a biotinylated antibody capture agent on the base layer of the microfluidic chip cavity. Compared with other microfluidic chips, this invention greatly improves the CTC enrichment and capture efficiency of peripheral blood and other body fluids from patients.

[0048] 3) This invention designs a combination of biotin-labeled multiple capture agents for CTCs of five SCLC subtypes (SCLC-A subtype capture agent, SCLC-N subtype capture agent, SCLC-A / N subtype capture agent, SCLC-P subtype capture agent and SCLC-I subtype capture agent) for molecular subtypes of SCLC (A, N, A / N, P and I subtypes), which can efficiently and specifically enrich and capture CTC cells of all molecular subtypes in blood samples and pleural effusion of SCLC patients in a single step.

[0049] 4) This invention designs a combination of multiple fluorescent probes (SCLC-A subtype fluorescent probe detector, SCLC-N subtype fluorescent probe detector, SCLC-A / N subtype fluorescent probe detector, SCLC-P subtype fluorescent probe detector, and SCLC-I subtype fluorescent probe detector) targeting the molecular subtypes of SCLC (A, N, A / N, P, and I subtypes). This allows for a one-time, non-invasive, early, sensitive, and dynamic detection of the patient's SCLC molecular subtype stage. Thus, it is the first in the world to develop a kit and method for identifying and detecting circulating tumor cells in SCLC that is closely related to clinical treatment. This kit is used to assist in the molecular subtyping of clinical SCLC, predict clinical efficacy, predict disease recurrence, and screen clinical drugs, filling a gap in domestic and international research.

[0050] 5) This invention provides a method for identifying and detecting circulating tumor cells (SCLC) based on microfluidic chips and multiplex immunofluorescence probes. This method can detect and diagnose SCLC and its molecular subtype earlier than clinical imaging techniques such as FDG-PET / CT and PET / MRI, as well as nuclear labeling imaging techniques such as PET / CT. It also avoids the limitations of IHC testing in tumor patient tissues, which has high sampling requirements (must be performed on tumor tissues for IHC identification), subjectivity and heterogeneity, and varying accuracy rates across different centers due to factors such as antibody quality and testing process (fixation and staining). This method can assist clinicians in providing early intervention guidance for medication and developing individualized precision treatment plans for tumor patients. It can also accurately perform non-invasive auxiliary dynamic efficacy monitoring and prognostic assessment for tumor patients in the postoperative recurrence and metastasis stage.

[0051] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. [Attached Image Description]

[0052] Figure 1 The results are the identification results of the five microfluidic-based SCLC molecular typing identification and detection kits in Example 1.

[0053] Figure 2 This is a schematic diagram of the microfluidic design of the microfluidic chip in Example 1;

[0054] Figure 3 This is a design diagram of the inlet and outlet structure of the microfluidic chip in Example 1;

[0055] Figure 4 This is an enlarged schematic diagram of the flow segment of the microfluidic chip in Embodiment 1, which has a double herringbone structure in the shunt channel.

[0056] Figure 5 This is a capture rate graph of the CTC microfluidic chip for graded dilution of MCF7 cells in Example 2.

Detailed Implementation Methods

[0057] Referring to Table 2 below, five CTC detection kits based on SCLC molecular typing using microfluidic chips and multiplex immunofluorescence probe technology were selected and identified separately. The specific operation steps are as follows:

[0058] a) Coating and blocking of biotin-labeled capture agents for microfluidic chips: ① Take the biotin-labeled capture agents from Table 2, add PBS buffer to a 60 μL system, mix well, and inject into the split lane of the microfluidic chip through the chip inlet and incubate at room temperature for 1.5 h; ② Wash the microfluidic chip twice with 100 μL of PBS buffer; ③ Fix with 100 μL of 2% PFA fixative at room temperature for 10 min; ④ Wash the microfluidic chip twice with 100 μL of PBS buffer; ⑤ Take 12 μL of FC receptor blocking solution, add 48 μL of 1×ADB antibody dilution buffer, mix well, inject into the microfluidic chip, and incubate at room temperature for 20 min;

[0059] b) Isolation of peripheral blood mononuclear cells (PBMCs) from SCLC patients: Blood was drawn from SCLC patients and PBMCs were isolated from SCLC patients by density gradient centrifugation using human peripheral blood lymphocyte separation medium.

[0060] c) Enrichment and capture of CTC cells in a microfluidic chip: Peripheral blood mononuclear cells (PBMCs) from SCLC patients were injected into a microfluidic chip to enrich and capture CTC cells;

[0061] d) Fluorescence immunoassay for CTC cells: ① Take the corresponding type and amount of detection reagent from Table 2, add 1×ADB antibody dilution buffer to a 60μL system, mix well, inject into the microfluidic chip, and incubate at room temperature for 1h; ② Wash the microfluidic chip twice with 100μL PBS buffer; ③ Take the fluorescent probe (various detection antibodies and key markers of SCLC molecule subtypes are labeled with different fluoresceins), add 1×ADB antibody dilution buffer to a 60μL system, mix well, inject into the microfluidic chip, and incubate at room temperature for 1h.

[0062] e) Staining of active nuclei of CTC cells: ① Wash the microfluidic chip twice with 100 μL of PBS buffer; ② Incubate with 200 μL of 30 μg / mL Hoechst 33342 DNA fluorescent staining solution for 10 min;

[0063] f) Scanning and interpretation of CTC cells: CTC cells were scanned, identified and analyzed using a four-color channel automated fluorescence scanning system.

[0064] The fabrication method of the microfluidic chip includes the following steps:

[0065] a) Fabrication of PDMS nano-substrate: SU-8 photoresist was uniformly coated on a silicon wafer that had been plasma-cleaned. The substrate was then subjected to pre-baking, UV exposure, post-baking, and development to obtain a photoresist nanoarray. Based on the photoresist nanoarray, PDMS adhesive was cast twice. Finally, holes were drilled and cut (see shape reference). Figures 2-4 After plasma cleaning, PDMS nano-substrate was obtained;

[0066] b) Preparation of functionalized modified glass slides: The surface of the glass slides after oxygen plasma treatment was silanized with 3-hydroxypropyltrimethoxysilane (3-MPTS), and then maleimide-PEG2-biotin, streptavidin (SA) and biotinylated monoclonal antibody were sequentially combined to obtain functionalized modified glass slides.

[0067] c) Combination: Bonding PDMS nanosubstrates to glass slides modified with functional groups.

[0068]

[0069]

[0070] Table 2. Main parameters of five microfluidic-based SCLC molecular typing-based circulating tumor cell identification and detection kits.

[0071] In addition, the cell lines and functions of the five microfluidic-based SCLC molecular subtyping-based circulating tumor cell identification and detection kits are shown in Table 3 below:

[0072] SCLC subtype classification Cell line name effect SCLC-A DMS153 Human SCLC-A positive test quality control products SCLC-N NCIH524 Human SCLC-N positive test quality control products SCLC-A / N CORL279 Human SCLC-A / N positive test quality control products SCLC-P NCIH526 Human SCLC-P positive test quality control products SCLC-I SW1271 Human SCLC-I positive test quality control products

[0073] Table 3. Cell lines and functions of five microfluidic-based SCLC molecular subtyping-based identification and detection kits.

[0074] The identification results of five SCLC molecular typing-based CTC detection kits using microfluidic chips and multiplex immunofluorescence probe technology are as follows: Figure 1 As shown in the figure. Furthermore, the prepared microfluidic chip was used to capture MCF7 control cells, and the test results are as follows. Figure 5 As shown.

[0075] The above embodiments are illustrative of the present invention and are not intended to limit the present invention. Any simple modifications to the present invention are within the scope of protection of the present invention.

Claims

1. A detection kit for CTCs based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology, characterized in that: The device includes a microfluidic chip, a biotin-labeled trapping agent, a balanced salt solution, a tissue cell fixative, a specific hybridization blocking solution, an active nuclear staining solution, and a diluent. It also includes a detection reagent composed of key subtype markers of SCLC labeled with different fluorescein groups, SCLC-related marker detection antibodies, and leukocyte-assisted identification markers, or key subtype markers of SCLC labeled with different fluorescein groups and leukocyte-assisted identification markers. The key subtype markers of SCLC include at least one of ASCL1, NEUROD1, POU2F3, and YAP1; the SCLC-related marker detection antibodies include at least one of PanCK, MYC, AVIL, CSV, and VIM. The microfluidic chip has several flow channels with flow segments in a double-row herringbone structure, and the flow channels are coupled sequentially with a silane modifier, a bifunctional crosslinking agent and streptavidin. The biotin-labeled capture agents are classified into SCLC-A subtype capture agents, SCLC-N subtype capture agents, SCLC-A / N subtype capture agents, SCLC-P subtype capture agents, and SCLC-I subtype capture agents according to SCLC molecular subtypes; the SCLC-A subtype capture agent is the biotinylated epithelial tumor marker EpCAM; the SCLC-N subtype capture agent is either the biotinylated epithelial tumor marker EpCAM and the stromal tumor marker vimentin CSV, or the biotinylated epithelial tumor marker EpCAM and the stromal tumor marker vimentin VIM; The SCLC-A / N subtype capture agent is either biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin CSV, or biotinylated epithelial tumor marker EpCAM and mesenchymal tumor marker vimentin VIM; the SCLC-P subtype capture agent is either biotinylated epithelial tumor marker EpCAM and small cell lung cancer P subtype characteristic marker POU2F3; the SCLC-I subtype capture agent is either biotinylated mesenchymal tumor marker vimentin CSV and small cell lung cancer I subtype highly expressed tumor marker receptor tyrosine kinase AXL. ASCL1, NEUROD1, POU2F3, and YAP1 were all labeled with primary antibodies and corresponding Alexa Fluor 647 red fluorescent pigments as secondary IgG antibodies. PanCK, MYC, AVIL, CSV, and VIM were all labeled with FITC or 488 green fluorescent pigments.

