CN116466089A - SCLC molecular typing-based CTC detection kit adopting microfluidic chip and multiple immunofluorescence probe technology and identification method thereof - Google Patents
SCLC molecular typing-based CTC detection kit adopting microfluidic chip and multiple immunofluorescence probe technology and identification method thereof Download PDFInfo
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Abstract
The invention discloses a detection kit based on SCLC molecular typing and an identification method thereof 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, a dilution solution, and also comprises a subtype key marker of SCLC marked by different fluorescein groups, an SCLC related marker detection antibody and a leukocyte auxiliary identification marker or a detection agent formed by the subtype key marker of SCLC marked by different fluorescein groups and a leukocyte auxiliary identification marker. The method can noninvasively, early and sensitively detect and analyze expression of four immune markers of ASCL1, NEUROD1, POU2F3 and YAP1 related to CTC in peripheral blood of a patient, is suitable for early screening of tumors, and can also conduct immune medication guidance, immune curative effect monitoring and prognosis evaluation of cancer.
Description
[ 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 with early metastasis and poor prognosis (Byers and Rudin, 2015), 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 alemtuzumab (Atezolizumab) or dulvacizumab (Durvalumab) in combination with etoposide + platinum-based first-line treatment of broad-phase small cell lung cancer, nivolumab (Nivolumab) ±ipilimumab) and pamglizumab (Pembrolizumab) for the treatment of small cell lung cancer that has 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 ecteinascidin derivative lurbingedin (RNA polymerase ii inhibitor) from pharamar has been filed on the market for secondary treatment of recurrent small cell lung cancer based on the results of secondary studies. However, unlike non-small cell lung cancer (NSCLC), its biomarker selection for targeted and immunotherapy significantly alters the traditional therapeutic profile (Zimmermann et al, 2018), and clinical targeted therapy studies of small cell lung cancer have focused mainly on non-selected populations, with generally disappointing findings, and no significant progress has been made since the advent of cisplatin and etoposide. 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 through 2020, numerous researchers compiled xenograft (PDX) data and mouse transgenic model (GEMMs) data from cell lines, patient sources 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 classifying SCLCs 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). 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, SCLC-A accounts for 41% and is sensitive to BCL-2 inhibitor or PARP inhibitor combined immune checkpoint inhibitor; SCLC-N type accounts for 8% and is sensitive to non-interstitial and non-epithelial Aurora kinase inhibitors; SCLC-A/N subtype accounts for 37%; SCLC-P type accounts for 7% and is sensitive to PARP inhibitors and nucleoside analogues; SCLC-type I represents 7% and is sensitive to immune checkpoint inhibitors. 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 2021 version of the CSCO guidelines indicated that CTCs, as a "liquid biopsy sample" representing a primary tumor, could be monitored in real-time, dynamically and noninvasively for the condition of a tumor patient. 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. Among them, membrane filtration can use cell size differentiation to isolate CTCs, but often has low filtration purity; the immune magnetic beads are mainly combined with the magnetic beads and the antibodies, and then are combined with antigens on the surface of CTC to capture cells, so that the cell activity is generally destroyed, and the follow-up steps such as cell culture and the like can not be further realized; the number of CTCs detected using the CellSearch system (expressing the epithelial markers EpCAM and cytokeratin CK 8/18/19) 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 se:Sub>A CTC detection and identification method based on epithelial marker EPCAM capture, and thus se:Sub>A clinician cannot be accurately assisted to provide accurate pathological classification and establishment of personalized treatment strategies for SCLC patients.
[ invention ]
The invention 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 method adopts se:Sub>A key marker fluorescent probe design combination (SCLC-A subtype, ASCL 1-Alexse:Sub>A 647 and PanCK488; SCLC-N subtype, NEUROD 1-Alexse:Sub>A 647 and Myc-488; SCLC-A/N subtype, ASCL 1-Alexse:Sub>A 647 and NEUROD 1-Alexse:Sub>A 647; SCLC-P subtype, POU2F 3-Alexse:Sub>A 647 and AVIL-488; SCLC-I subtype YAP 1-Alexse:Sub>A 647 and CSV-FITC or VIM-488) for identification of the white blood cell, and simultaneously adds an auxiliary white blood cell identification marker CD45-PE, so that the method can perform CTC multiplex immunofluorescent probe combination identification and analysis based on microfluidic chip enrichment for five parts of peripheral samples (1-2 ml/part) of the same patient, and can detect the SCLC molecular subtype period of the patient in se:Sub>A non-invasive blood sample, early and sensitive dynamic manner. The application designs a high-efficiency microfluidic CTC enrichment chip and a corresponding combination of a CTC capture reagent and a multiplex immunofluorescence probe for identifying five molecular subtype of SCLC aiming at five molecular subtype characteristic molecular markers (ASCL 1, NEUROD1, POU2F3 and YAP 1) of SCLC A, N, A/N, P and I, and can utilize the existing methodsThe 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 marker of SCLC comprises 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.
