CN113009145A - Microfluidic chip for detecting cell secretory protein and preparation method thereof - Google Patents
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Abstract
The invention discloses a micro-fluidic chip for detecting protein secreted by therapeutic cells and a preparation method thereof, wherein the chip comprises a microcavity array chip and an antibody bar code substrate, and the antibody bar code substrate covers the microcavity array chip; the microcavity array chip is a biological affinity chip, a graphene nano material is modified on the microcavity wall of the microcavity array chip, a functional capture antibody is printed on the antibody barcode substrate, and the functional capture antibody is positioned at the top of the microcavity. The microfluidic chip disclosed by the invention can improve the cell culture environment, is beneficial to cell growth, has high antibody capturing efficiency, good uniformity and more accurate analysis result, and the preparation method has the advantages of simple process and low cost, and is suitable for large-scale production.
Description
Technical Field
The invention relates to the field of single cell analysis, in particular to a micro-fluidic chip for detecting therapeutic cell secretory protein and a preparation method thereof.
Background
The biotherapy cell therapy is a fourth means for treating tumors except for surgery, radiotherapy and chemotherapy and is a necessary means for future tumor therapy, peripheral blood mononuclear cells are separated traditionally, cells with high-efficiency antitumor activity are amplified in a large quantity under the induction of various factors and then are infused back into a patient body through veins, intradermal injection, intervention and the like to achieve the purposes of enhancing the immune function of the patient and killing tumor cells, and common cell therapy means comprise CIK, DC, TIL, DC + CIK cells and the like.
When the tumor is found clinically, the tumor is generally in the middle and late stages, at the moment, the tumor cells in the body of a patient are dominant, the immune function of the body is seriously damaged, the cell function is damaged in the microenvironment of the immune system, the efficiency of activating and treating the cells is low, and the capability of attacking the cancer cells is not enough and is not accurate enough; in addition, tumor cells escape from immune cell attack by escape mechanisms that either underexpress or do not express MHC molecules. Therefore, the technology of targeting CAR-T, CAR-NK and treating stem cells of anti-tumor cells is developed as a result of the need of manufacturing a cell weapon with precise guidance and precise strike to overcome the tumor killing mechanism mediated by MHC. However, these therapeutic cells were found to have toxic side effects on humans, and the Kaufman group transplanted human ovarian cancer cells into immunosuppressed mice, injected CAR-NK cells into animal models, and used CAR-T cells as controls, and found that organs such as liver, lung, and kidney in CAR-T cell treated animals were more severely damaged than animals receiving CAR-NK cell treatment. The level of inflammatory cytokines in vivo is increased, the treatment cells cause discomfort to mice, the symptom is weight loss, the treatment cell technology is applied to clinic, the single cell function analysis is needed to be further carried out, and the potential mechanism of the treatment cell work is fully understood.
Over the past few years, innovative strategies for cell isolation and protein secretion analysis have advanced, enabling single cell isolation. Among them, droplet microfluidics has proven to be a promising platform for single cell sorting and analysis. This technique essentially involves encapsulating individual cells in droplets loaded with specific fluorescent antibodies or enzymes to capture and detect secreted cytokines. Although this technique has the function of single cell separation. However, subsequent cytokine quantification requires the use of flow cytometry, which adds complexity to the overall system and does not allow for in situ detection, particularly of adherent cells. Although the flow cytometer used in the method can quickly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, the flow cytometer is a zero-time-resolution instrument, can only measure indexes of the cells such as total nucleic acid amount, total protein amount and the like at a certain time point, cannot dynamically monitor the cells, cannot perform subsequent analysis and treatment on the cells at a single-cell level in real time, has zero detail resolution, is expensive, and is not suitable for the public.
For some applications, flow cytometry is still somewhat limited. The cells must be in suspension, which means that the tissue needs to be dissociated, resulting in loss of cell function and cell-cell interaction and tissue structure. Subpopulations with similar marker expression are difficult to distinguish and overlap of emission spectra between fluorescent dyes may result in increased noise levels, rendering low intensity samples unusable for detection. Furthermore, the sorting system of flow cytometry may have a non-negligible impact on cell viability. In addition, the minimum sample size of a flow cytometer system is several hundred microliters to several milliliters. The long tubing length makes rare samples unusable, especially when the entire sample needs to be analyzed. Finally, due to the complex system of non-disposable components, aseptic procedures are generally difficult to achieve with flow cytometry.
Polydimethylsiloxane (PDMS) has the advantages of good optical performance, thermal stability, biocompatibility and the like, and is one of main processing materials of a microfluidic chip. In recent years, chips processed by using PDMS as a material, namely PDMS microfluidic chips, have gradually become low-cost, portable and environment-friendly biochemical trace detection tools. The chip based on PDMS material has been successfully applied in cell manipulation, gene expression detection in cells, cell culture, immunofluorescence analysis, etc. In the aspect of cell culture, the traditional method is to modify an organic bioactive material, such as BSA and the like, on the surface of PDMS, but researches show that the modified organic bioactive material has the problems of poor stability, easy contamination and the like, and the graphene nano material is a novel inorganic biological substrate material, has the effect of promoting cell adhesion and proliferation, and is a new nano material and is rarely used in the aspect of cell culture.
