CN120446482B - Microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and preparation method - Google Patents

Abstract

The invention belongs to the technical field of biochemical detection, and particularly relates to a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and a preparation method thereof; according to the kit, an EpCAM antibody, a Vimentin antibody and a double-site PSMA capturing combination are fixed in a chip channel through a streptavidin-biotin system, so that double enrichment of epithelial and interstitial CTCs is realized, PSMA targets are identified through the combination of an aptamer and the antibody, and the detection specificity and sensitivity are improved. The competitive short peptide is further introduced to block the non-specific binding site on the surface of the leucocyte, so that false positive is reduced. Combining multiple fluorescent staining system and double antibody nucleic acid probe signal amplification strategy to realize accurate identification and quantification of CTC and PSMA positive cells. The scheme is suitable for early screening, curative effect evaluation and recurrence monitoring of the prostate cancer, and has good clinical application prospect.

Description

Micro-fluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and preparation method thereof

Technical Field

The invention relates to the technical field of biochemical detection, in particular to a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit and a preparation method thereof.

Background

Prostate cancer is one of the malignant tumors of the male urinary system, often lacking obvious symptoms in early stages, and often has progressed to locally advanced or distant metastatic stages. In recent years, circulating tumor cells (circulatingtumorcells, CTCs) become important markers for early screening of tumors, efficacy monitoring and recurrence assessment, as they can non-invasively reflect primary or metastatic lesion characteristics. Among them, prostate specific membrane antigen (prostate-specificmembraneantigen, PSMA) as a transmembrane glycoprotein with high expression of prostate cancer specificity has been widely used for targeted diagnosis and treatment research of prostate cancer, and has important value in CTC detection field.

Traditional CTC detection methods comprise density gradient centrifugation, immunomagnetic bead capture, molecular labeling methods and the like, but the methods generally have the problems of low separation purity, complicated processing steps, limited capture efficiency and the like, and are difficult to meet the requirements of clinical high-throughput and high-sensitivity detection. In recent years, development of microfluidic chip technology provides a new approach for efficient enrichment and separation of CTC. The micro-fluidic chip has the advantages of small sample consumption, automatic operation process, small cell damage and the like, and can realize multi-marker immunocapture and high-throughput cell treatment.

Aiming at the detection requirement of the prostate cancer CTC, an immune enrichment detection kit which takes PSMA as a main recognition target point and combines a microfluidic chip technology is developed, so that not only can the specific recognition and efficient capture of the CTC be realized, but also the accuracy and the repeatability of the clinical sample detection can be improved, and the kit is expected to play an important role in early diagnosis, recurrence monitoring and curative effect evaluation of the prostate cancer.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit which comprises the following biochemical combination components:

a) A biotin-modified cell capture agent comprising the following three classes of biotinylated molecules and formulated in a molar ratio of (1-3): (1-2): 1:

the first kind of mouse anti-human EpCAM monoclonal antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO.1 and the amino acid sequence of the light chain variable region of SEQ ID NO. 2;

The second kind is a mouse anti-human Vimentin monoclonal antibody, the heavy chain variable region amino acid sequence is SEQ ID NO. 3, and the light chain variable region amino acid sequence is SEQ ID NO. 4;

a third class, a dual site anti-PSMA capture combination, comprising:

biotin-modified murine anti-human PSMA monoclonal antibody having a heavy chain variable region amino acid sequence of SEQ ID NO. 5, a light chain variable region amino acid sequence of SEQ ID NO. 6, and

A 30nt PSMA aptamer modified by biotin, the nucleic acid sequence of which is SEQ ID NO. 7;

b) The immune specificity blocker is a competitive PSMA antibody short peptide, and the sequence is SEQ ID NO. 8, and is used for selectively saturating non-specific PSMA antibody binding sites on the surface of white blood cells before enrichment.

In a preferred embodiment, the method further comprises the following biochemical combination components:

c) The bottom layer of the microfluidic chip is fixed with streptavidin and can form a covalent outer layer with the biotinylated molecules;

d) A multiplex fluorescence detection composition comprising:

FITC-anti-CK antibody, alexaFluor 647-anti-Vimentin antibody, PE-anti-CD 45 antibody, DAPI;

The PSMA double-site signal amplification pair comprises a first detection antibody coupling oligonucleotide P1 and a second detection antibody coupling oligonucleotide P2, wherein the P1/P2 self-assembles to form a fluorescent-nucleic acid probe when the distance is less than or equal to 40nm, so that PSMA specific double confirmation is realized;

e) Pretreatment agent, separating liquid, fixing liquid, penetrating agent and 5% BSA blocking liquid.

