CN116466089B

CN116466089B - SCLC molecular typing-based CTC detection kit adopting microfluidic chip and multiple immunofluorescence probe technology and identification method thereof

Info

Publication number: CN116466089B
Application number: CN202310467024.7A
Authority: CN
Inventors: 张开山; 于杰; 田华
Original assignee: HANGZHOU WATSON BIOTECH Inc
Current assignee: HANGZHOU WATSON BIOTECH Inc
Priority date: 2023-04-27
Filing date: 2023-04-27
Publication date: 2025-10-31
Anticipated expiration: 2043-04-27
Also published as: CN116466089A

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

SCLC molecular typing-based CTC detection kit adopting microfluidic chip and multiple immunofluorescence probe technology and identification method thereof

[ Field of technology ]

The invention relates to the technical field of molecular typing of Small Cell Lung Cancer (SCLC), in particular to the technical field of a detection kit and an identification method thereof based on SCLC molecular typing CTC (circulating tumor cells) by adopting a microfluidic chip and multiple immunofluorescence probe technology.

[ Background Art ]

Small Cell Lung Cancer (SCLC) is an aggressive neuroendocrine tumor (Byers and Rudin, 2015) with early metastasis and poor prognosis, accounting for 13-17% of all lung cancer types. Because SCLC has high deterioration degree, distant metastasis easily occurs in early stage, and the prognosis is usually in late stage and extremely bad. Chemotherapy and radiation therapy are currently the most traditional treatments for small cell lung cancer. In recent years, immunotherapy has progressed rapidly, and the U.S. FDA has approved the use of either alemtuzumab (Atezolizumab) or divaruzumab (Durvalumab) in combination with etoposide + platinum-based first-line treatment of broad-phase small cell lung cancer, nivolumab (Nivolumab) ±ipilimumab (Ipilimumab) and palbocizumab (Pembrolizumab) for the treatment of small cell lung cancer that had previously undergone platinum-based chemotherapy and disease progression following at least one other therapy. Despite the increased immunotherapy in platinum-based first-line chemotherapy, progression Free Survival (PFS) and total survival (OS) prolongation in SCLC patients is not evident (Chung et al, 2020; paz-Ares et al, 2019). The high responsiveness of SCLC to platinum-based chemotherapy regimens, including complete remission, in the 70 s and 80 s of the 20 th century led to believing that SCLC could heal soon. Forty years later, the 5-year survival rate of SCLC patients remains at 5% due to inherent or most common acquired therapeutic resistance. There have also been advances in targeted drug therapies for small cell lung cancer, such as temozolomide (Temozolomide) in combination with the PARP inhibitor olaparib (Olaparib) to treat recurrent small cell lung cancer, while An Luoti ni has been approved in china for tri-line therapy of recurrent small cell lung cancer. The ascin derivative Lurbinectedin (RNA polymerase II inhibitor) from PHARMAMAR has been filed on the market based on the results of a secondary study and can be used for the secondary treatment of recurrent small cell lung cancer. Unlike non-small cell lung cancer (NSCLC), however, its biomarker selection for targeted and immunotherapy significantly alters the traditional therapeutic profile (Zimmermann et al, 2018), the clinical targeted therapy study of small cell lung cancer is mainly focused on non-selected populations, the results of which are generally disappointing, and there has been no significant progress since cisplatin and etoposide have been made. The driving factors inherent in small cell lung cancer are still unclear from chemosensitivity to chemoresistance and rapid progression. Therefore, the molecular typing and subtype characteristics of SCLC are more clearly and precisely defined to determine the precise selection and efficacy assessment of targeted therapies and immunotherapies, which are the current urgent problems to be solved.