Disclosure of Invention
In order to solve the technical problems, the invention provides a micro-fluidic chip for detecting protein secreted by therapeutic cells and a preparation method thereof, so as to achieve the purposes of improving the cell culture environment, facilitating cell growth, having high antibody capturing efficiency and good uniformity and more accurate analysis result.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a micro-fluidic chip for detecting protein secreted by therapeutic cells comprises a microcavity array chip and an antibody bar code substrate, wherein the antibody bar code substrate covers the microcavity array chip; the microcavity array chip is a biological affinity chip, a graphene nano material is modified on the microcavity wall of the microcavity array chip, a functional capture antibody is printed on the antibody barcode substrate, and the functional capture antibody is positioned at the top of the microcavity.
In the scheme, the micro-cavity array chip is prepared by baking polydimethylsiloxane and a curing agent at a high temperature according to a mass ratio of 5:1-20:1 and then demolding.
In the scheme, the microcavity array chip contains 3000-4000 microcavities, and the length, width and depth of the microcavity size are 1800-2500 μm, 40-120 μm and 20-50 μm respectively.
In the scheme, the antibody barcode substrate comprises 20 antibody channels, the width of each channel is 10-80 μm, and the distance between the channels is not less than 10 μm.
A preparation method of a microfluidic chip for detecting secretory protein of therapeutic cells comprises the following steps:
(1) adopting polydimethylsiloxane and a curing agent according to the mass ratio of 5:1-20:1, baking at high temperature, and demoulding to prepare a microcavity array chip;
(2) the method comprises the steps of hydroxylating Plasma of the microcavity array chip, and then carrying out amination on the microcavity array chip by using a graphene nano material coupling agent to form an aminated microcavity array chip;
(3) carrying out graphene nanomaterial modification assembly on the microcavity array chip by using a graphene nanomaterial modifier, and carrying out bioaffinity modification on the microcavity array chip by using a biological modifier to form a bioaffinity microcavity array chip;
(3) carrying out graphene nanomaterial modification on the antibody barcode substrate, and then printing a functional capture antibody on the antibody barcode substrate;
(4) soaking the antibody bar code substrate and the biological affinity microcavity array chip by using a cell buffer solution and then drying;
(5) when the micro-fluidic chip is used, therapeutic cells are loaded into the micro-cavity of the biological affinity micro-cavity array chip and sealed by using the antibody bar code substrate to form the therapeutic cell micro-fluidic chip.
Through the technical scheme, the micro-fluidic chip for detecting the cell secretory protein has the following beneficial effects:
1. compared with the traditional PDMS biological material, the micro-fluidic chip for detecting the cell secretory protein has the advantages that the graphene nano material is used for modifying the micro-cavity, the cell culture environment is improved, the growth of cells is facilitated (the cell growth rate in three days reaches 1200%), the single cells are in a normal growth state, the function analysis result is more accurate, and the graphene nano material is used for modifying the antibody bar code substrate, so that the efficiency and the uniformity of fixing and capturing the antibody are doubled.
2. The preparation method of the microfluidic chip for detecting the cell secretory protein has the advantages of simple operation and low cost.
3. The micro-fluidic chip for detecting the secretory protein of the therapeutic cell has wide detection range, can be used for detecting blood cell samples, seawater cell samples, in-vitro culture cell line samples and the like from suspended cells to adherent cells, can observe the activity of the cells in real time and simultaneously detect various secretory proteins of single cells, saves various instruments and equipment required by the previous single cell experiment, and greatly simplifies the flow of the analysis experiment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of a disassembled structure of a microfluidic chip for detecting protein secreted from therapeutic cells according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method for manufacturing a microfluidic chip for detecting a protein secreted from a therapeutic cell according to an embodiment of the present invention;
FIG. 3 is a cytometric thermograph of the CAR-T therapeutic cell secretion signal detection results according to embodiments of the present invention.
In the figure, 1, a microcavity array chip; 2. a microcavity; 3. an antibody barcode substrate; 4. a functional capture antibody.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The invention provides a micro-fluidic chip for detecting protein secreted by therapeutic cells, which has a structure shown in figure 1, and comprises a microcavity array chip 1 and an antibody bar code substrate 3, wherein the antibody bar code substrate 3 covers the microcavity array chip 1; the microcavity array chip 1 is a bioaffinity chip, a graphene nanomaterial is modified on the wall of a microcavity 2, a functional capture antibody 4 is printed on an antibody barcode substrate 3, and the functional capture antibody 4 is positioned at the top of the microcavity 2.
In the embodiment, the microcavity array chip 1 is prepared by baking polydimethylsiloxane and a curing agent at 80 ℃ for 2 hours in a mass ratio of 10:1 and demolding.
