CN115254209A - Preparation method of PDMS-PDA-MOF micro-fluidic chip for single cell sequencing - Google Patents
Preparation method of PDMS-PDA-MOF micro-fluidic chip for single cell sequencing Download PDFInfo
- Publication number
- CN115254209A CN115254209A CN202210519804.7A CN202210519804A CN115254209A CN 115254209 A CN115254209 A CN 115254209A CN 202210519804 A CN202210519804 A CN 202210519804A CN 115254209 A CN115254209 A CN 115254209A
- Authority
- CN
- China
- Prior art keywords
- pdms
- pda
- mofs
- preparing
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000012621 metal-organic framework Substances 0.000 claims abstract description 121
- 239000004205 dimethyl polysiloxane Substances 0.000 claims abstract description 68
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims abstract description 68
- 239000000758 substrate Substances 0.000 claims abstract description 64
- 229920001690 polydopamine Polymers 0.000 claims abstract description 60
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 35
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 24
- -1 amino modified PDMS Chemical class 0.000 claims abstract description 17
- 229960003638 dopamine Drugs 0.000 claims abstract description 12
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 6
- 239000004005 microsphere Substances 0.000 claims abstract description 4
- 238000010030 laminating Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 27
- 238000012986 modification Methods 0.000 claims description 21
- 230000004048 modification Effects 0.000 claims description 21
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical group CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 10
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 238000001338 self-assembly Methods 0.000 claims description 8
- 229910052710 silicon Inorganic materials 0.000 claims description 8
- 239000010703 silicon Substances 0.000 claims description 8
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 8
- 125000003342 alkenyl group Chemical group 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 229960003151 mercaptamine Drugs 0.000 claims description 5
- PTBDIHRZYDMNKB-UHFFFAOYSA-N 2,2-Bis(hydroxymethyl)propionic acid Chemical compound OCC(C)(CO)C(O)=O PTBDIHRZYDMNKB-UHFFFAOYSA-N 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000013208 UiO-67 Substances 0.000 claims description 4
- 229920002545 silicone oil Polymers 0.000 claims description 4
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 4
- 229920002554 vinyl polymer Polymers 0.000 claims description 4
- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 claims description 4
- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 claims description 4
- 229920001971 elastomer Polymers 0.000 claims 1
- 239000000806 elastomer Substances 0.000 claims 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical class C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 abstract description 64
- 235000013870 dimethyl polysiloxane Nutrition 0.000 abstract description 49
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 abstract description 49
- 239000002131 composite material Substances 0.000 abstract 1
- 238000002715 modification method Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 239000004816 latex Substances 0.000 description 6
- 229920000126 latex Polymers 0.000 description 6
- 239000003921 oil Substances 0.000 description 6
- 238000007385 chemical modification Methods 0.000 description 4
- 230000004913 activation Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000006845 Michael addition reaction Methods 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000002199 base oil Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000010517 secondary reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012719 thermal polymerization Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B81—MICROSTRUCTURAL TECHNOLOGY
- B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
- B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
- B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Manufacturing & Machinery (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Dispersion Chemistry (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
Abstract
The invention relates to a preparation method of a PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing, which is characterized by firstly preparing an amino modified PDMS substrate; carrying out self-polymerization on a DA (dopamine) solution on an amino modified PDMS substrate under an alkaline condition to obtain a PDA (polydopamine) modified PDMS substrate; modifying MOFs on the surface of the composite material to obtain a microspheric PDMS-PDA-MOFs elastic agent with a microstructure which is internally and externally coated; and curing, laminating and baking the PDMS-PDA-MOFs elastic agent in a mold to finally obtain the PDMS-PDA-MOFs microfluidic chip. Solves the problems of preparation and application of PDMS in the field of microfluidic chips.
Description
Technical Field
The invention relates to a preparation method of a PDMS-PDA-MOFs microfluidic chip, in particular to a preparation method of a PDMS-PDA-MOFs microfluidic chip for single cell sequencing.
Background
The heterogeneity of individual cells in a cell population plays a major role in the development and progression of disease, but most traditional genetic analysis methods currently mask individual cell differences. Single cell sequencing can exhibit intrinsic heterogeneity of individual cells and reveal complex and rare cell populations. In recent years, different microfluidic technologies have appeared for single cell research, becoming the leading edge of the field.