In 2019-2020, numerous researchers compiled xenograft (PDX) data from cell lines, patient sources, and mouse transgenic model (GEMMs) data and conducted comprehensive SCLC genome studies, suggesting heterogeneous SCLC molecular subtype classification based on mRNA expression profiling differentiation program control. That is, classifying the SCLC subtype into a Neuroendocrine (NE) and a non-neuroendocrine (non-NE) type according to the Neuroendocrine (NE) phenomenon prevalent in SCLC increases the SCLC non-NE cluster cell variant subtype which has not been found before, and supplements the SCLC subtype defined by the transcription factor POU2F 3. Furthermore, since there are still many patients not belonging to these three subtypes, the fourth subtype driven by the transcription factor YAP1 provides a partial solution for this part of the unclassified tumor. Recent consensus suggests that SCLC is divided into five subtypes, ASCL high expression (SCLC-A), NEUROD1 high expression (SCLC-N), ASCL co-expression with NEUROD1 (SCLC-A/N), POU2F3 high expression (SCLC-P) and YAP1 high expression (SCLC-I). According to molecular characteristics of SCLC of Chinese population and correlation analysis of tissue samples and cell line expression profiles, the sensitivity difference of different molecular subtypes of SCLC to different medicines is found. Wherein, the SCLC-A type accounts for 41 percent, is sensitive to A BCL-2 inhibitor or A PARP inhibitor combined immune checkpoint inhibitor, the SCLC-N type accounts for 8 percent, is sensitive to non-interstitial and non-epithelial aurorA kinase inhibitors, the SCLC-A/N subtype accounts for 37 percent, the SCLC-P type accounts for 7 percent, is sensitive to the PARP inhibitor and the nucleoside analogue, the SCLC-I type accounts for 7 percent, and is sensitive to the immune checkpoint inhibitor. Current clinical laboratory studies are exploring the relationship of different SCLC molecular subtypes to immunobiology. The challenge faced by SCLC clinical treatment is how to select appropriate markers with optimal strategies, thereby accurately identifying the benefited population, and the accurate identification of SCLC molecular subtypes helps to provide patients with personalized accurate treatment regimens.

SCLC is a very invasive disease and there are limits to the available alternatives. Although the patient initially responds to the treatment, it quickly recurs. To date, there are no approved targeted drugs for SCLC or biomarkers to direct treatment. Temozolomide (temozolomide) has a therapeutic effect on recurrent SCLC. Small cell lung cancer patients rarely undergo surgery and the commonly available tissue samples are insufficient for biomarker analysis. Most SCLC patients (about 70%) manifest themselves as extensive SCLC (ES-SCLC) at diagnosis, with the remaining 30% being restricted SCLC (LS-SCLC). The prognosis for small cell lung cancer is poor, and the median total survival (OS) in ES-SCLC patients is 10 months, which can be up to 4 years. Platinum-based chemotherapy in combination with etoposide or irinotecan is the first line treatment of the SCLC standard. Recently, immune Checkpoint Inhibitors (ICIs), alone or in combination with chemotherapy, have been approved for the treatment of SCLC. Despite the high initial response to chemotherapy (ici alone or in combination), most patients frequently experience recurrent and metastatic disease quickly and with poor prognosis. Only approved two-wire drug topotecan has a lower response rate and shorter survival. Unlike non-small cell lung cancer (NSCLC) and other cancer types, small cell lung cancer has little choice of other treatment modalities, nor has a targeted treatment regimen been targeted for treatment of advanced patients. The highly invasive nature of SCLC and the lack of effective positive therapies indicate that there is an urgent need for targeted biomarker analysis and identification, and these markers can aid in the selection of SCLC personalized treatment regimens and the development of targeted drugs.

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, study drug resistance mechanisms, and discover new targets for treatment. Although ctDNA detection is a relatively common method in the early detection of clinical tumors at present, a great deal of research in SCLC has been focused on CTCs. Circulating tumor cells (Circulating Tumor Cell, CTCs) are the collective name for tumor cells that shed from primary and metastatic tumors and enter the peripheral blood circulation (one CTC per 106-107 leukocytes), and are the primary cause of cancer development and metastasis. The CSCO guidelines of the 2021 edition indicate that CTCs, as a "liquid biopsy sample" representing a primary tumor, can be used to monitor the condition of a tumor patient in real-time, dynamically, and noninvasively. Researches prove that SCLC cell division period is short, increment is fast, blood circulation is easy to enter, remote transfer occurs, and the detection rate of CTC in SCLC population is 67-86%. The detection of CTCs is helpful for accurately judging clinical stages of diseases, so that proper clinical schemes can be selected, personalized treatment of SCLC patients can be guided, tumor recurrence and metastasis can be monitored, treatment efficacy can be judged, prognosis survival can be predicted, and the method is a means for analyzing drug-resistant molecular mechanisms and solving tumor heterogeneity. Previous studies have also shown that SCLC patients have a relatively higher number of CTCs than NSCLC patients, and extensive stage SCLC (ES-SCLC) patients have a relatively higher number of CTCs than restricted stage (LS-SCLC) SCLC patients. In recent years, technological advances in CTC isolation methods, as well as the possibility of single CTC molecule characterization studies, have helped assess the potential role of CTCs as biomarkers for SCLC efficacy detection and monitoring disease progression in order to study tumor heterogeneity and drug resistance mechanisms of treatment. Furthermore, the study of CTC-derived xenograft (CDX) models may also provide complementary information on the therapeutic sensitivity/resistance mechanisms for genomic analysis and CTC counting, studying tumor heterogeneity and drug resistance mechanisms in CDX in vivo.