The microcavity array chip 1 contains 3381 microcavities 2, the length, width and depth of the microcavities are 2100um, 100um and 30um respectively, and the sizes of the microcavities are enough for the cells to grow in an adherent manner normally.
The antibody barcode substrate 3 contains 20 antibody channels, which are 20 μm wide and 20 μm apart, and can capture 20 secreted signals at most simultaneously.
A method for preparing a microfluidic chip for detecting protein secreted by therapeutic cells, as shown in fig. 2, comprises the following steps:
(1) adopting polydimethylsiloxane and a curing agent according to the mass ratio of 10:1, baking for 2 hours at 80 ℃, and demolding to prepare a microcavity array chip;
(2) the method comprises the steps of hydroxylating Plasma of the microcavity array chip, and then carrying out amination on the microcavity array chip by using a graphene nano material coupling agent to form an aminated microcavity array chip;
(3) carrying out graphene nanomaterial modification assembly on the microcavity array chip by using a graphene nanomaterial modifier, and carrying out bioaffinity modification on the microcavity array chip by using a biological modifier to form a bioaffinity microcavity array chip;
(3) carrying out graphene nanomaterial modification on the antibody barcode substrate, and then printing a functional capture antibody on the antibody barcode substrate;
(4) soaking the antibody bar code substrate and the biological affinity microcavity array chip by using a cell buffer solution and then drying;
(5) when the micro-fluidic chip is used, therapeutic cells are loaded into the micro-cavity of the biological affinity micro-cavity array chip, and the micro-fluidic chip is formed by covering and sealing the antibody bar code substrate.
When cell secretion signal detection is carried out, therapeutic cells CAR-T are grown in a microcavity and incubated for a period of time, secretion signals of IL8, IL12 and HSP70 are captured by an antibody barcode substrate, then the antibody barcode substrate is removed, paired fluorescence detection antibodies IL8, IL12 and HSP70 are assembled and put into a fluorescence scanner for scanning detection, and the detection result is shown in figure 3. The amount of three secreted proteins of CAR-T3200 single-cell IL8 IL12 HSP70 is detected, the detection result is visually analyzed by using a clustering heat map method, and the CAR-T cells are further classified in multiple stages. colorbar shows the amount of secreted protein, log normalized to 0-55 levels and increasing in order from 0 to 5, left side shows the CAR-T cell subpopulation level, increasing level once from outside to inside, outermost side is one-stage subpopulation, and so on. The upper panel shows the CAR-T cell secretory protein association, with HSP70 being more relevant to IL12 than IL 8.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A micro-fluidic chip for detecting protein secreted by therapeutic cells is characterized by comprising a microcavity array chip and an antibody bar code substrate, wherein the antibody bar code substrate covers the microcavity array chip; the microcavity array chip is a biological affinity chip, a graphene nano material is modified on the microcavity wall of the microcavity array chip, a functional capture antibody is printed on the antibody barcode substrate, and the functional capture antibody is positioned at the top of the microcavity.
2. The microfluidic chip for detecting protein secreted from therapeutic cells according to claim 1, wherein the microcavity array chip is prepared by mixing polydimethylsiloxane and a curing agent according to the ratio of 5:1-20:1, baking at high temperature, and demolding to obtain the product.
3. The micro-fluidic chip for detecting the protein secreted by the therapeutic cell as claimed in claim 1, wherein the micro-cavity array chip comprises 3000-4000 micro-cavities, and the length, width and depth of the micro-cavities are 1800-2500 μm, 40-120 μm and 20-50 μm, respectively.
4. The microfluidic chip for detecting the secreted protein of the therapeutic cell according to claim 1, wherein the antibody barcode substrate comprises 20 antibody channels, the width of each antibody channel is 10-80 μm, and the distance between the antibody channels is not less than 10 μm.
5. A method for preparing a microfluidic chip for detection of a secreted protein from a therapeutic cell according to claim 1, comprising the steps of:
(1) adopting polydimethylsiloxane and a curing agent according to the weight ratio of 5:1-20:1, baking at high temperature, and demolding to prepare the microcavity array chip;
(2) the method comprises the steps of hydroxylating Plasma of the microcavity array chip, and then carrying out amination on the microcavity array chip by using a graphene nano material coupling agent to form an aminated microcavity array chip;
(3) carrying out graphene nanomaterial modification assembly on the microcavity array chip by using a graphene nanomaterial modifier, and carrying out bioaffinity modification on the microcavity array chip by using a biological modifier to form a bioaffinity microcavity array chip;
(3) carrying out graphene nanomaterial modification on the antibody barcode substrate, and then printing a functional capture antibody on the antibody barcode substrate;
(4) soaking the antibody bar code substrate and the biological affinity microcavity array chip by using a cell buffer solution and then drying;
(5) when the micro-fluidic chip is used, therapeutic cells are loaded into the micro-cavity of the biological affinity micro-cavity array chip and sealed by using the antibody bar code substrate to form the therapeutic cell micro-fluidic chip.
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