Compared to conventional techniques, microfluidic techniques have several advantages in analyzing samples: firstly, the structure and the function of the microfluidic chip are designed flexibly, and the requirement of single cell analysis can be met. Second, typical microfluidic channels have dimensions of tens to hundreds of microns, and can handle solution volumes from picoliters to nanoliters, thereby reducing sample loss and high sensitivity, and enabling high throughput single cell analysis. In addition, the integration of the multifunctional unit and the microfluidic chip can realize automation, and measurement errors caused by manual operation are prevented.
Most microfluidic devices use microfluidic principles to isolate individual cells, hydrodynamic cell traps, pneumatic membrane valves and oil drop-based isolation. The most popular microfluidic isolation method at present is to encapsulate the single cells in an inert carrier oil using microdroplets to form a closed space, reducing the risk of sample contamination. Water-in-oil droplets are generated by taking water as a dispersed phase and taking an oil phase as a continuous phase. This type of droplet tends to be a hydrophobic channel material. PDMS is the most common chip processing material at present, is a low-temperature thermal polymerization curing polymer material, has strong moulding after molding, has certain tolerance to solvents, and has the advantages of low price, good optical transparency, good biocompatibility, air permeability, convenient operation, high efficiency and the like when used for manufacturing chips. Therefore, a series of means for detecting samples of the microfluidic chip can be developed based on the characteristics, the bonding mode of the PDMS chip is various, the operation is simple and convenient, simple reversible bonding can be realized by utilizing a physical effect, the chip can be repeatedly used, and irreversible bonding can also be realized by utilizing methods such as chemical modification. However, higher hydrophobicity is required to form stable water-in-oil droplets, an unmodified PDMS microfluidic chip is not sufficient to provide such high hydrophobicity, and the PDMS microfluidic chip also has the disadvantages of easy deformation and collapse of channels, low loading capacity, and the like. Surface modification and modification are required for application. .
Generally, a modification method of a high polymer material can be adopted, and the modification method is divided into the following modification ranges: surface modification methods and bulk modification methods, wherein the surface modification methods are most widely used and are subdivided into two main types, namely physical modification methods and chemical modification methods.
Chemical modification methods can be divided into: wet modification and surface grafting by covalent bonding. The wet modification is to make the solution to be modified contact the surface of PDMS directly, so that the component to be modified on the surface of PDMS is adsorbed on the surface of PDMS by physical adsorption or electrostatic force. Common wet modification methods include layer-by-layer self-assembly, sol-gel coating, dynamic surfactant modification, protein adsorption and the like. The common characteristic of the modification methods is that the modification methods are simpler overall. However, since the PDMS surface is not linked to the modification layer by covalent chemical bonds, the modification layer has poor stability and is easily lost with the increase of the usage time. The surface modification by covalent bond is to bond the modification layer to the PDMS surface by covalent bond through chemical reaction. If the modification layer is also a type of polymer, such surface modification is also referred to as surface grafting. The modification method has the greatest advantages that the modification layer is stable, the surface property after modification is long in retention time, and the method is a common method for chemical modification of the PDMS chip.
In the prior art, poly-dopamine (PDA) is used to modify the surface of PDMS, and dopamine is self-polymerized into poly-dopamine on the inner surface of the PDMS microfluidic chip channel under an alkaline condition to form a layer of PDA coating, thereby modifying the surface of the PDMS microfluidic chip. However, the method has certain problems, such as difficult polymerization operation in the microchannel, uneven coating thickness, unstable performance of the modified microfluidic chip, complicated preparation steps and the like.
In addition, the MOFs are porous crystal materials formed by self-assembling metal ions or metal clusters and organic ligands through coordination bonds, can be used for depositing on the surface of a solid matrix or synthesizing MOFs particles later to form a proper micro-level rough structure, enriches the roughness of the surface of the material, and can modify low-surface-energy chemical substances to enhance the surface hydrophobic property. The existing PDMS microfluidic chip channel surface modification method is not easy to form a stable modification layer, so that MOFs are not applied to surface modification of PDMS microfluidic chips.