Currently, the existing CTC detection methods in the market mainly comprise a membrane filtration method, an immunomagnetic bead technology and a microfluidic chip detection method. The membrane filtration method can separate CTC by utilizing the difference of cell sizes, but the filtration purity is low, the immunomagnetic beads are mainly combined with magnetic beads and antibodies, cells are captured by combining with antigens on the surface of the CTC, the cell activity is usually destroyed, the follow-up such as cell culture can not be further realized, and the number of CTCs (for expressing epithelial markers EpCAM and cytokeratin CK 8/18/19) detected by a CellSearch system has independent prognostic significance in SCLC. However, the evolution of the pathogenesis of small cell lung cancer from SCLC-A subtype to SCLC-I subtype is the conversion process of tumor cells from epithelial type to interstitial type EMT, so that the accurate classification and identification of SCLC five molecular subtypes cannot be carried out by simply adopting A CTC detection and identification method based on epithelial marker EPCAM capture, and thus A clinician cannot be accurately assisted to provide accurate pathological classification and establishment of personalized treatment strategies for SCLC patients.

[ Invention ]

The application aims to solve the problems in the prior art and provides a detection kit based on SCLC molecular typing of CTC by adopting a microfluidic chip and multiple immunofluorescence probe technologies and an identification method thereof. The application adopts the design combination of the fluorescent probes of the key markers for identifying five molecular subtypes of SCLC (SCLC-A subtype, ASCL < 1 > -AlexA647 and PanCK; SCLC-N subtype, NEUROD < 1 > -AlexA647 and Myc-488; SCLC-A/N subtype, ASCL < 1 > -AlexA647 and NEUROD < 1 > -AlexA647; SCLC-P subtype, POU < 2F < 3 > -AlexA647 and AVIL-488; SCLC-I subtype YAP < 1 > -AlexA647 and CSV-FITC or VIM-488), and simultaneously adds the auxiliary white blood cell identification marker CD45-PE, can carry out the identification and analysis of the multi-immunity fluorescent probe combination of the CTC based on the enrichment of micro-fluidic chip aiming at the peripheral blood sample (1-2 ml/part) halving five parts of the same patient, and can detect the molecular subtype of SCLC of the patient in A non-invasive, early and sensitive dynamic way. The application designs a high-efficiency microfluidic CTC enrichment chip and a corresponding CTC capture reagent and five SCLC molecular subtype identification multiplex immunofluorescence probe combination aiming at five molecular subtype characteristic molecular markers (ASCL, NEUROD1, POU2F3 and YAP 1) of SCLC A, N, A/N, P and I, and can utilize the existing molecular subtype identification multiplex immunofluorescence probe combinationThe CTC detection system platform is used for internationally developing SCLC circulating tumor cell identification and detection kits and methods closely related to clinical treatment, is used for assisting molecular typing identification of clinical SCLC, prediction of clinical curative effect, early warning of disease recurrence, screening of clinical medicines and the like, and fills up the blank at home and abroad.

In order to achieve the aim, the invention provides a detection kit of SCLC molecular typing-based CTC by adopting a microfluidic chip and multiple immunofluorescence probe technology, which comprises a microfluidic chip, a biotin-marked capturing agent, a balanced salt solution, a tissue cell fixing solution, a specific hybridization blocking solution, an active nuclear dye solution and a diluent, and also comprises a subtype key marker of SCLC marked by different fluorescein groups, an SCLC related marker detection antibody and a leucocyte auxiliary identification marker or a detection agent formed by the subtype key marker of SCLC marked by different fluorescein groups and a leucocyte auxiliary identification marker;

the subtype key markers of SCLC include at least one of ASCL1, NEUROD1, POU2F3 and YAP 1;

the SCLC-related marker detection antibody comprises at least one of PanCK, MYC, AVIL, CSV and VIM.