2. The detection kit for CTCs based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology as described in claim 1, characterized in that: The balanced salt solution is PBS buffer; The tissue cell fixative is PFA fixative; The specific hybridization blocking solution is an FC receptor blocking solution; The active nuclear staining solution is Hoechst 33342 DNA fluorescent staining solution; The diluent is 1×ADB antibody dilution buffer.

3. The detection kit for CTCs based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology as described in claim 1, characterized in that: The detection reagents are classified into SCLC-A subtype fluorescent probe detection reagents, SCLC-N subtype fluorescent probe detection reagents, SCLC-A / N subtype fluorescent probe detection reagents, SCLC-P subtype fluorescent probe detection reagents, SCLC-I subtype fluorescent probe detection reagents, and leukocyte fluorescent probe detection reagents according to the type of analyte and SCLC molecular typing. The SCLC-A subtype fluorescent probe detection reagent consists of ASCL1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled PanCK. The SCLC-N subtype fluorescent probe detection reagent consists of NEUROD1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled MYC. The SCLC-A / N subtype fluorescent probe detection group consists of ASCL1 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, NEUROD1 primary antibody, and Alexa647 fluorescently labeled IgG secondary antibody. The SCLC-P subtype fluorescent probe detection reagent consists of POU2F3 primary antibody, Alexa647 fluorescently labeled IgG secondary antibody, and 488 fluorescently labeled AVIL. The SCLC-I subtype fluorescent probe detection reagent is either YAP1 primary antibody, Alexa647 fluorescein-labeled IgG secondary antibody, and FITC fluorescein-labeled CSV, or YAP1 primary antibody, Alexa647 fluorescein-labeled IgG secondary antibody, and 488 fluorescein-labeled VIM. The leukocyte fluorescent probe detection agent is CD45-PE.

4. A non-diagnostic identification method using the detection kit for CTCs based on SCLC molecular typing, employing microfluidic chips and multiplex immunofluorescence probe technology as described in claim 3, characterized in that... Includes the following steps: a) Coating and blocking of biotin-labeled trapping agents in microfluidic chips: The biotin-labeled trapping agent was diluted with a balanced salt solution and injected into the split lane through the chip inlet and incubated. After washing the microfluidic chip with a balanced salt solution, tissue cell fixation solution was injected and fixed. After washing the microfluidic chip with a balanced salt solution, a specific hybridization blocking solution diluted with a diluent was injected and incubated. b) Isolation of peripheral blood mononuclear cells (PBMCs) from SCLC patients: Blood was drawn from SCLC patients and PBMCs were isolated from SCLC patients by density gradient centrifugation using human peripheral blood lymphocyte separation medium. c) Enrichment and capture of CTC cells in a microfluidic chip: Peripheral blood mononuclear cells (PBMCs) from SCLC patients were injected into a microfluidic chip to enrich and capture CTC cells; d) Multiplex fluorescent immunoassay probe hybridization of CTC cells: The reagents in the detection kit, except for the Alexa647 fluorescently labeled IgG secondary antibody, were diluted and injected into the microfluidic chip and incubated. After washing the microfluidic chip with balanced salt solution, the Alexa647 fluorescently labeled IgG secondary antibody diluted with diluent was added and incubated. d) Viable nuclear staining of CTC cells: After cleaning the microfluidic chip with balanced salt solution, viable nuclear staining solution diluted with diluent was injected and incubated. f) Scanning and interpretation analysis of CTC cells: CTC cells were scanned, identified and analyzed using a four-color channel automated fluorescence scanning system.

5. The non-diagnostic identification method of the CTC detection kit based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology as described in claim 4, characterized in that: In step a), the biotin-labeled capture agent is diluted 50 to 100 times and incubated at room temperature for 0.5 to 2 hours; the tissue cell fixative is fixed for 5 to 15 minutes at room temperature; and the specific hybridization blocking solution is diluted 200 to 400 times and incubated at room temperature for 10 to 30 minutes.

6. The non-diagnostic identification method of the CTC detection kit based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology as described in claim 4, characterized in that: In step d), the detection reagent is diluted 50 to 200 times and incubated at room temperature for 0.5 to 1.5 hours.

7. The non-diagnostic identification method of the CTC detection kit based on SCLC molecular typing using microfluidic chip and multiplex immunofluorescence probe technology as described in claim 4, characterized in that: In step e), the incubation time of the active nuclear stain solution at room temperature is 5 to 15 minutes.

Images