Disclosure of Invention
The invention provides a preparation method of a PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing, which solves the problem of application of PDMS in the field of micro-fluidic chips.
The invention adopts the following technical scheme: a preparation method of PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing comprises preparing amino modified PDMS substrate; carrying out self-polymerization on DA (dopamine) solution on an amino modified PDMS substrate under an alkaline condition to form a layer of PDA film with super strong adhesion, obtaining a PDMS substrate modified by PDA (poly-dopamine), and then growing MOFs on the surface of the PDMS substrate in a self-assembly manner to obtain a microspheric PDMS-PDA-MOFs elastic agent with a microstructure coated inside and outside; and curing, laminating and baking the PDMS-PDA-MOFs elastic agent in a mold to finally obtain the PDMS-PDA-MOFs microfluidic chip.
Further, the method comprises the following steps:
(1) Preparation of amino-modified PDMS substrate: mixing vinyl silicone oil and PDMS according to a mass ratio of 2 (1), heating for 1h at 50 ℃ for activation by 0.5g in total to obtain a PDMS substrate containing alkenyl; dissolving cysteamine (0.1 g) and dimethylolpropionic acid (0.005 g) in ethanol (20 mL) to form a solution A, then dissolving a PDMS substrate containing alkenyl in the solution A, and reacting for 0.5-2h under ultraviolet light; washing with ethanol to remove unreacted substances to obtain an amino modified PDMS substrate;
(2) Performing PDA modification on the amino modified PDMS substrate: preparing a DA (dopamine) solution with the concentration of 2mg/ml by using a Tris hydrochloric acid solution with the pH =8.5, pouring the solution into the amino modified PDMS substrate obtained in the step 1, uniformly mixing, pouring the mixture into a volume of 1-2ml, then placing the mixture in an environment at 25 ℃, acting for 24 hours, and combining the mixture by virtue of a covalent bond, wherein the PDMS has lower surface energy, so that the PDA is uniformly coated on the surface of the amino modified PDMS substrate, washing the PDMS substrate for several times by using deionized water, and washing off DA (dopamine) which is not adhered and is not firmly adhered on the PDMS to obtain the PDMS substrate modified by the PDA (polydopamine);
(3) Preparing a PDMS-PDA-MOFs elastic agent: adding MOFs powder into a solvent for ultrasonic dispersion to obtain an MOFs suspension, adding the MOFs suspension into a PDA (poly dopamine) -modified PDMS substrate to promote the self-assembly growth of the MOFs on the surface of the PDA (poly dopamine) -modified PDMS substrate, placing the substrate in an environment at 37 ℃, and acting for 20 hours to obtain a PDMS-PDA-MOFs elastic agent;
(4) Preparing PDMS-PDA-MOFs with micro-channels: placing a silicon wafer with a mould in a clean glass dish, mixing a PDMS-PDA-MOFs (polydimethylsiloxane-PDA-metal organic frameworks) elastic agent and a curing agent in a mass ratio of 10; obtaining PDMS-PDA-MOFs with the micro-channel after stripping;
(5) Preparing a PDMS-PDA-MOFs microfluidic chip: placing a silicon wafer in a clean glass dish, mixing a PDMS-PDA-MOFs elastic agent and a curing agent according to a mass ratio of 10.
Further, the wavelength of the ultraviolet light in the step (1) is 365nm, and the effective distance is 10cm.
Further, the solid-to-liquid ratio of the MOFs powder to the solvent in the step (3) is 1g: (80-120) ml.
Further, the curing agent is DBP (dibutyl phthalate).
Further, in the step (3), the solvent is dichloromethane (CH 2Cl 2), ethanol (ethanol, C2H5 OH) or Dimethylformamide (DMF).
Further, the ultrasonic dispersion time in the step (3) is 45-75 min.
Further, the volume ratio of the MOFs suspension liquid to the PDA (poly dopamine) -modified PDMS substrate in the step (3) is 1: (15 to 25).
Further, the cooling time in the step (4) is 1.5-2.5 h.
Furthermore, the MOFs are ZIF-8, uiO-66 or UiO-67.