In a preferred embodiment, the molar ratio of the murine anti-human EpCAM monoclonal antibody, the murine anti-human Vimentin monoclonal antibody, the biotin-modified murine anti-human PSMA monoclonal antibody, and the biotin-modified 30nt PSMA aptamer is (2-3): 1:0.5:0.5;

When carrying out HABA-Avidin colorimetric quantification on the four biotinylated molecules, the number of biotin residues combined on each molecule of antibody or aptamer is controlled to be 3-8mol/mol, wherein the average number of antibody molecules is 5-6, the number of aptamers is 3-4, and the total protein concentration after the capture agent is compounded is And pH 7.2.+ -. 0.1.

In a preferred embodiment, the short peptide of the competitive PSMA antibody SEQ ID NO. 8 is 14.+ -.2 amino acid residues with an affinity constantPSMA homology sites on human leukocyte surface are less than 100nM, but PSMA affinity for tumor cells is greater than 5. Mu.M, and the competitive PSMA antibody short peptide is incubated at 0.2-0.5. Mu.M final concentration for 1-3min prior to enrichment.

In a preferred embodiment, the separation solution is PBS,20mM, pH7.2-7.6, containing 0.6% -1.0% (m/v) BSA, 1mM EDTA and 50 μ MZnCl ₂;

The separation was sterilized by filtration through a 0.22 μm pes membrane twice and stored at 4 ℃ to avoid precipitation of metal ions.

In a preferred embodiment, the PSMA double-site signal amplification pair consists of a 6-FAM labeled oligonucleotide P1 and a Quasar670 labeled oligonucleotide P2, wherein the complementary region of the P1 and the P2 is 15nt and is positioned at the C terminal ends of the two PSMA detection antibodies;

when the target protein is combined with the P1-antibody and the P2-antibody simultaneously, the FRET distance is not more than 35nm, and green to near-red signal conversion is realized.

In a preferable technical scheme, the asymmetric bifurcation angle of the upper layer of the microfluidic chip is 45-60 degrees, and the width decreasing ratio of the sub-channels is 1 (0.8-0.9): 0.6-0.8;

The inner wall of the channel is coated with streptavidin after being transiently activated by 3-aminopropyl triethoxysilane (APTES), and the coating quantity is more than or equal to 1 mug cm ^-2;

The chips were sterilized with 75% ethanol and stored dry at 2-8 ℃.

The invention also provides a preparation method of the microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, which comprises the following steps:

s1, synthesizing or expressing an antibody variable region shown as SEQ ID NO. 1-6, and carrying out IgG1 and Ig kappa recombinant expression and protein A purification;

s2, biotinylating the antibody and the SEQ ID NO. 7 aptamer by using NHS-biotin;

s3, mixing the mouse anti-human EpCAM monoclonal antibody, the mouse anti-human Vimentin monoclonal antibody and the double-site anti-PSMA capturing combination according to the proportion to form a cell capturing agent modified by biotin;

s4, performing plasma treatment on the PDMS-glass microfluidic chip, and then injecting streptavidin to form a fixed layer;

s5, injecting a capture agent into a chip channel and coupling the capture agent with streptavidin to complete immune activation;

S6, preparing the competitive PSMA antibody short peptide, wherein the pretreatment agent, the separation solution, the fixing solution, the penetrating agent, the 5% BSA blocking solution and the multiple fluorescence detection composition;

And S7, sequentially sub-packaging the chip and all the liquid components, filling nitrogen in vacuum, and packaging to complete the finished product kit.

In a preferred embodiment, the final protein concentration of the capture agent in step S3 is 0.5-1.0mg/mL and the final aptamer concentration is 100-200nM.

In a preferred embodiment, the streptavidin coating concentration is 10 μg/mL and incubated at 4℃for 1h.

Advantageous effects

According to the invention, by constructing a double-target immunocapture strategy and combining EpCAM and Vimentin antibodies, the heterogeneity of epithelial and mesenchymal Circulating Tumor Cells (CTC) is effectively taken into account, and high-efficiency enrichment is realized.

The invention also blocks the PSMA homologous sites on the surface of the leucocytes by arranging the competitive short peptide, effectively avoids non-specific combination of non-tumor cells, improves background interference, and improves imaging contrast and judgment accuracy of positive cells by cooperative application of multiple fluorescent staining and a signal amplification system.

The whole technical path of the invention combines the capturing efficiency, the specificity and the flux, and is suitable for early screening, curative effect monitoring and prognosis evaluation of the prostate cancer.