Preferably, a plurality of shunting lanes with flow sections in a double-row herring bone structure are arranged in the microfluidic chip.

Furthermore, 8 grooves with double herring bone structures are arranged in the microfluidic chip, and the grooves are provided with flow distribution channels which are staggered and connected in series according to a specific angle.

Preferably, the modified silane agent, the bifunctional crosslinking agent and the streptavidin are coupled in sequence in the split lanes.

The preparation method of the microfluidic chip comprises the steps of uniformly coating photoresist on a silicon wafer subjected to plasma cleaning, heating, drying, sequentially exposing, heating and developing to obtain a photoresist nano array, pouring PDMS (polydimethylsiloxane) adhesive based on the photoresist nano array, punching and cutting the photoresist, performing plasma cleaning and bonding to obtain the PDMS substrate, preparing ② functionalized probe-loaded glass slide, performing silanization treatment on the surface of the glass slide subjected to oxygen plasma treatment by using 3-aminopropyl triethoxy, sequentially combining a bifunctional amine-sulfhydryl cross-linking agent and Streptavidin (SA), and ③ bonding, wherein the bonding of the microfluidic flow channel substrate and the functionalized glass slide loaded by a functionalized probe in the peripheral non-flow channel edge region is performed after oxygen plasma treatment.

Preferably, the biotin-labeled capture agents are classified into an SCLC-A subtype capture agent, an SCLC-N subtype capture agent, an SCLC-A/N subtype capture agent, an SCLC-P subtype capture agent and an SCLC-I subtype capture agent by SCLC molecule;

The SCLC-A subtype capture agent is A biotinylated epithelial tumor marker EpCAM;

The SCLC-N subtype capturing agent is biotinylation epithelial tumor marker EpCAM and interstitial tumor marker vimentin CSV or biotinylation epithelial tumor marker EpCAM and interstitial tumor marker vimentin;

the SCLC-A/N subtype capturing agent is biotinylation epithelial tumor marker EpCAM and interstitial tumor marker vimentin CSV or biotinylation epithelial tumor marker EpCAM and interstitial tumor marker vimentin VIM;

the SCLC-P subtype capturing agent is a biotinylation epithelial tumor marker EpCAM and a small cell lung cancer P subtype characteristic marker POU2F3;

The SCLC-I subtype capturing agent is a tumor marker receptor tyrosine kinase AXL with high expression of a matrix tumor marker vimentin CSV and a small cell lung cancer I subtype.

Preferably, the balanced salt solution is PBS buffer solution, the tissue cell fixing solution is PFA fixing solution, the specific hybridization blocking solution is FC receptor blocking solution, the active nuclear dye solution is Hoechst33342DNA fluorescent dye solution, and the dilution solution is 1 xADB antibody dilution buffer solution.

Preferably, the detection agent is classified into an SCLC-A subtype fluorescent probe detection agent, an SCLC-N subtype fluorescent probe detection agent, an SCLC-A/N subtype fluorescent probe detection agent, an SCLC-P subtype fluorescent probe detection agent, an SCLC-I subtype fluorescent probe detection agent and A leukocyte fluorescent probe detection agent according to the kind of the identifier and the molecular classification of SCLC;

the SCLC-A subtype fluorescent probe detection agent is ASCL primary antibody, alexA647 fluorescein labeled IgG secondary antibody and 488 fluorescein labeled PanCK;

the SCLC-N subtype fluorescent probe detection agent is NEUROD1 primary antibody, alexa647 fluorescein labeled IgG secondary antibody and 488 fluorescein labeled MYC;

The SCLC-A/N subtype fluorescent probe detection group comprises ASCL first antibody, alexA647 fluorescein labeled IgG second antibody, NEUROD1 first antibody and AlexA647 fluorescein labeled IgG second antibody;

The SCLC-P subtype fluorescent probe detection agent is POU2F3 primary antibody, alexa647 fluorescein labeled IgG secondary antibody and 488 fluorescein labeled AVIL;

The SCLC-I subtype fluorescent probe detection agent is 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.