Compared with the prior art, the preparation method of the PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing has the following beneficial effects:
(1) And preparing the PDMS-PDA-MOFs elastic agent by utilizing a PDA template guiding method, wherein the PDA can guide the assembly of PDMS and MOFs and coat the MOFs. The PDA is used for modifying the PDMS substrate material, and the DA can be self-polymerized on the surface of PDMS particles in a solvent environment to form a layer of PDA film with super strong adhesion. In addition, the ortho-position of the phenolic hydroxyl group of the PDA can react with the hydroxyl group or the sulfydryl group, the amino group, the capturable metal ion and the chelated metal ion can be used as a nuclear growth site, strong adhesive force and secondary reaction capacity are provided for subsequent modification of the surface of the PDMS, the PDMS can be used as a secondary platform, the MOFs ligand can be promoted to grow in a self-assembly mode on the particle surface, the MOFs are prevented from nucleating automatically, and the MOFs on the surface of the PDMS can be coated.
(2) The integrated method is adopted to cast the PDMS with the modified surface to prepare the micro-fluidic chip, and the preparation method is simple and convenient.
(3) The method comprises the steps of reacting ultraviolet light irradiation induced sulfydryl and olefin with cysteamine to obtain an amino-containing PDMS surface, and grafting PDA to an amino-modified PDMS substrate successfully through Schiff base reaction/Michael addition reaction under the room temperature alkaline condition to ensure the uniform coating of the PDA.
(4) The coating microstructure ensures the stability of performance, and the homogeneous PDMS-PDA-MOFs elastic agent exists in the coating microsphere structure, so that the structural instability caused by uneven coating due to macroscopic surface treatment can be avoided.
Drawings
FIG. 1 is a schematic view of the microstructure of PDMS-PDA-MOFs material of the preparation method of PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to the present invention;
FIG. 2 is a schematic diagram of a chip channel of a microfluidic chip of the preparation method of the PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments.
Example one
A method for preparing PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing comprises introducing DA solution into a channel, and allowing the DA solution to undergo self-polymerization in the channel under alkaline condition to form PDA film adhered to the surface of the PDMS channel; and modifying the upper MOFs.
The method comprises the following steps:
(1) Preparation of amino-modified PDMS substrate: mixing vinyl silicone oil and PDMS according to a mass ratio of 2 (1), heating for 1h at 50 ℃ for activation by 0.5g in total to obtain a PDMS substrate containing alkenyl; dissolving cysteamine (0.1 g) and dimethylolpropionic acid (0.005 g) in ethanol (20 mL) to form a solution A, then dissolving a PDMS substrate containing alkenyl in the solution A, and reacting for 0.5-2h under ultraviolet light; washing with ethanol to remove unreacted substances to obtain an amino modified PDMS substrate;
(2) Modification of PDA on amino modified PDMS substrate: preparing a DA (dopamine) solution with the concentration of 2mg/ml by using a Tris hydrochloric acid solution with the pH =8.5, pouring the solution into the amino modified PDMS substrate obtained in the step 1, uniformly mixing, pouring the mixture into a volume of 1ml, then placing the mixture in an environment at 25 ℃, acting for 24 hours, and combining the mixture by virtue of a covalent bond, wherein the PDMS has lower surface energy, so that the PDA is uniformly coated on the surface of the amino modified PDMS substrate, washing the PDMS substrate for several times by using deionized water, and washing off the DA (dopamine) which is not adhered and is not firmly adhered on the PDMS to obtain the PDMS substrate modified by the PDA (polydopamine);
(3) Preparing a PDMS-PDA-MOFs elastic agent: adding MOFs powder into a solvent for ultrasonic dispersion to obtain an MOFs suspension, adding the MOFs suspension into a PDA (poly dopamine) -modified PDMS substrate to promote the self-assembly growth of the MOFs on the surface of the PDA (poly dopamine) -modified PDMS substrate, placing the substrate in an environment at 37 ℃, and acting for 20 hours to obtain a PDMS-PDA-MOFs elastic agent;
(4) Preparing PDMS-PDA-MOFs with microchannels: placing a silicon wafer with a mold in a clean glass dish, mixing a PDMS-PDA-MOFs (polydimethylsiloxane-metal-organic frameworks) elastic agent and a curing agent according to a mass ratio of 10; stripping to obtain PDMS-PDA-MOFs with microchannels;
(5) Preparing a PDMS-PDA-MOFs microfluidic chip: placing a silicon wafer in a clean glass dish, mixing a PDMS-PDA-MOFs (polydimethylsiloxane-PDA-metal-organic frameworks) elastic agent and a curing agent according to a mass ratio of 10 to prepare a mixed solution, then pouring the mixed solution into the glass dish, wherein the volume of the glass dish is 0.5ml, removing bubbles in a vacuum drying oven for 1h, baking the mixed solution at 90 ℃ for 2h, cooling the PDMS-PDA-MOFs for curing to obtain flat PDMS-PDA-MOFs, closely attaching the flat PDMS-PDA-MOFs to PDMS-PDA-MOFs with a microchannel, placing the PDMS-PDA-MOFs in an oven, baking the flat PDMS-PDA-MOFs at 90 ℃ for 1h, cooling and taking out to obtain the PDMS-PDA-MOFs microfluidic chip.