Preferably, the preparation method of the microfluidic chip comprises the following steps: (1) manufacturing a microfluidic flow channel PDMS substrate: uniformly coating photoresist on a silicon wafer subjected to plasma cleaning, heating and drying, sequentially exposing, heating and developing to obtain a photoresist nano array, pouring PDMS (polydimethylsiloxane) adhesive on the basis of the photoresist nano array, punching and cutting the photoresist, performing plasma cleaning, and bonding to obtain a PDMS substrate; (2) preparation of functional probe-loaded glass slides: silanization treatment is carried out on the surface of the glass slide after oxygen plasma treatment by using 3-aminopropyl triethoxy, and then a bifunctional amine-sulfhydryl cross-linking agent and Streptavidin (SA) are respectively combined in sequence; (3) bonding: and (3) performing oxygen plasma treatment on the microfluidic flow channel PDMS substrate and the functionalized glass slide loaded by the functionalized probe, and then bonding the peripheral non-flow channel edge regions.
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-se:Sub>A subtype capture agent is se:Sub>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; the tissue cell fixing liquid is PFA fixing liquid; the specific hybridization blocking solution is FC receptor blocking solution; the active nuclear dye solution is Hoechst33342DNA fluorescent dye solution; the dilution was 1 x ADB antibody dilution buffer.
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 se:Sub>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 ASCL1 primary antibody, alexse:Sub>A 647 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 an ASCL1 primary antibody, an Alexse:Sub>A 647 fluorescein labeled IgG secondary antibody, se:Sub>A NEUROD1 primary antibody and an Alexse:Sub>A 647 fluorescein labeled IgG secondary 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 sets of SCLC molecular subtype marker probes can be simultaneously identified by multiple immunofluorescence probe combinations aiming at the peripheral blood sample (1-2 ml/serving) halved into five parts of 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 features of the multiplex immunodetector probes each label fluorescein of different emission wavelengths distinguishable from each other for identification of SCLC molecular subtypes: ASCL1, NEUROD1, POU2F3 and YAP1 all adopt primary antibodies and corresponding Alexa Fluor 647 red fluorescein labeled serial IgG secondary antibodies; panCK, MYC, AVIL and CSV (or VIM) are marked by FITC (isothiocyanate) or 488 green fluorescein; CD45 is labeled with 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) Coating and blocking of biotin-labeled capture agent of microfluidic chip: injecting a capturing agent marked by biotin into a shunting lane from a chip inlet after being diluted by balanced salt solution, incubating, injecting tissue cell fixing solution into a microfluidic chip after the microfluidic chip is cleaned by using the balanced salt solution, fixing, injecting specific hybridization blocking solution diluted by diluent after the microfluidic chip is cleaned by using the balanced salt solution, and incubating;
b) Peripheral blood mononuclear cell PBMC isolation of SCLC patients: extracting blood of an SCLC patient and adopting a human peripheral blood lymphocyte separation liquid to perform density gradient centrifugation to separate peripheral blood mononuclear cell PBMC of the SCLC patient;
c) Enrichment and capture of microfluidic chip of CTC cells: injecting peripheral blood mononuclear cell PBMCs of SCLC patients into the microfluidic chip to enrich the captured CTC cells;
d) Multiplex fluorescent immunity in situ probe hybridization of CTC cells: diluting the reagent except Alexa647 fluorescein marked serial IgG secondary antibodies in the detection agent, injecting the diluted reagent into the microfluidic chip for incubation, and then adding the diluted Alexa647 fluorescein marked IgG secondary antibodies into the microfluidic chip for incubation after washing the microfluidic chip by using a balanced salt solution;
e) Active nuclear staining of CTC cells: injecting the reactive nuclear dye solution diluted by the diluent after the microfluidic chip is cleaned by using the balanced salt solution, and incubating;
f) Scanning and interpretation analysis of CTC cells: and adopting a four-color channel automatic fluorescence scanning system to scan, identify and analyze the CTC cells.
Preferably, in step a), the biotin-labeled capture reagent is diluted 50-100-fold and incubated at room temperature for 0.5-2 h; the fixing time of the tissue cell fixing solution at room temperature is 5-15 min; the specific hybridization blocking solution is diluted by 200-400 times and incubated for 10-30 min at room temperature.