The five groups of SCLC molecular subtype marker probes can be used for simultaneously carrying out multiple immunofluorescence probe combination identification on five peripheral blood samples (1-2 ml/serving) halved by the same patient.

The biotin-labeled capture agent combinations and detector combinations for SCLC five molecular subtype one-time CTC enrichment isolation and identification are referenced in table 1 below:

TABLE 1 separation and identification of five molecular subtypes of SCLC one-time CTC enrichment, biotin-labeled Capture agent combination and detection agent group

Wherein, the fluorescein labeling characteristics of the multiplex immunodetection agent probe respectively label the fluorescein with different emission wavelengths which can be distinguished from each other aiming at SCLC molecular subtype identification, ASCL, NEUROD1, POU2F3 and YAP1 are respectively labeled by primary antibodies and corresponding Alexa Fluor 647 red fluorescein-labeled serial IgG secondary antibodies, panCK, MYC, AVIL and CSV (or VIM) are respectively labeled by FITC (isothiocyanate) or 488 green fluorescein, and CD45 is labeled by PE (phycoerythrin) orange fluorescein. The SCLC five-molecule typed CTC cells identified by adopting the combination of multiple immunofluorescence probes marked by different luciferins can be completely distinguished under different optical filters by a fluorescence microscope.

The identification method of the detection kit based on SCLC molecular typing CTC by adopting a microfluidic chip and multiple immunofluorescence probe technology comprises the following steps:

a) The method comprises the steps of coating and sealing a biotin-marked capturing agent of a microfluidic chip, diluting the biotin-marked capturing agent by using a balanced salt solution, injecting the diluted capturing agent into a shunting lane from a chip inlet, incubating, injecting tissue cell fixing solution into the microfluidic chip after the microfluidic chip is washed by using the balanced salt solution, fixing the tissue cell fixing solution, injecting specific hybridization blocking solution diluted by a diluent after the microfluidic chip is washed by using the balanced salt solution, and incubating;

b) Peripheral blood mononuclear cell PBMC of SCLC patient is separated by extracting blood of SCLC patient and performing density gradient centrifugation with human peripheral blood lymphocyte separation liquid to obtain peripheral blood mononuclear cell PBMC of SCLC patient;

c) Enrichment and capture of a microfluidic chip of CTC cells, namely injecting peripheral blood mononuclear cell PBMC of an SCLC patient into the microfluidic chip to enrich and capture the CTC cells;

d) Multiple fluorescence immunity in situ probe hybridization of CTC cells, namely, diluting the reagent except Alexa647 fluorescein labeled serial IgG secondary antibodies in the detection agent, injecting the diluted reagent into a microfluidic chip for incubation, and then adding the diluted Alexa647 fluorescein labeled IgG secondary antibodies into the microfluidic chip for incubation after washing the microfluidic chip by using balanced salt solution;

e) The reactive nucleus staining of CTC cells, namely injecting reactive nucleus staining solution diluted by a diluent after the micro-fluidic chip is washed by using a balanced salt solution, and incubating;

f) And (3) scanning and interpretation analysis of CTC cells, namely scanning, identifying and analyzing the CTC cells by adopting a four-color channel automatic fluorescence scanning system.

Preferably, in the step a), the biotin-labeled capturing agent is diluted by 50-100 times and incubated for 0.5-2 hours at room temperature, the tissue cell fixing solution is fixed for 5-15 min at room temperature, and the specific hybridization blocking solution is diluted by 200-400 times and incubated for 10-30 min at room temperature.

Preferably, in the step d), the detection agent is diluted by 50-200 times and incubated for 0.5-1.5 h at room temperature.

Preferably, in the step e), the incubation time of the active nuclear dye solution at room temperature is 5-15 min.

The invention has the beneficial effects that:

1) The invention designs and develops a microfluidic matrix CTC enrichment chip based on a groove herring double-row herringbone structure, the geometrical structure design effectively enhances the contact probability of CTC surface antigens and a capturing agent, and further realizes high-efficiency and specific CTC enrichment by adjusting the flow rate and the shearing force direction and the size of microfluid under the condition of keeping the small dosage of a patient blood sample (0.2-1 ml), thereby greatly improving the capturing efficiency of CTC cells in body fluids such as peripheral blood, cerebrospinal fluid, pleuroperitoneal cavity ridge fluid, urine and the like of the patient, keeping the integrity of the enriched CTC cell morphology, reducing the retention of White Blood Cells (WBC) and greatly improving the CTC enrichment purity of the microfluidic chip.

2) According to the invention, the substrate layer in the inner cavity of the microfluidic chip is sequentially subjected to silanization, a bifunctional protein cross-linking agent, streptavidin (SA) coupling modification and biotinylation antibody capturing agent coating, so that CTC immune enrichment based on cascade signal amplification reaction of streptavidin and biotin is realized, and compared with other microfluidic chips, CTC enrichment capturing efficiency of peripheral blood and other body fluids of a patient is greatly improved.

3) According to the invention, five multi-capture reagent combinations (SCLC-A subtype capture reagent, SCLC-N subtype capture reagent, SCLC-A/N subtype capture reagent, SCLC-P subtype capture reagent and SCLC-I subtype capture reagent) of the SCLC molecular typing (A, N, A/N, P and I subtype) marked by the CTC biotin of the SCLC subtype are designed, so that the CTC cells of all molecular subtypes in A blood sample and chest fluid of an SCLC patient can be enriched and captured at one time with high efficiency and specificity.

4) The invention designs A multiple fluorescent probe combination (SCLC-A subtype fluorescent probe detection agent, SCLC-N subtype fluorescent probe detection agent, SCLC-A/N subtype fluorescent probe detection agent, SCLC-P subtype fluorescent probe detection agent and SCLC-I subtype fluorescent probe detection agent) of characteristic markers for SCLC molecular subtype identification (A, N, A/N, P and I subtype), which can detect the SCLC molecular subtype period of A patient at one time in A noninvasive, early and sensitive dynamic way, thereby developing SCLC circulating tumor cell identification and detection kit and method closely related to clinical treatment for assisting the molecular subtype identification of clinical SCLC, the prediction of clinical curative effect, the disease recurrence early warning, the screening of clinical medicines and the like on the international basis, and filling the gap between the country and the foreign.

5) According to the identification and detection method of the SCLC molecular parting circulating tumor cells based on the micro-fluidic chip and the multiplex immunofluorescence probe technology, SCLC and molecular parting thereof can be found and confirmed earlier than clinical imaging FDG-PET/CT and PET/MRI and nuclear marker imaging PET/CT, the defects that the sampling requirement is higher (IHC detection identification is needed to be carried out on tumor tissues) in IHC detection of tumor patients, the limitation that the detection accuracy of each center is different due to the influence of factors such as a certain subjectivity and heterogeneity, the quality of detection antibodies and the detection process (fixation and dyeing) and the like are present are avoided, and therefore, a clinician can be assisted to intervene in the drug administration guidance and individuation accurate treatment scheme formulation of the tumor patients early, and noninvasive auxiliary dynamic curative effect monitoring and prognosis evaluation can be carried out on tumor patients in postoperative recurrence and metastasis stage accurately.

The features and advantages of the present invention will be described in detail by way of example with reference to the accompanying drawings.

[ Description of the drawings ]

FIG. 1 shows the identification results of five circulating tumor cells based on SCLC molecular typing by microfluidic technology in the first example;

Fig. 2 is a schematic view of a microfluidic design of a microfluidic chip according to the first embodiment;

Fig. 3 is a design diagram of an inlet and outlet structure of a microfluidic chip according to the first embodiment;

FIG. 4 is an enlarged schematic view of a flow section of herring bone structure in a herring shape of a split lane of the microfluidic chip of the first embodiment;

Fig. 5 is a graph of CTC microfluidic chip capture rate of gradient dilution of MCF7 as a quality control cell in example two.

[ Detailed description ] of the invention

Referring to the following table 2, five detection kits based on SCLC molecular typing CTCs using microfluidic chip and multiplex immunofluorescence probe technology are taken and identified respectively, and the specific operation steps are as follows:

a) The coating and sealing of biotin-marked capturing agents of the microfluidic chip comprises the steps of ① respectively taking the biotin-marked capturing agents in table 2, adding PBS buffer solution to a 60 mu L system, injecting the mixture into a shunting lane of the microfluidic chip from a chip inlet after uniform mixing and incubating for 1.5 hours at room temperature, ② respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution, ③ respectively cleaning the microfluidic chip twice by adopting 100 mu L of 2% PFA fixing solution at room temperature for 10 minutes, ④ respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution, ⑤ respectively taking 12 mu L of FC receptor blocking solution, adding 48 mu L of 1 xADB antibody dilution buffer solution, injecting the mixture into the microfluidic chip after uniform mixing and incubating for 20 minutes at room temperature;

d) The fluorescence immunity in situ hybridization of CTC cells comprises ① respectively taking detection agents of corresponding types and dosage in Table 2, adding 1 xADB antibody dilution buffer solution to 60 mu L system, mixing, injecting into microfluidic chip, incubating at room temperature for 1h, ② respectively cleaning microfluidic chip twice with 100 mu L PBS buffer solution, ③ respectively taking fluorescent probes (various detection antibodies and subtype key markers of SCLC molecules are respectively marked with different luciferins), adding 1 xADB antibody dilution buffer solution to 60 mu L system, mixing, injecting into microfluidic chip, and incubating at room temperature for 1h;

e) The active nuclei of CTC cells are stained by ① adopting 100 mu L of PBS buffer solution to respectively wash the microfluidic chip twice, ② adopting 200 mu L of 30 mu g/mL Hoechst 33342DNA fluorescent dye solution to incubate for 10min;

The preparation method of the microfluidic chip comprises the following steps:

a) Uniformly coating SU-8 photoresist on a silicon wafer subjected to plasma cleaning, sequentially performing pre-baking, ultraviolet exposure, post-baking and development to obtain a photoresist nano array, sequentially pouring PDMS photoresist twice on the basis of the photoresist nano array, and performing plasma cleaning after punching and photoresist cutting (the shape is shown in figures 2-4) to obtain the PDMS nano substrate;

b) Preparing a functional group modified slide, namely performing silanization treatment on the surface of the slide subjected to oxygen plasma treatment by using 3-hydroxypropyl trimethoxy silane (3-MPTS), and sequentially combining maleimide-PEG 2-biotin, streptavidin (SA) and a biotinylation monoclonal antibody to obtain the functional group modified slide;

c) Combining PDMS nano substrate and functional group modified slide.

TABLE 2 five major parameters of SCLC molecular typing-based circulating tumor cell identification and detection kit employing microfluidic technology

In addition, five cell lines and actions aimed at by the SCLC molecule-based differentiation and detection kit for circulating tumor cells using microfluidic technology are shown in table 3 below:

SCLC subtype classification	Cell line name	Action
			SCLC-A	DMS153	Quality control product for human SCLC-A positive detection
SCLC-N	NCIH524	Quality control product for human SCLC-N positive detection
			SCLC-A/N	CORL279	Quality control product for human SCLC-A/N positive detection
SCLC-P	NCIH526	Quality control product for human SCLC-P positive detection
			SCLC-I	SW1271	Quality control product for human SCLC-I positive detection

TABLE 3 identification and Effect of five circulating tumor cells based on SCLC molecular typing by microfluidic technique on cell lines and Effect directed by the kit

The identification results of five detection kits based on SCLC molecular typing CTC by adopting microfluidic chip and multiplex immunofluorescence probe technology are shown in figure 1. In addition, the prepared microfluidic chip was used for capturing and testing the quality control cell MCF7, and the test results are shown in fig. 5.

The above embodiments are illustrative of the present invention, and not limiting, and any simple modifications of the present invention fall within the scope 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 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;

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 as described in claim 3, employing microfluidic chips and multiplex immunofluorescence probe technology, characterized by comprising 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 washing 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 detection kit for CTC 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 50-100 times and incubated at room temperature for 0.5-2 hours; the fixation time of the tissue cell fixative at room temperature is 5-15 minutes; and the specific hybridization blocking solution is diluted 200-400 times and incubated at room temperature for 10-30 minutes.

6. The non-diagnostic identification method of the detection kit for CTC based on SCLC molecular typing using microfluidic chip and multiple 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 h.

7. The non-diagnostic identification method of the detection kit for CTC 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 staining solution at room temperature is 5 to 15 min.