And finally, inserting a latex tube at the inlet of the micro-channel of the PDMS-PDA-MOFs microfluidic chip, wherein the inner diameter of the latex tube is 0.38mm, and the outer diameter of the latex tube is 1.09mm.
In this embodiment, the wavelength of the ultraviolet light in step (1) is 365nm, and the effective distance is 10cm.
In this embodiment, the solid-to-liquid ratio of the MOFs powder to the solvent in step (3) is 1g:80ml.
In this example, the curing agent was DBP (dibutyl phthalate).
In this embodiment, the solvent in step (3) is dichloromethane (CH 2Cl 2), and in some embodiments, ethanol (ethanol, C2H5 OH) or dimethylformamide (n, n-dimethylformamide, DMF) may also be used.
In this example, the ultrasonic dispersion time in step (3) was 45min.
In this embodiment, the volume ratio of the MOFs suspension to the PDA (polydopamine) -modified PDMS substrate in step (3) is 1:15.
in this example, the cooling time in step (4) was 1.5.
In this embodiment, the MOFs are ZIF-8, and in some embodiments, uiO-66 or UiO-67 may also be used.
And (3) adopting a contact angle measuring instrument to represent the contact angles of water and oil on the surface of the prepared PDMS-PDA-MOFs so as to analyze the wettability of the PDMS-PDA-MOFs. The specific process is as follows: a drop of 5 microliters of deionized water was dropped on the PDMS-PDA-MOFs surface, a photograph was immediately taken with a water contact angle measuring instrument and the water contact angle value was calculated, and each material was tested for 5 different points, as shown in Table 1, and the calculated average value was the contact angle of the material, which was 141.7 degrees, indicating its superhydrophobicity.
Table 1 contact Angle test
Example two
A method for preparing PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing comprises introducing DA solution into a channel, and allowing the DA solution to undergo self-polymerization in the channel under alkaline condition to form PDA film adhered to the surface of the PDMS channel; the upper MOFs were further modified.
The method comprises the following steps:
(1) Preparation of amino-modified PDMS substrate: mixing vinyl silicone oil and PDMS according to a mass ratio of 2 (1), heating for 1h at 50 ℃ for activation by 0.5g in total to obtain a PDMS substrate containing alkenyl; dissolving cysteamine (0.1 g) and dimethylolpropionic acid (0.005 g) in ethanol (20 mL) to form a solution A, then dissolving a PDMS substrate containing alkenyl in the solution A, and reacting for 2h under ultraviolet light; washing with ethanol to remove unreacted substances to obtain an amino modified PDMS substrate;
(2) Modification of PDA on amino modified PDMS substrate: preparing a DA (dopamine) solution with the concentration of 2mg/ml by using a Tris hydrochloric acid solution with the pH =8.5, pouring the solution into the amino modified PDMS substrate obtained in the step 1, uniformly mixing, pouring the solution into a volume of 2ml, then placing the solution in an environment at 25 ℃, acting for 24 hours, and combining the solution by virtue of a covalent bond, wherein the PDMS has lower surface energy, so that the PDA is uniformly coated on the surface of the amino modified PDMS substrate, washing the PDMS substrate for several times by using deionized water, and washing off the DA (dopamine) which is not adhered and is not firmly adhered on the PDMS to obtain the PDMS substrate modified by the PDA (polydopamine);
(3) Preparing a PDMS-PDA-MOFs elastic agent: adding MOFs powder into a solvent for ultrasonic dispersion to obtain an MOFs suspension, adding the MOFs suspension into a PDA (poly dopamine) -modified PDMS substrate to promote the self-assembly growth of the MOFs on the surface of the PDA (poly dopamine) -modified PDMS substrate, placing the substrate in an environment at 37 ℃, and acting for 20 hours to obtain a PDMS-PDA-MOFs elastic agent;
(4) Preparing PDMS-PDA-MOFs with micro-channels: placing a silicon wafer with a mold in a clean glass dish, mixing a PDMS-PDA-MOFs (polydimethylsiloxane-metal-organic frameworks) elastic agent and a curing agent according to a mass ratio of 10; stripping to obtain PDMS-PDA-MOFs with microchannels;
(5) Preparing a PDMS-PDA-MOFs microfluidic chip: placing a silicon wafer in a clean glass dish, mixing a PDMS-PDA-MOFs elastic agent and a curing agent according to the mass ratio of 10.