Drawings

FIG. 1 is a schematic flow chart of a method according to a first embodiment of the invention;

FIG. 2 is a schematic representation of the comparative experimental results (total number of CTCs after enrichment) of the present invention;

FIG. 3 is a graph showing comparative experimental results (PSMA ⁺ CTC ratio) of the present invention;

FIG. 4 is a graph showing the results of comparative experiments (residual leukocyte levels) according to the present invention;

FIG. 5 is a graph showing the results of comparative experiments (leukocyte removal rate) according to the present invention;

FIG. 6 is a graph showing the comparative experimental results (false positive rate) of the present invention.

Detailed Description

The present invention will be further described in detail with reference to the following examples, which are only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

Example one (T1)

The embodiment provides a specific configuration and component parameters of a microfluidic chip PSMA prostate cancer circulating tumor cell immune enrichment detection kit, which are suitable for enrichment of CTC (circulating tumor cells) and specific identification of PSMA positive cells in a blood sample of a prostate cancer patient. The steps are as shown in fig. 1, including:

S1, configuration of a capturing agent:

the cell capturing agent is prepared from three types of biotinylation molecules according to a molar ratio of 3:1:1, and the three types of biotinylation molecules are respectively as follows:

The amino acid sequence of the heavy chain variable region of the mouse anti-human EpCAM monoclonal antibody is SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the mouse anti-human Vimentin monoclonal antibody is SEQ ID NO.3, and the amino acid sequence of the light chain variable region is SEQ ID NO. 4;

PSMA dual site capture combination:

the biotin modified mouse anti-human PSMA monoclonal antibody has the heavy chain variable region amino acid sequence of SEQ ID NO. 5, the light chain variable region amino acid sequence of SEQ ID NO. 6, the biotin modified 30nt aptamer and the nucleic acid sequence of SEQ ID NO. 7.

Each antibody is modified by NHS-biotin, the binding degree of HABA-Avidin colorimetric method is 5-6 mol/mol, and the aptamer modification site is 3-4 mol/mol. After the combination, the final concentration of the protein is 0.8 mg/mL, the final concentration of the aptamer is 150 nM, the buffer system is PBS, and the pH value is 7.2+/-0.1, and the mixture is stored in a 4 ℃ light-shielding environment.

S2, competitive blocker configuration:

The PSMA short peptide is used as an immune specificity blocking agent, the sequence of the PSMA short peptide is SEQ ID NO. 8, the PSMA short peptide contains 14 amino acid residues in total, and the affinity constant of the PSMA short peptide to human leucocyte PSMA homologous sites is determined Less than 100 nM, and binding affinity for prostate cancer cell PSMA protein exceeding 5 μm. The short peptide was added to the sample at a concentration of 0.3 μm prior to the enrichment procedure and incubated for 2 minutes to block non-specific binding sites on leukocytes, reducing false positives.

S3, constructing a microfluidic chip:

The chip is of PDMS-glass structure, after oxygen plasma treatment, 10 mug/mL of streptavidin is introduced, and the chip is left to stand for 1 hour at 4 ℃ to complete coating. The upper layer channel of the chip has an asymmetric bifurcation structure, the branching angle is 50 degrees, the channel width is gradually decreased to 400 mu m, 320 mu m and 240 mu m, the channel depth is 50 mu m, and the inner wall is activated by APTES. The chip is sterilized by 75% ethanol and then dried and stored at 2-8 ℃.

S4, multiplex fluorescence detection composition:

The enriched cells were sequentially fixed with 4% paraformaldehyde, 0.1% TritonX-100 permeabilized and 5% BSA blocked, and then labeled with the following fluorochromes, respectively:

FITC-anti-CK antibody, alexaFluor 647-anti-Vimentin antibody, PE-anti-CD 45 antibody, DAPI nuclear stain.

The PSMA specific recognition is realized through a double-site signal amplification system, wherein a first detection antibody is coupled with an oligonucleotide P1 (6-FAM mark), a second detection antibody is coupled with an oligonucleotide P2 (Quasar 670 mark), two complementary regions 15nt are connected to the C end of the antibody, when the target PSMA protein is combined, the distance between the two probes is lower than 35 nm, FRET occurs, the transfer from green to near-red fluorescence is realized, and a PSMA positive judgment standard is formed.

S5, preparing functional liquid:

The separation solution was prepared by adding 0.8% BSA, 1 mM EDTA, 50. Mu.M ZnCl ₂ to PBS buffer (20 mM, pH 7.4), double-sterilizing with 0.22 μm PES film, and storing at 4 ℃;

Pretreatment agent containing ammonium chloride, sodium bicarbonate and EDTA, pH7.3;

4% of fixing solution PFA;

0.1% TritonX-100% permeate;

blocking solution 5% BSA.

S6, enrichment flow:

Pretreating a peripheral blood sample of a1 mL patient, injecting the peripheral blood sample into a chip, flowing for 30 minutes under the action of a circulating cell separator, finishing elution, fixation, permeation and fluorescent staining after the cells are combined, finally recognizing CK ⁺/CD45^- cells as CTC (cell culture) under a fluorescent microscope, and further screening PSMA-FRET positive cells as prostate specific CTC for counting.

Example two (T2)

Compared with the first embodiment, the embodiment provides an improved micro-fluidic chip PSMA prostate cancer circulating tumor cell immunity enrichment detection kit, which is mainly characterized in that the proportion of PSMA related molecules in a cell capturing agent is increased, different biotin modification modes are respectively adopted for an aptamer and an antibody, the concentration of a blocking agent is optimized, and the complementary length and fluorescence pair combination of an oligonucleotide probe are adjusted in the design of a signal amplification pair.

S1, configuration of a capturing agent:

The following four biotinylated molecules were formulated in a molar ratio of 2:1:2:1:

the mouse anti-human EpCAM monoclonal antibody (heavy chain variable region SEQ ID NO:1, light chain variable region SEQ ID NO: 2) is modified by NHS-biotin, and the binding degree is 6 mol/mol;

A murine anti-human Vimentin monoclonal antibody (heavy chain variable region SEQ ID NO:3, light chain variable region SEQ ID NO: 4), modified with NHS-biotin, binding at 5 mol/mol;

The mouse anti-human PSMA monoclonal antibody (heavy chain variable region SEQ ID NO:5, light chain variable region SEQ ID NO: 6) adopts click chemistry method to carry out terminal biotin modification, and the binding degree is about 3 mol/mol;

30nt PSMA aptamer (SEQ ID NO: 7) modified with terminal Azide and coupled with DBCO-PEG4-Biotin, the average degree of binding was 4 mol/mol.

The total protein concentration after compounding is controlled to be 0.6 mg/mL, the aptamer concentration is 200 nM, and the mixture is prepared in PBS buffer solution (pH 7.2), the storage temperature is 4 ℃, and the mixture is stored in a dark place.

S2, competitive blocker setting:

The short peptide sequence is still SEQ ID NO. 8, but the incubation conditions are adjusted:

The concentration was increased to 0.5 μm and the incubation time was shortened to 1 min to more rapidly saturate the leukocyte non-specific binding sites, thus compressing the overall treatment cycle.

S3, processing and structure of the micro-fluidic chip:

the chip is of a two-layer PDMS structure, the bottom layer adopts a through channel mode, and a herring bone structure is not used;

The upper layer is designed into an inclined confluence channel, the angle is 55 degrees, and the inner diameter width is kept consistent to 300 mu m;

after the inner wall of the channel is modified by 3-aminopropyl triethoxysilane (APTES), 10 mug/mL of streptavidin is coated, and the channel is incubated for 90 minutes at 4 ℃ to form a stable binding layer;

after the coupling is completed, the solution is rinsed with sterile PBS and stored dry at 2-8deg.C.

S4, multiplex fluorescence detection composition and signal amplification:

In this embodiment, the PSMA identification component uses an optimally designed nucleic acid probe system:

the P1 oligonucleotide was fluorescently labeled with 6-FAM;

The P2 oligonucleotide was fluorescently labeled with Cy 5;

The two complementary regions were adjusted to 18 nt to extend hybridization stability, and P1 and P2 were coupled to the C-terminus of different PSMA detection antibodies, respectively.

The design can still form stable signals when the polymerization density of PSMA protein is low, so that the detection probability of a weak positive sample is improved, and the false negative is reduced.

The fluorescent detection combinations were as follows, CK antibody (AlexaFluor 488), vimentin antibody (TexasRed), CD45 antibody (AlexaFluor 750), DAPI nuclear stain, PSMA double-antibody site combination+FRET fluorescence pair (FAM-Cy 5) for targeting specific enhanced recognition.

S5, preparing functional liquid:

The separation was replaced with HEPES buffer system (20 mM HEPES, pH 7.4) in the proportions still containing 0.8% BSA, 1 mM EDTA and 50. Mu.M ZnCl ₂;

the concentration of the fixing solution is adjusted to 2% PFA, so that the structural damage is reduced;

the blocking solution was 10% BSA for enhancing the signal-to-noise ratio of the high background samples.