Preferably, in step d), the detection agent is diluted 50-200 times and incubated at room temperature for 0.5-1.5 h.
Preferably, in step e), the incubation time of the active nuclear dye solution at room temperature is between 5 and 15 minutes.
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 subtype CTC biotin marks are designed aiming at SCLC molecular typing (A, N, A/N, P and I subtype), so that the CTC cells of all molecular subtypes in se:Sub>A blood sample and chest spinal fluid of an SCLC patient can be enriched and captured at one time with high efficiency and specificity.
4) The invention designs se:Sub>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 se:Sub>A patient at one time in se:Sub>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) Coating and blocking of biotin-labeled capture agent of microfluidic chip: (1) taking biotin-marked capturing agents in table 2 respectively, adding PBS buffer solution to a 60 mu L system, injecting the capturing agents into a shunting lane of a microfluidic chip from a chip inlet after uniformly mixing, and incubating for 1.5h at room temperature; (2) respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution; (3) fixing with 100uL of 2% PFA fixing solution at room temperature for 10min; (4) respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution; (5) taking 12 mu L of FC receptor blocking solution, adding 48 mu L of 1 xADB antibody dilution buffer solution, uniformly mixing, injecting into a microfluidic chip, and incubating for 20min at room temperature;
b) Peripheral blood mononuclear cell PBMC isolation of SCLC patients: extracting blood of an SCLC patient and adopting a human peripheral blood lymphocyte separation liquid to perform density gradient centrifugation to separate peripheral blood mononuclear cell PBMC of the SCLC patient;
c) Enrichment and capture of microfluidic chip of CTC cells: injecting peripheral blood mononuclear cell PBMCs of SCLC patients into the microfluidic chip to enrich the captured CTC cells;
d) Fluorescent immunoin situ hybridization of CTC cells: (1) respectively taking the detection agents with the corresponding types and the corresponding amounts in the table 2, adding the 1 xADB antibody dilution buffer solution to a 60 mu L system, injecting the mixture into a microfluidic chip after uniform mixing, and incubating for 1h at room temperature; (2) respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution; (3) taking fluorescent probes (various detection antibodies and subtype key markers of SCLC molecules are respectively marked by adopting different luciferins), adding 1 xADB antibody dilution buffer solution to a 60 mu L system, injecting the mixture into a microfluidic chip after uniform mixing, and incubating for 1h at room temperature;
e) Active nuclear staining of CTC cells: (1) respectively cleaning the microfluidic chip twice by adopting 100 mu L of PBS buffer solution; (2) incubation for 10min with 200. Mu.L of 30. Mu.g/mL Hoechst33342DNA fluorescent dye;
f) Scanning and interpretation analysis of CTC cells: and adopting a four-color channel automatic fluorescence scanning system to scan, identify and analyze the CTC cells.
The preparation method of the microfluidic chip comprises the following steps:
a) Preparation of PDMS nano substrate: 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 (polydimethylsiloxane) 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 a PDMS nano substrate;
b) Preparing a functional group modified slide: silanization treatment is carried out on the surface of the glass slide after oxygen plasma treatment by using 3-hydroxypropyl trimethoxy silane (3-MPTS), and maleimide-PEG 2-biotin, streptavidin (SA) and a biotinylation monoclonal antibody are combined in sequence to obtain a functional group modified glass slide;
c) Combination: and bonding the PDMS nano substrate with the 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 (10)
1. The detection kit of the SCLC molecular typing-based CTC by adopting a microfluidic chip and a multiplex immunofluorescence probe technology is characterized in that:
the fluorescent dye 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, a diluent, and also comprises a subtype key marker of SCLC marked by different fluorescein groups, an SCLC related marker detection antibody and a leukocyte auxiliary identification marker or a detection agent formed by the subtype key marker of SCLC marked by different fluorescein groups and the leukocyte auxiliary identification marker;
the subtype key marker of SCLC comprises 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.
2. The SCLC molecule typing-based CTC detection kit using a microfluidic chip and multiplex immunofluorescence probe technique as claimed in claim 1, characterized in that: the microfluidic chip is internally provided with a plurality of shunting lanes with flow sections in double-row herring bone structures.
3. The SCLC molecule typing-based CTC detection kit using a microfluidic chip and multiplex immunofluorescence probe technique as claimed in claim 2, characterized in that: and the branched lanes are sequentially coupled with a modified silane agent, a bifunctional crosslinking agent and streptavidin.