And finally, inserting a latex tube at the inlet of the micro-channel of the PDMS-PDA-MOFs microfluidic chip, wherein the inner diameter of the latex tube is 0.38mm, and the outer diameter of the latex tube is 1.09mm.
In this embodiment, the wavelength of the ultraviolet light in step (1) is 365nm, and the effective distance is 10cm.
In this example, the solid-to-liquid ratio of the MOFs powder to the solvent in step (3) is 1g:120ml of the solution.
In this example, the curing agent was DBP (dibutyl phthalate).
In this embodiment, the solvent in step (3) is Dimethylformamide (DMF), and in some embodiments, ethanol (ethanol, C2H5 OH) or dichloromethane (CH 2Cl 2) may also be used.
In this example, the ultrasonic dispersion time in step (3) was 75min.
In this embodiment, the volume ratio of the MOFs suspension to the PDA (polydopamine) -modified PDMS substrate in step (3) is 1:25.
in this example, the cooling time in step (4) was 2.5 hours.
In this embodiment, the MOFs is UiO-67, and in some embodiments, uiO-66 or ZIF-8 may also be used.
And (3) characterizing the contact angles of water and oil on the surface of the prepared PDMS-PDA-MOFs by using a contact angle measuring instrument so as to analyze the wettability of the PDMS-PDA-MOFs. The specific process is as follows: a drop of 5 microliters of deionized water was dropped on the PDMS-PDA-MOFs surface, a photograph was immediately taken with a water contact angle meter and the water contact angle value was calculated, 5 different points were measured for each material, and as shown in Table 2, the calculated average value was the contact angle of the material, which was 152.7 degrees, indicating its superhydrophobicity.
Table 2 contact Angle test
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A preparation method of PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing is characterized in that firstly, an amino modified PDMS substrate is prepared; carrying out self-polymerization on DA (dopamine) solution on an amino modified PDMS substrate under an alkaline condition to form a layer of PDA film with super strong adhesion, obtaining a PDMS substrate modified by PDA (poly-dopamine), and then growing MOFs on the surface of the PDMS substrate in a self-assembly manner to obtain a microspheric PDMS-PDA-MOFs elastic agent with a microstructure coated inside and outside; and curing, laminating and baking the PDMS-PDA-MOFs elastic agent in a mold to finally obtain the PDMS-PDA-MOFs microfluidic chip.
2. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 1, comprising the following steps:
(1) Preparation of amino-modified PDMS substrate: mixing vinyl silicone oil and PDMS (polydimethylsiloxane) according to a mass ratio of (2); dissolving cysteamine (0.1 g) and dimethylolpropionic acid (0.005 g) in ethanol (20 mL) to form a solution A, then dissolving a PDMS substrate containing alkenyl in the solution A, and reacting for 0.5-2h under ultraviolet light; washing with ethanol to remove unreacted substances to obtain an amino modified PDMS substrate;
(2) Performing PDA modification on the amino modified PDMS substrate: preparing a DA (dopamine) solution with the concentration of 2mg/ml by using a Tris hydrochloric acid solution with the pH =8.5, pouring the solution into the amino modified PDMS substrate obtained in the step 1, uniformly mixing, pouring the solution into a volume of 1-2ml, then placing the mixture in an environment at 25 ℃, acting for 24 hours, and combining the solution by virtue of a covalent bond, wherein due to the fact that PDMS has low surface energy, PDA is uniformly coated on the surface of the amino modified PDMS substrate, washing the surface of the amino modified PDMS substrate for several times by using deionized water, and washing off the DA (dopamine) which is not adhered and is not firmly adhered on the PDMS to obtain the PDMS substrate modified by PDA (poly dopamine);
(3) Preparing a PDMS-PDA-MOFs elastic agent: adding MOFs powder into a solvent for ultrasonic dispersion to obtain an MOFs suspension, adding the MOFs suspension into a PDA (poly dopamine) -modified PDMS substrate to promote the self-assembly growth of the MOFs on the surface of the PDA (poly dopamine) -modified PDMS substrate, placing the substrate in an environment at 37 ℃, and acting for 20 hours to obtain a PDMS-PDA-MOFs elastic agent;
(4) Preparing PDMS-PDA-MOFs with micro-channels: placing a silicon wafer with a mold in a clean glass dish, mixing a PDMS-PDA-MOFs (polydimethylsiloxane-moving oxygen-silicon-free functional elastomers) and a curing agent according to a mass ratio of 10; stripping to obtain PDMS-PDA-MOFs with microchannels;
(5) Preparing a PDMS-PDA-MOFs microfluidic chip: placing a silicon wafer in a clean glass dish, mixing a PDMS-PDA-MOFs elastic agent and a curing agent according to a mass ratio of 10.
3. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the wavelength of the ultraviolet light in step (1) is 365nm, and the effective distance is 10cm.
4. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the solid-to-liquid ratio of said MOFs powder and said solvent in step (3) is 1g: (80-120) ml.
5. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the curing agent is DBP (dibutyl phthalate).
6. The method for preparing a PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2 or 4, wherein the solvent in step (3) is dichloromethane (CH 2Cl 2), ethanol (ethanol, C2H5 OH) or dimethylformamide (n, n-dimethylformamide, DMF).
7. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the ultrasonic dispersion time in step (3) is 45-75 min.
8. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the volume ratio of said MOFs suspension to the PDA (polydopamine) -modified PDMS substrate in step (3) is 1: (15 to 25).
9. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 2, wherein the cooling time in step (4) is 1.5-2.5 h.
10. The method for preparing PDMS-PDA-MOFs microfluidic chip for single cell sequencing according to claim 1 or 2, wherein the MOFs is ZIF-8, uiO-66 or UiO-67.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210519804.7A CN115254209B (en) | 2022-05-12 | 2022-05-12 | Preparation method of PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210519804.7A CN115254209B (en) | 2022-05-12 | 2022-05-12 | Preparation method of PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115254209A true CN115254209A (en) | 2022-11-01 |
| CN115254209B CN115254209B (en) | 2024-06-18 |
Family
ID=83758848
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210519804.7A Active CN115254209B (en) | 2022-05-12 | 2022-05-12 | Preparation method of PDMS-PDA-MOFs micro-fluidic chip for single cell sequencing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115254209B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114539410A (en) * | 2022-03-15 | 2022-05-27 | 苏州量化细胞生物科技有限公司 | CLDN 18.2-binding antibodies, probes and use in single cell sequencing of CLDN 18.2-expressing cells |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101817495A (en) * | 2010-03-25 | 2010-09-01 | 湖南大学 | Micro fluid control chip and preparation method and application thereof |