S6, detecting flow:

Taking a blood sample of a patient of 1 mL, treating the blood sample by using a red blood cell lysate, re-suspending cells by using a pre-cooling separation solution, adding blocking short peptide, and incubating for 1 minute;

Then injecting the micro-fluidic chip, maintaining the flow rate at 200 mu L/min under the action of a circulating separator, and enriching for 20 minutes;

And (3) sequentially carrying out fixation, permeation, sealing and fluorescent staining after elution, finally, collecting images through a multichannel fluorescent microscope, and carrying out PSMA-FRET signal secondary confirmation on the CK ⁺/CD45^- cell population.

Comparative example 1 (C1)

The comparative example aims at simulating a detection scheme lacking key innovative features, and the first comparative example adopts the following components and preparation steps to construct a microfluidic chip PSMA prostate cancer circulating tumor cell detection combination:

s1, respectively obtaining the following antibodies:

the amino acid sequence of the heavy chain variable region of the biotin-modified mouse anti-human EpCAM monoclonal antibody is SEQ ID NO.1, and the light chain is SEQ ID NO. 2;

The biotin modified mouse anti-human PSMA monoclonal antibody has the heavy chain variable region of SEQ ID NO. 5 and the light chain of SEQ ID NO. 6.

S2, mixing the two antibodies according to a molar ratio of 2:1 to form a trapping agent compound solution, setting the final concentration of protein to be 0.8mg/mL, adjusting the pH to 7.2, and storing in a 4 ℃ light-shielding environment for later use.

S3, finishing biotinylation reaction of the antibody by using an NHS-biotin reagent, and controlling the biotin modification degree to be 5-6 residues per molecule of the antibody.

S4, selecting a PDMS-glass double-layer microfluidic chip, adopting standard plasma to activate, injecting 10 mug/mL streptavidin solution into the bottom layer, incubating for 1h at 4 ℃, washing and drying, then injecting the capturing agent solution, and standing for 30min to realize antibody fixation.

S5, the chip upper layer structure adopts a conventional herringbone channel (the channel width is 200 mu m and the depth is 80 mu m), has no gradual change bifurcation design, does not carry out surface activation modification, and does not overlap an aptamer or other signal amplification mechanisms.

S6, collecting 5mL of peripheral blood sample of the prostate cancer patient, treating the peripheral blood sample by using a red blood cell lysis buffer solution, and separating the peripheral blood sample by using a chip, wherein the separation solution is PBS (20 mM, pH 7.4), only 0.5% BSA is contained as a non-specific blocking component, and EDTA or Zn ²⁺ plasma stabilizing components are not added.

S7, cell fixation (4% paraformaldehyde), permeation (0.2% Triton X-100) and blocking (5% BSA) are sequentially carried out, triple staining is carried out, namely, FITC-anti-CK antibody, PE-anti-CD 45 antibody and DAPI are used for dying nuclei, and a Vimentin antibody channel is not arranged.

Comparative example two (C2)

The second comparative example is a kit model for deleting a double-site PSMA recognition system, and aims to verify the key effects of the double-site recognition and signal amplification strategy in the first and second embodiments of the invention on improving the PSMA recognition efficiency and reducing the false negative.

S1, selecting three types of biotinylation molecules for chip capture:

The murine anti-human EpCAM antibody has a heavy chain variable region of SEQ ID NO. 1 and a light chain of SEQ ID NO. 2;

the heavy chain variable region of the mouse anti-human Vimentin antibody is SEQ ID NO. 3, and the light chain is SEQ ID NO. 4;

the heavy chain variable region of the murine anti-human PSMA antibody is SEQ ID NO. 5, and the light chain is SEQ ID NO. 6.

S2, the antibody is not introduced into a PSMA aptamer, a double-site capture structure is not constructed, and only a PSMA antibody recognition mechanism is adopted. The three types of biotinylation molecules are mixed according to the mass ratio of 1:1:1, the final concentration is controlled at 0.7mg/mL, and the pH value is 7.4.

S3, modifying with NHS-biotin to ensure that each molecule of antibody is combined with about 4-5 biotin residues, and confirming the combined amount by a HABA method.

S4, the microfluidic chip is of a standard PDMS structure, no upper herring bone channel is designed, and no micro vortex induction structure is designed. The bottom layer of the chip was plasma activated and injected with streptavidin (10. Mu.g/mL), incubated at 4℃for 30min, followed by injection of capture reagent to complete immobilization.

S5, APTES or other surface affinity enhancement treatment is not carried out on the inner wall of the chip channel, and a nucleic acid signal amplification probe system is not configured.

S6, after 5mL of the blood sample is treated by the erythrocyte lysis buffer solution, the chip is adopted to enrich CTC. The competing PSMA antibody short peptide of SEQ ID NO. 8 was not used during sample processing, i.e., potential PSMA homology structures on leukocytes in blood were not pre-blocked.

S7, in the dyeing detection process, FITC-anti-CK antibody, alexaFluor 647-anti-Vimentin antibody, PE-anti-CD 45 antibody and DAPI are adopted. No PSMA-recognized diabody signal amplification system was set up, using a panel of Cy 5-labeled anti-PSMA antibodies, lacking a two-site confirmation and FRET mechanism.

Comparative example III (C3)

The three purposes of this comparative example are to verify the technical effect brought by the "streptavidin-biotin" directed coupling mode. Although the types and proportions of capture molecules in the kit remain the same as in the examples of the present invention, the manner of immobilization of capture molecules is changed to chemical cross-linked adsorption.

The method comprises the following specific steps:

s1, preparing a non-biotinylated molecule mixed solution:

The murine anti-human EpCAM antibody (SEQ ID NO: 1/2), the murine anti-human Vimentin antibody (SEQ ID NO: 3/4), the murine anti-human PSMA antibody (SEQ ID NO: 5/6) and 30nt PSMA aptamer (SEQ ID NO: 7) were mixed in the molar ratio of 2:1:1:1, the final protein concentration was 0.8 mg/mL, the aptamer concentration was 150 nM, PBS buffer (pH 7.2) and stored at 4℃for further use.

S2, preparing a micro-fluidic chip modified by chemical crosslinking:

A linear chip (without herringbone turbulence) with a PDMS-glass structure is adopted, the inner wall of the chip is treated by oxygen plasma for 60 seconds, then 2.5% glutaraldehyde solution (PBS, pH 7.0) is injected, and the chip is kept stand at 37 ℃ for 10 minutes and then thoroughly rinsed with deionized water.

S3, fixing a non-biotinylation capture agent:

And (3) injecting the mixed solution obtained in the step (S1) into a chip channel, standing at 37 ℃ and incubating for 1 hour, and forming a covalent bond by glutaraldehyde-amine reaction to complete the random directional fixation of the capture molecules. Thereafter, the chips were washed 3 times with PBS and stored in a wet state.

S4, blocking treatment of leucocytes before enrichment:

Blood from a patient with 4 mL prostate cancer was treated with a red blood cell lysate, and the competitive short peptide of SEQ ID NO. 8 was added at a final concentration of 0.3. Mu.M and incubated for 2 minutes to block PSMA homology sites on leukocytes.

S5, enrichment and dyeing detection:

injecting the sample into the chip at a flow rate of 200 μL/min, introducing for 30min, fixing cells with 4% paraformaldehyde for 10 min, allowing 0.1% Triton X-100 to permeate for 3 min, and blocking with 5% BSA for 15 min;

sequentially adding FITC labeled anti-FITC-anti-CK antibody, alexaFluor 488-anti-Vimentin antibody, PE-anti-CD 45 antibody and DAPI dye for fluorescence labeling, and only adopting Cy5 labeled single-point anti-PSMA antibody without using PSMA double-site P1/P2 signal amplification probes.

Design of comparative experiment

The experimental purpose is to verify the detection performance difference of the micro-fluidic chip PSMA prostate cancer CTC immune enrichment detection kit under different schemes, and to highlight whether the kit has obvious advantages in the aspects of CTC enrichment efficiency, prostate specific identification, false positive control and biochemical suitability.

The experimental samples are 8 cases of blood samples of patients with advanced diagnosis of prostate cancer, and 5mL peripheral blood are collected in each case. Samples were divided into 5 groups after treatment with red blood cell lysate and pre-blocking treatment with white blood cells, and were treated with examples T1 and T2 and comparative examples C1, C2 and C3, respectively.

The experimental grouping and processing modes are shown in table 1:

Table 1 comparative experimental group

The experimental operation steps are as follows:

CTC enrichment treatment the microfluidic chip was injected according to each set of protocols, maintaining the same flow rate (200 μl/min) and incubation time (30 min).

Immobilization, permeabilization and blocking the same fixative (4% paraformaldehyde), permeabilizer (0.1% Triton X-100) and 5% BSA blocking solution were used.

Multiplex fluorescent staining FITC-anti-CK antibody, alexaFluor 647-anti-Vimentin antibody, PE-anti-CD 45 antibody, DAPI and PSMA double site probes (T1, T2) or single site antibody (group C) were used in unison.

Fluorescence microscope detection and counting, namely identifying and classifying CTCs in each group of chips one by one, and recording the following indexes respectively:

Number of epithelial CTCs (CK ⁺/CD45^-);

number of interstitial CTCs (Vimentin ⁺/CD45^-);

PSMA positive CTC number (P1/P2 probe signal or Cy5 label);

Background white blood cell residual (CD 45 ⁺ total cells);

false positive ratio (number of non-CTC cells of CD45 ⁺/PSMA⁺).

Evaluation index:

CTC enrichment efficiency (cells/mL): total CTC count/sample volume;

PSMA-positive CTC ratio (%) PSMA ⁺ CTC number/CTC total number;

The white blood cell removal (%) is the ratio of the initial CD45 ⁺ quantity to the enriched CD45 ⁺ quantity;

false positive (%) number of cells stained with PSMA probe/total number of cells detected, not CTCs.

The following are comparative experimental data corresponding to example one (T1), example two (T2) and comparative examples one (C1), comparative examples two (C2), comparative example three (C3), unifying samples from 8 clinical prostate cancer patients, with each group of average calculations, and the specific data are shown in table 2:

table 2 comparative experimental data

The results show that the average number of CTCs enriched for the T1 and T2 groups was 62.8 and 69.4/mL, respectively, significantly better than C1 (38.5/mL), C2 (59.6/mL) and C3 (31.2/mL), as shown in fig. 2, where the T2 group performed best in terms of capture efficiency. This result is attributable to the dual-site PSMA capture strategy employed in the present invention and EpCAM/Vimentin dual antibody synergy, effectively covering both epithelial and mesenchymal CTCs, improving the overall capture capacity in heterogeneous settings.

In terms of specific recognition, as shown in fig. 3, the PSMA-positive CTCs of the T2 group were up to 89.2% higher than those of the other groups, especially the C1 group was only 56.4%. This difference demonstrates that the aptamer-antibody combination designed according to the invention not only improves recognition sensitivity, but also significantly depresses false positive signals by a spatial double-confirmation mechanism. In addition, as shown in fig. 6, the T2 group was the lowest in false positive rate index, which is only 0.8%, far superior to C1 (3.6%) and C2 (7.9%), further verifying the contribution of the signal self-assembled probe system in recognition accuracy.

In terms of leukocyte background control, as shown in fig. 4 and 5, T1 and T2 achieved 99.6% and 99.7% leukocyte removal rates, respectively, with the C2 group lacking competitive short peptide blockade, being only 89.5%, with a significant increase in residual background signal. This shows that the PSMA homologous site blocking peptide shown in SEQ ID NO. 8 introduced in the invention can effectively prevent non-specific binding, and improve the subsequent detection sensitivity and the background purification degree.

From the analysis, the first and second embodiments are significantly superior to each comparative example in terms of capturing efficiency, specificity recognition, false positive control and leukocyte depletion, wherein the second embodiment has the most excellent overall performance due to the adoption of a more optimized capturing ratio and signal amplification mechanism, and fully proves the technical advantages of the biochemical combination strategy in the detection of prostate cancer CTCs.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (9)

1. The micro-fluidic chip PSMA prostate cancer circulating tumor cell immunity enrichment detection kit is characterized by comprising the following biochemical combination components:

a) A biotin-modified cell capture agent comprising the following three classes of biotinylated molecules and formulated in a molar ratio of (1-3): (1-2): 1:

the first kind of mouse anti-human EpCAM monoclonal antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO.1 and the amino acid sequence of the light chain variable region of SEQ ID NO. 2;

The second kind is a mouse anti-human Vimentin monoclonal antibody, the heavy chain variable region amino acid sequence is SEQ ID NO. 3, and the light chain variable region amino acid sequence is SEQ ID NO. 4;

a third class, a dual site anti-PSMA capture combination, comprising:

biotin-modified murine anti-human PSMA monoclonal antibody having a heavy chain variable region amino acid sequence of SEQ ID NO. 5, a light chain variable region amino acid sequence of SEQ ID NO. 6, and

A 30nt PSMA aptamer modified by biotin, the nucleic acid sequence of which is SEQ ID NO. 7;

b) The immune specificity blocker is a competitive PSMA antibody short peptide, and the sequence is SEQ ID NO. 8, and is used for selectively saturating non-specific PSMA antibody binding sites on the surface of white blood cells before enrichment.

2. The microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment detection kit of claim 1, further comprising the following biochemical combination components:

c) The bottom layer of the microfluidic chip is fixed with streptavidin and can form a covalent outer layer with the biotinylated molecules;

d) A multiplex fluorescence detection composition comprising:

FITC-anti-CK antibody and AlexaFluor 647-anti-Vimentin antibody and PE-anti-CD 45 antibody and DAPI;

The PSMA double-site signal amplification pair comprises a first detection antibody coupling oligonucleotide P1, a second detection antibody coupling oligonucleotide P2, P1 and P2 which are self-assembled to form a fluorescent-nucleic acid probe when the distance is less than or equal to 40nm to realize PSMA specific double-confirmation, wherein the PSMA double-site signal amplification pair comprises a 6-FAM labeled oligonucleotide P1 and a Quasar670 labeled oligonucleotide P2, and the complementary region of the P1 and the P2 is 15nt and is positioned at the C ends of two PSMA detection antibodies;

When the target protein is combined with the P1-antibody and the P2-antibody simultaneously, the FRET distance is not more than 35nm, so that green-to-near-red signal conversion is realized;

e) Pretreatment agent, separating liquid, fixing liquid, penetrating agent and 5% BSA blocking liquid.

3. The microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment assay kit of claim 1, wherein:

The molar ratio of the mouse anti-human EpCAM monoclonal antibody to the mouse anti-human Vimentin monoclonal antibody to the biotin-modified mouse anti-human PSMA monoclonal antibody to the biotin-modified 30nt PSMA aptamer is (2-3) 1:0.5:0.5;

When carrying out HABA-Avidin colorimetric quantification on the four biotinylated molecules, the number of biotin residues bound on each molecule of antibody or aptamer is controlled to be 3-8mol/mol, wherein the average number of antibody molecules is 5-6, and the number of aptamers is 3-4;

the total protein concentration after the capture agent is compounded is And pH 7.2.+ -. 0.1.

4. The microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment assay kit of claim 1, wherein:

the competitive PSMA antibody short peptide SEQ ID NO.8 is 14+/-2 amino acid residues, and the affinity constant thereof PSMA homology sites on human leukocyte surface are less than 100nM, but PSMA affinity for tumor cells is greater than 5. Mu.M, and the competitive PSMA antibody short peptide is incubated at 0.2-0.5. Mu.M final concentration for 1-3min prior to enrichment.

5. The microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment assay kit of claim 2, wherein:

the separating liquid is PBS,20mM, pH7.2-7.6, wherein the separating liquid contains 0.6% -1.0% (m/v) BSA, 1mM EDTA and 50 mu MZnCl ₂;

The separation was sterilized by filtration through a 0.22 μm pes membrane twice and stored at 4 ℃ to avoid precipitation of metal ions.

6. The microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment assay kit of claim 5, wherein:

the upper layer asymmetric bifurcation angle of the micro-fluidic chip is 45-60 degrees, and the width decreasing ratio of the sub-channels is 1 (0.8-0.9): (0.6-0.8);

The inner wall of the channel is coated with streptavidin after being transiently activated by 3-aminopropyl triethoxysilane, and the coating amount is the same as that of the channel ;

The chips were sterilized with 75% ethanol and stored dry at 2-8 ℃.

7. The method for preparing the microfluidic chip PSMA prostate cancer circulating tumor cell immunoenrichment detection kit according to any one of claims 1 to 6, comprising:

s1, synthesizing or expressing an antibody variable region shown as SEQ ID NO. 1-6, and carrying out IgG1 and Ig kappa recombinant expression and protein A purification;

s2, biotinylating the antibody and the SEQ ID NO. 7 aptamer by using NHS-biotin;

s3, mixing the mouse anti-human EpCAM monoclonal antibody, the mouse anti-human Vimentin monoclonal antibody and the double-site anti-PSMA capture combination into a biotin modified cell capture agent in a molar ratio of (1-3): 1-2): 1;

s4, performing plasma treatment on the PDMS-glass microfluidic chip, and then injecting streptavidin to form a fixed layer;

s5, injecting a capture agent into a chip channel and coupling the capture agent with streptavidin to complete immune activation;

S6, preparing a competitive PSMA antibody short peptide, wherein the sequence of the competitive PSMA antibody short peptide is SEQ ID NO. 8, and preparing a pretreatment agent, a separation solution, a fixing solution, a penetrating agent, a 5% BSA blocking solution and a multiplex fluorescence detection composition;

And S7, sequentially sub-packaging the chip and all the liquid components, filling nitrogen in vacuum, and packaging to complete the finished product kit.

8. The method of claim 7, wherein the final concentration of the capturing agent in step S3 is 0.5-1.0mg/mL and the final concentration of the aptamer is 100-200nM.

9. The method according to claim 8, wherein the streptavidin coating concentration is 10 μg/mL and the incubation is performed at 4 ℃ for 1h.