4. The SCLC molecule typing-based CTC detection kit using a microfluidic chip and multiplex immunofluorescence probe technique as claimed in claim 1, characterized in that: 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 according to the molecular classification of SCLC;
the SCLC-se:Sub>A subtype capture agent is se:Sub>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.
5. The SCLC molecule typing-based CTC detection kit using a microfluidic chip and multiplex immunofluorescence probe technique as claimed in claim 1, characterized in that:
the balanced salt solution is PBS buffer solution;
the tissue cell fixing liquid is PFA fixing liquid;
the specific hybridization blocking solution is FC receptor blocking solution;
the active nuclear dye solution is Hoechst33342DNA fluorescent dye solution;
the dilution was 1 x ADB antibody dilution buffer.
6. The SCLC molecule typing-based CTC detection kit using a microfluidic chip and multiplex immunofluorescence probe technique as claimed in claim 1, characterized in that: the detection agents are classified into SCLC-A subtype fluorescent probe detection agents, SCLC-N subtype fluorescent probe detection agents, SCLC-A/N subtype fluorescent probe detection agents, SCLC-P subtype fluorescent probe detection agents, SCLC-I subtype fluorescent probe detection agents and leucocyte fluorescent probe detection agents according to the types of the identified substances and SCLC molecules;
the SCLC-A subtype fluorescent probe detection agent is ASCL1 primary antibody, alexse:Sub>A 647 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 an ASCL1 primary antibody, an Alexse:Sub>A 647 fluorescein labeled IgG secondary antibody, se:Sub>A NEUROD1 primary antibody and an Alexse:Sub>A 647 fluorescein labeled IgG secondary 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.
7. The identification method of the SCLC molecular typing-based CTC detection kit using the microfluidic chip and multiplex immunofluorescence probe technique according to claim 6, comprising the steps of:
a) Coating and blocking of biotin-labeled capture agent of microfluidic chip: injecting a capturing agent marked by biotin into a shunting lane from a chip inlet after being diluted by balanced salt solution, incubating, injecting tissue cell fixing solution into a microfluidic chip after the microfluidic chip is cleaned by using the balanced salt solution, fixing, injecting specific hybridization blocking solution diluted by diluent after the microfluidic chip is cleaned by using the balanced salt solution, and incubating;
b) Peripheral blood mononuclear cell PBMC isolation of SCLC patients: extracting blood of an SCLC patient and adopting a human peripheral blood lymphocyte separation liquid to perform density gradient centrifugation to separate peripheral blood mononuclear cell PBMC of the SCLC patient;
c) Enrichment and capture of microfluidic chip of CTC cells: injecting peripheral blood mononuclear cell PBMCs of SCLC patients into the microfluidic chip to enrich the captured CTC cells;
d) Multiplex fluorescent immunity in situ probe hybridization of CTC cells: diluting the reagent except Alexa647 fluorescein marked serial IgG secondary antibodies in the detection agent, injecting the diluted reagent into the microfluidic chip for incubation, and then adding the diluted Alexa647 fluorescein marked IgG secondary antibodies into the microfluidic chip for incubation after washing the microfluidic chip by using a balanced salt solution;
e) Active nuclear staining of CTC cells: injecting the reactive nuclear dye solution diluted by the diluent after the microfluidic chip is cleaned by using the balanced salt solution, and incubating;
f) Scanning and interpretation analysis of CTC cells: and adopting a four-color channel automatic fluorescence scanning system to scan, identify and analyze the CTC cells.
8. The identification method of the detection kit for CTCs based on SCLC molecular typing by using a microfluidic chip and multiple immunofluorescence probe technique according to claim 7, wherein the identification method comprises the following steps: in step a), the biotin-labeled capture reagent is diluted 50-100 fold and incubated at room temperature for 0.5-2 h; the fixing time of the tissue cell fixing solution at room temperature is 5-15 min; the specific hybridization blocking solution is diluted by 200-400 times and incubated for 10-30 min at room temperature.
9. The identification method of the detection kit for CTCs based on SCLC molecular typing by using a microfluidic chip and multiple immunofluorescence probe technique according to claim 7, wherein the identification method comprises the following steps: in step d), the detection agent is diluted 50-200 times and incubated at room temperature for 0.5-1.5 h.
10. The identification method of the detection kit for CTCs based on SCLC molecular typing by using a microfluidic chip and multiple immunofluorescence probe technique according to claim 7, wherein the identification method comprises the following steps: in step e), the incubation time of the active nuclear dye solution at room temperature is 5-15 min.
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