| CN102162815A (en) * | 2011-01-07 | 2011-08-24 | 北京大学 | Plasma separating chip and preparation method thereof |
| CN102411060A (en) * | 2011-12-06 | 2012-04-11 | 东南大学 | Microfluidic chip with high-aspect-ratio micro-fluidic channel and fabrication method thereof |
| CN106268991A (en) * | 2016-08-17 | 2017-01-04 | 西北工业大学 | A kind of manufacture method of PDMS micro-fluidic chip |
| CN107119012A (en) * | 2017-06-04 | 2017-09-01 | 西南大学 | A kind of new method for preparing nano-roughened PDMS substrates |
| CN111763612A (en) * | 2020-06-10 | 2020-10-13 | 宁波大学 | Single cell gene detection chip and manufacturing method and detection method thereof |
-
2022
- 2022-05-12 CN CN202210519804.7A patent/CN115254209B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101817495A (en) * | 2010-03-25 | 2010-09-01 | 湖南大学 | Micro fluid control chip and preparation method and application thereof |
| CN102162815A (en) * | 2011-01-07 | 2011-08-24 | 北京大学 | Plasma separating chip and preparation method thereof |
| CN102411060A (en) * | 2011-12-06 | 2012-04-11 | 东南大学 | Microfluidic chip with high-aspect-ratio micro-fluidic channel and fabrication method thereof |
| CN106268991A (en) * | 2016-08-17 | 2017-01-04 | 西北工业大学 | A kind of manufacture method of PDMS micro-fluidic chip |
| CN107119012A (en) * | 2017-06-04 | 2017-09-01 | 西南大学 | A kind of new method for preparing nano-roughened PDMS substrates |
| CN111763612A (en) * | 2020-06-10 | 2020-10-13 | 宁波大学 | Single cell gene detection chip and manufacturing method and detection method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114539410A (en) * | 2022-03-15 | 2022-05-27 | 苏州量化细胞生物科技有限公司 | CLDN 18.2-binding antibodies, probes and use in single cell sequencing of CLDN 18.2-expressing cells |
| CN114539410B (en) * | 2022-03-15 | 2023-09-05 | 苏州量化细胞生物科技有限公司 | CLDN18.2 binding antibodies, probes and use in single cell sequencing of CLDN18.2 expressing cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115254209B (en) | 2024-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4191608B2 (en) | Microfluidic devices and surface modification processes for solid phase affinity binding assays | |
| US20200009564A1 (en) | Biocompatible Micropillar Array Substrate and Methods for Fabricating Such Substrate | |
| US20170298314A1 (en) | Nano-droplet plate | |
| CN115254209A (en) | Preparation method of PDMS-PDA-MOF micro-fluidic chip for single cell sequencing | |
| WO2017205876A9 (en) | Methods and apparatus for coated flowcells | |
| EP3778024B1 (en) | Device and method for size-selective particle separation, trapping, and manipulation of micro and nanoparticles for molecular detection | |
| Yuan et al. | Protein A‐based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme‐linked immunosorbent assay | |
| CN104627953B (en) | A kind of be base material with SU-8 photoresist and PDMS micro-fluidic chip bonding method | |
| CN108579828A (en) | A kind of Surface modification of microfluidic chip method that flow velocity is controllable | |
| CN112630282A (en) | Preparation of array electrode chip combined with micro-fluidic clamp and construction method of electrochemical immunosensor | |
| CN1928534B (en) | Method for preparing micro-flowing injection type chemical luminous chip | |
| CN1286889C (en) | Chip simply non-reversion binding method using poly dimethyl siloxanes as substrate material | |
| Song et al. | Polymers for microfluidic chips | |
| EP1605264B1 (en) | An integrating analysis chip with minimized reactors and its application | |
| CN108489942B (en) | Preparation method of zinc oxide-sodium polyacrylate composite nanorod array in microchannel | |
| CN109370891B (en) | Biological chip and preparation method thereof | |
| CN115260503A (en) | Preparation method of elastic agent and application of elastic agent in microfluidic chip | |
| CN103060927A (en) | Magnetic controllable writing method of water-soluble protein based on super hydrophobic interface | |
| Chen et al. | Surface-tension-confined droplet microfluidics☆ | |
| KR101106022B1 (en) | Cell-based chemotaxis kit and fabricating method thereof | |
| CN104096609A (en) | Colloidal crystal paper chip and preparation method | |
| CN104549590A (en) | Technology for modifying inner wall of micro-channel in metal micro-fluidic chip | |
| CN105092555A (en) | Micro-fluidic surface enhanced Raman test chip and preparation method thereof | |
| CN106191260A (en) | A kind of biochip substrate | |
| CN111019827A (en) | Hydrogel oocyte in-vitro three-dimensional culture micro-fluidic chip based on different cross-linking degrees and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |