CN117920368A - Microfluidic chip device, preparation method and microsphere loading method - Google Patents

Microfluidic chip device, preparation method and microsphere loading method Download PDF

Info

Publication number
CN117920368A
CN117920368A CN202410109276.7A CN202410109276A CN117920368A CN 117920368 A CN117920368 A CN 117920368A CN 202410109276 A CN202410109276 A CN 202410109276A CN 117920368 A CN117920368 A CN 117920368A
Authority
CN
China
Prior art keywords
layer
chip
microspheres
microfluidic chip
micropores
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.)
Pending
Application number
CN202410109276.7A
Other languages
Chinese (zh)
Inventor
卢晓慧
尹胜男
姚禅钰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen University
Original Assignee
Shenzhen University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shenzhen University filed Critical Shenzhen University
Priority to CN202410109276.7A priority Critical patent/CN117920368A/en
Publication of CN117920368A publication Critical patent/CN117920368A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

The invention provides a microfluidic chip device, a preparation method and a microsphere loading method, and relates to the technical field of microfluidic chips. A microfluidic chip device comprising a chip comprising: a first layer, wherein a micropore array is arranged in the middle of the upper part of the first layer, 200000-300000 micropores are arranged in the micropore array, and the tops of the micropores are open; and the second layer is arranged above the first layer and is connected with the first layer in a bonding way, the lower part of the second layer is provided with a runner, the two ends of the runner are provided with an inlet and an outlet, the middle part of the runner is provided with a detection area, the inlet, the outlet and the detection area are communicated with the runner, and the bottom of the detection area is opened and covers the micropore array. The microspheres in the micropore array of the microfluidic chip device provided by the invention are in a monodisperse state, no aggregation occurs, and the loading rate of the microspheres in the whole micropore array reaches more than 90%.

Description

Microfluidic chip device, preparation method and microsphere loading method
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a microfluidic chip device, a preparation method and a microsphere loading method.
Background
With the development of science and technology, the continuous progress of medicine and the improvement of the level of life analysis instruments, scientists have gradually moved to a new height for exploration of the life analysis field, and have higher requirements for detection of biomarkers. In order to explore the heterogeneity between molecules and cells, the traditional detection system is difficult to collect a large number of molecular average signals to meet the actual demands, while the digital analysis can explore the information of single molecules, even achieve absolute quantitative analysis under certain conditions, a powerful quantitative method is provided for the detection of biomarkers in biomedical research, and the method has huge potential practical value and higher commercialization prospect.
The precondition of digital analysis is that a large number of micro-nano reaction carriers are prepared, the carriers are required to be uniformly and regularly distributed in size, a large number of magnetic microspheres are directly used for reaction, finally, the magnetic microspheres are dripped on a glass slide for detection, and the magnetic microspheres are agglomerated, but not in a monodisperse state and cannot be used for digital analysis. The existing preparation methods of digital microarray analysis platforms are mostly only suitable for one-pot reactions, and require relatively expensive special instruments.
Therefore, a microfluidic chip device capable of uniformly dispersing and cleaning magnetic microspheres is needed to be studied to prepare a microarray substrate, so that a better reaction carrier is provided for digital analysis.
Disclosure of Invention
In view of the above analysis, the present invention aims to provide a microfluidic chip device, a preparation method and a microsphere loading method, which are used for solving at least one of the following problems: the microspheres can be uniformly dispersed and not agglomerated, and can be cleaned in the subsequent analysis operation.
The aim of the invention is mainly realized by the following technical scheme:
In a first aspect, the present invention provides a microfluidic chip device comprising a chip comprising: a first layer, wherein a micropore array is arranged in the middle of the upper part of the first layer, 200000-300000 micropores are arranged in the micropore array, and the top of each micropore is opened; and the second layer is arranged above the first layer and is connected with the first layer in a bonding way, the lower part of the second layer is provided with a runner, two ends of the runner are provided with an inlet and an outlet, the middle part of the runner is provided with a detection area, the inlet, the outlet and the detection area are communicated with the runner, and the bottom of the detection area is opened and covers the micropore array.
Preferably, the microfluidic chip device further comprises a glass slide arranged below the first layer, and the glass slide is bonded and connected with the first layer.
Preferably, the microfluidic chip device further comprises a magnet support, and the magnet support is arranged below the chip and connected with the chip in a detachable connection mode.
Preferably, the diameter of the inlet and the outlet is 1-3mm, the length of the flow channel on either side of the detection area is 4-6mm, the width and the height of the flow channel are 100-300 micrometers, the diameter of the detection area is 4-6mm, the height of the detection area is 100-300 micrometers, and the diameter of the micropore array is 3-5mm; the diameter of the micropores is 4-6 microns, the depth of the micropores is 3-5 microns, and the distance between every two adjacent micropores is 2-4 microns.
In a second aspect, the present invention provides a method for preparing a microfluidic chip device, comprising the steps of: s1: preparing a second layer; s2: preparing a first layer; s3: bonding the second layer to the first layer by oxygen plasma bonding, the detection region of the second layer being aligned with the array of microwells of the first layer, to produce the chip.
Preferably, step S1 comprises the steps of: s1-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching; s1-2: uniformly mixing dimethyl siloxane and an initiator in a mass ratio of (9-11) 1, vacuumizing to remove bubbles, and pouring into the die prepared in the step S1-1; s1-3: and vacuumizing again to remove bubbles invisible to naked eyes, heating and curing, taking out cured polydimethylsiloxane, and punching at an inlet and an outlet respectively to obtain the second layer.
Preferably, step S2 comprises the steps of: s2-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching; s2-2: uniformly mixing dimethyl siloxane and an initiator in a mass ratio of (9-11) 1, vacuumizing to remove bubbles, and pouring into the die prepared in the step S2-1; s2-3: and vacuumizing again to remove bubbles invisible to naked eyes, heating and curing, and taking out the cured polydimethylsiloxane to obtain the first layer.
Preferably, the method further comprises the following steps: s4: the chip was bonded to the slide with oxygen plasma bonding.
In a third aspect, the present invention provides a microsphere loading method, using the microfluidic chip device, comprising the steps of: step 1: injecting a solution containing magnetic microspheres from the inlet of the chip, wherein the magnetic microspheres are distributed in the micropore array area; step 2: after standing, the magnetic microspheres enter micropores by gravity; and step 3: washing the magnetic microsphere without holes on the chip at a speed of 0.05-0.2 mu L/min for 30-120min.
Preferably, the microsphere loading method further comprises step 4: placing the chip obtained in the step 3 and loaded with the magnetic microspheres on the magnet support, and enabling the magnetic microspheres to be in magnetic attraction connection with the magnet support, so that the microfluidic chip device is used for subsequent analysis; and/or the concentration of the magnetic microspheres in the magnetic microsphere-containing solution is 4.8X10 4/μl to 1.2X10 5/μl, and the total number of the magnetic microspheres in the magnetic microsphere-containing solution is 1-3 times the number of the micropores.
Compared with the prior art, the invention has at least one of the following beneficial effects:
A) The microfluidic chip device provided by the invention has the advantages that the solution containing the microspheres flows into the micropore array in the detection area through the inlet and the runner, the microspheres enter the micropores in the micropore array, the microspheres in the micropore array are in a monodisperse state and cannot be agglomerated, and the microfluidic chip device can be used for digital analysis.
B) According to the microsphere loading method provided by the invention, by optimizing the concentration and the flow rate of the injected magnetic microspheres, only one microsphere is loaded in each micropore, and the loading rate of the microspheres in the whole micropore array is more than 90%.
C) According to the microsphere loading method provided by the invention, the chip and the glass slide are used as the microarray substrate to be placed on the magnet bracket after the microsphere is loaded, so that the micro holes cannot be punched after repeated reaction and cleaning in the subsequent analysis experiment of the microspheres.
C) The microfluidic chip device provided by the invention is expected to provide a new reaction platform for digital analysis of subsequent multi-step reactions, and is suitable for a wider absolute quantitative analysis method of biomarkers.
Drawings
Fig. 1 is a perspective view of a microfluidic chip device provided by the present invention.
Fig. 2 is a schematic diagram of a chip structure of a microfluidic chip device according to the present invention.
Fig. 3 is a schematic diagram of a microwell array of a microfluidic chip device provided by the present invention.
FIG. 4 is a schematic representation of a microwell array before and after microsphere access.
FIG. 5 is a microscopic field image of the microspheres before and after entry into the well, without washing.
Fig. 6 is a physical diagram of a microfluidic chip device provided by the invention.
FIG. 7 is a microscopic field plot of the microwell array before and after washing after different numbers of microspheres are injected into the chip.
Reference numerals illustrate:
100-chip; 110-a first layer; a 111-microwell array; 112-microwells; 120-a second layer; 121-flow channel; 122-inlet; 123-outlet; 124-detection zone; 200-slide glass; 300-magnet support.
Detailed Description
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. Furthermore, the various embodiments described herein are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments. The term "or" as used herein refers to a non-exclusive or, unless otherwise indicated. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present invention.
Conventional biomarker detection methods typically acquire biomarker information by acquiring an average analog signal, such as total fluorescence intensity, generated by a large number of biomarkers, and indirectly perform a relative quantitative analysis of the biomarkers by establishing a calibration curve between the analog signal generated by a standard and the concentration of the biomarker to be detected. Unlike traditional detection means, digital quantitative analysis has unique advantages, and can realize absolute quantitative analysis of biomarkers without relying on a calibration curve to realize the quantification of the biomarkers in a sample. In digital quantitative analysis, a reaction system is divided into a large number of micro-reactors, target molecules of a biomarker to be detected are diluted to a certain concentration, most of the reactors contain one copy number of target molecules at most through a certain technical means, and after the reaction is finished, the micro-reactors loaded with one or zero target molecules output 1 or 0 respectively. In a large number of independent microreactors, only the microreactors showing positive signals are counted, and the total signal intensity of a reaction system is not subjected to statistical analysis, so that absolute quantitative analysis of target molecules can be realized by combining the number of the positive microreactors with poisson distribution.
In recent years, through the continuous efforts of researchers, some commercial instruments based on digital absolute quantitative analysis of biomarkers have emerged, with the most widely used microdroplet digital PCR technique (ddPCR) and single molecule array technique (Simoa).
Digital PCR techniques produce thousands of water-in-oil droplet structures by a droplet generator, where each droplet contains at most one copy number of target DNA molecules whose distribution follows the poisson distribution law. Then each droplet is used as a micro-reactor for PCR amplification, after the amplification is finished, each droplet is detected one by one, only the droplet containing the target molecule can generate a fluorescent signal, and the fluorescent signal is output as '1', and the droplet without the target molecule has no fluorescent signal and is output as '0'. Counting all the microdroplets to obtain the number of positive microdroplets and negative microdroplets, and obtaining the initial copy number or initial concentration of the target nucleic acid molecules to be detected according to the poisson distribution statistical rule.
The single molecule array technology (Simoa) is used for fixing the capture antibody on the microsphere, and generating a sandwich immune reaction with the antigen and a biotin (biotin) marked detection antibody; when the concentration of antigen in the solution is extremely dilute, the ratio of antigen to spheres is less than 1:1, and then the ratio of spheres containing the sandwich immune complexes follows the poisson distribution rule, and only one or zero immune complexes exist on each sphere; adding STV modified galactosidase to bind to biotin, resulting in one or zero galactosidase per microsphere; the microspheres are added into the micropores on the surface of the optical fiber bundle, and each micropore can only contain one microsphere by controlling the size of the micropores. The sealing is then performed with a tailored oil, which ensures a high fluorescence intensity locally for each microwell, since the individual enzymes are confined within one microwell. And (3) performing fluorescence imaging on the micropore array, counting the number of positive micropores, and performing immunoassay to realize the digital ultrahigh-sensitivity detection of the antigen.
However, the micro-droplet used in ddPCR technology or the micro-pore array on the surface of the optical fiber bundle used in Simoa technology is only suitable for one-pot reaction, and the cleaning can not be performed after oil sealing. ddPCR technology requires the use of special, relatively expensive, specialized equipment (ddRCR instruments). The steps of etching micropores on the surface of the optical fiber bundle, loading microspheres by a centrifugal method, oil sealing and the like are quite complicated. Therefore, it remains a great challenge to build an integrated, easy to clean, inexpensive digital reaction device.
1-4, The present invention provides a microfluidic chip device comprising a chip 100, the chip 100 comprising:
A first layer 110, wherein a micropore array 111 is arranged in the middle of the upper part of the first layer 110, 200000-300000 micropores 112 are arranged in the micropore array 111, and the top of each micropore 112 is open;
The second layer 120 is arranged above the first layer 110, and is connected with the first layer 110 in a bonding way, the lower part of the second layer 120 is provided with a runner 121, two ends of the runner 121 are provided with an inlet 122 and an outlet 123, the middle part of the runner 121 is provided with a detection area 124, the inlet 122, the outlet 123 and the detection area 124 are communicated with the runner 121, and the bottom of the detection area 124 is opened and covers the micropore array 111.
The micro-fluidic chip device provided by the invention can be used for cleaning the microspheres in the micro-pore array, and cleaning liquid flows in from the inlet and flows out from the outlet after passing through the micro-pore array, so that the micro-fluidic chip is wider in application.
As a specific embodiment of the present invention, the microfluidic chip device further includes a glass slide 200 disposed under the first layer 110. It should be noted that the first layer 110, the second layer 120, and the glass slide 200 of the chip 100 may be bonded by oxygen plasma bonding.
As an embodiment of the present invention, the microfluidic chip device provided by the present invention further includes a magnet holder 300, and the chip 100 is detachably connected to the magnet holder 300. The magnet holder 300 may be a container or device containing a magnet, for example, it may be arranged that the magnet is placed in a housing or container with a receiving cavity. In some cases, the magnet may or may not be in contact with the slide 200, as the magnetic microspheres in the final chip 100 may be magnetically coupled to the magnet.
As an embodiment of the present invention, the flow channel 121 may be groove-shaped, and open at the bottom of the second layer 120. In another embodiment of the present invention, the flow path 121 may be provided in an internal channel shape without opening at the bottom. Similarly, the inlet 122 and the outlet 123 may or may not be open at the bottom of the second layer 120, provided that the flow channels 121, the inlet 122, and the outlet 123 allow fluid to enter the detection region 124 and enter the array of micro-holes 111.
As a specific embodiment of the present invention, the diameter of the inlet 122 and outlet 123 may be 1-3mm, preferably 2mm; the length of the flow channel 121 on either side of the detection zone 124 is 4-6mm, preferably 5mm; the width and height of the flow channels 121 may be from 100 to 300 microns, preferably 200 microns; the middle detection zone 124 may be circular and may have a diameter of 4-6mm, preferably 5mm; the height of the detection zone 124 may be 100-300 microns, preferably 200 microns.
As a specific embodiment of the present invention, the microwell array 111 may be circular, may have a diameter of 3-5mm, preferably 4mm, the microwells 112 may be cylindrical holes, may have a diameter of 4-6 microns, preferably 4.5 microns, the microwells 112 may have a depth of 3-5 microns, preferably 3.8 microns, the spacing between adjacent microwells 112 may be 2-4 microns, preferably 3 microns, and the number of microwells may be 200000-300000, preferably 222211.
In a second aspect, the present invention provides a method for preparing a microfluidic chip device, comprising the steps of:
S1: preparation of the second layer 120;
s2: preparation of the first layer 110;
s3: bonding the second layer 120 to the first layer 110 by oxygen plasma bonding, the detection region 124 of the second layer 120 being aligned with the array of microwells 111 of the first layer 110;
S4: the chip 100 is bonded to the slide 200 by oxygen plasma bonding.
As a specific embodiment of the present invention, step S1 includes the steps of:
S1-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching;
s1-2: mixing dimethyl siloxane (DMS) and initiator (9-11) 1 (w: w), vacuumizing to remove bubbles, and pouring into the mold;
S1-3: after removing bubbles invisible to the naked eye by vacuum again, heating and curing, taking out the cured Polydimethylsiloxane (PDMS), and punching holes at the inlet 122 and the outlet 123 by using a puncher respectively to obtain the second layer 120.
As a specific embodiment of the present invention, the initiator may be SYLGARD184 silicone rubber.
As a specific embodiment of the present invention, step S2 includes the steps of:
S2-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching;
S2-2: mixing dimethyl siloxane (DMS) and initiator (9-11) 1 (w: w), vacuumizing to remove bubbles, and pouring into the mold;
S2-3: after evacuating again to remove bubbles invisible to the naked eye, heating and curing, and taking out the cured Polydimethylsiloxane (PDMS) from the mold to obtain the first layer 110.
As a specific embodiment of the invention, the preparation method of the microfluidic chip device provided by the invention further comprises the following steps:
s5: preparation of a magnet support: the preparation process is as follows: drawing a magnet support design drawing by adopting AutoCAD software, cutting a polymethyl methacrylate (PMMA) plate with the thickness of 2mm into a required shape according to the design drawing by a laser cutting machine, finally assembling the PMMA slice into a required support by using methylene dichloride, and fixing the magnet on the support.
As a specific embodiment of the present invention, the specific operation of step S1-1 is as follows: taking a monocrystalline silicon wafer, heating overnight at 135 ℃, activating by a plasma cleaner after cooling, pouring about 5g of SU8-3050 photoresist, and homogenizing by a spin coating machine (spin coating parameters are 100rpm,10s;2350rpm,55s;100rpm,10 s); heating at 65deg.C for 1min, transferring to 95deg.C, and heating for 20min to volatilize solvent in the photoresist; cooling, exposing in a photoetching machine (exposure energy is 130mJ/cm 2), and performing post-exposure baking (heating at 65 ℃ for 1min, transferring to 95 ℃ and heating for 6 min); and (3) after cooling, washing the uncrosslinked photoresist with a developing solution to obtain the chip mould meeting the design requirements.
As a specific embodiment of the present invention, the specific operation of step S2-1 is as follows: heating monocrystalline silicon wafer at 135 deg.C overnight, cooling, activating with a plasma cleaner, pouring SU8-3050 photoresist about 5g, and homogenizing with a spin coating machine (spin coating parameters are 1000rpm,20s;20000rpm,360s;1000rpm,20 s); heating at 65deg.C for 1min, transferring to 95deg.C, and heating for 20min to volatilize solvent in the photoresist; cooling, exposing in a photoetching machine (exposure energy is 130mJ/cm 2), and performing post-exposure baking (heating at 65 ℃ for 1min, transferring to 95 ℃ and heating for 6 min); and (3) after cooling, washing the uncrosslinked photoresist with a developing solution to obtain the chip mould meeting the design requirements.
In a third aspect, the present invention provides a microsphere loading method, comprising the steps of:
step 1: injecting a solution containing magnetic microspheres through an inlet 121 of the chip 100, wherein the micro-pore array 111 is fully distributed with the microspheres;
Step 2: standing for 20-30min, and allowing the microspheres to enter the micropores by gravity;
step 3: washing the microspheres without holes on the chip twice at the speed of 0.05-0.2 mu L/min for 30-120min each time;
Step 4: and placing the chip 100 loaded with the microspheres on a magnet support, and enabling the microspheres to be in magnetic attraction connection with the magnet support, so that the microfluidic chip device is used for subsequent analysis.
As one embodiment of the present invention, the concentration of the microspheres in the solution containing magnetic microspheres is 4.8X10 4/μL to 1.2X10 5/μL, and the total number of the injected magnetic microspheres is 1 to 3 times the number of micropores.
As an embodiment of the present invention, the solution containing magnetic microspheres may be a solution of magnetic microspheres dispersed in1 XPBST (1 XPBS containing 0.1% Tween-20) with 1 XPBS (10 mM, pH 7.4, containing 137mM NaCl and 2.7mM KCl).
As one embodiment of the present invention, deionized water is used to rinse the microspheres.
Fig. 5 (a) is a microscopic field diagram of the microwell array before the microsphere is introduced, fig. 5 (b) is a microscopic field diagram after the microsphere is introduced but before the excess microsphere is washed, and fig. 5 (c) is a microscopic field diagram after the microwell array in which the microsphere is introduced is washed. As can be seen from fig. 5, each microwell enters at most one microsphere, and the washing process only washes away the microspheres that are not in the wells, but does not overflow the microspheres from the wells.
According to the microsphere loading method provided by the invention, by optimizing the concentration and the flow rate of the injected magnetic microspheres, only one microsphere is loaded in each micropore, and the loading rate of the microspheres in the whole micropore array is more than 90%. The chip and the glass slide are used as a microarray substrate to be placed on a bracket with a magnet after the microsphere is loaded, so that the micro holes can not be punched after repeated reaction and cleaning in the subsequent analysis experiment. The microfluidic chip device provided by the invention is expected to provide a new reaction platform for digital analysis of subsequent multi-step reactions, and is suitable for a wider absolute quantitative analysis method of biomarkers.
The following detailed description of the preferred embodiments of the invention illustrates the principles of the invention and is not intended to limit the scope of the invention.
Example 1
Preparation of chips
S1: preparation of the second layer 120; s1 comprises the following specific steps:
S1-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching; the method comprises the following specific steps:
Drawing a design drawing of the second chip layer 120 by adopting AutoCAD software, wherein the diameters of an inlet 122 and an outlet 123 of a runner 121 are 2mm, the length of the runner 121 at any side of a detection area 124 is 5mm, the width and the height of the runner 121 are 200 micrometers, the diameter of a middle circular detection area 124 is 5mm, the height is 200 micrometers, and a chromium mask plate is prepared according to the design drawing of the second chip layer 120;
The die for die inversion adopts wet photoetching, and SU8-3050 photoresist is used for etching monocrystalline silicon wafers, and the specific operation is as follows: the monocrystalline silicon piece is taken, heated overnight at 135 ℃, activated by a plasma cleaner after cooling, and then the SU8-3050 photoresist is poured for about 5g, and is homogenized by a homogenizer (spin coating parameters are 100rpm,10s;2350rpm,55s;100rpm,10 s). Heating at 65 ℃ for 1min, transferring to 95 ℃ for 20min to volatilize solvent in the photoresist, cooling, exposing in a photoetching machine (the exposure energy is 130mJ/cm 2), performing post-exposure baking (heating at 65 ℃ for 1min, transferring to 95 ℃ for 6 min), cooling, and washing out uncrosslinked photoresist by using a developing solution to obtain the chip mould meeting the design requirements;
S1-2: mixing dimethyl siloxane (DMS) and an initiator thereof at a ratio of 10:1 (w: w), vacuumizing to remove bubbles, and pouring into the mold;
S1-3: vacuumizing again to remove bubbles invisible to naked eyes, heating at 95 ℃ for 30min for curing, taking out the cured PDMS, and punching at an inlet and an outlet by using a puncher respectively.
S2: preparation of the first layer 110; s2, the specific steps are as follows:
S2-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching; the method comprises the following specific steps:
Drawing a design drawing of a first layer 110 of the chip by adopting AutoCAD software, wherein the diameter of a micropore array 111 is 4mm, the diameter of micropores 112 is 4.5 microns, the height is 3.8 microns, the distance between adjacent micropores 112 is 3 microns, the number of micropores is 222211, and preparing a chromium plate mask plate according to the design drawing of the upper layer of the chip;
The die for die inversion adopts wet photoetching, and SU8-3050 photoresist is used for etching monocrystalline silicon wafers, and the specific operation is as follows: taking monocrystalline silicon piece, heating overnight at 135 ℃, cooling, activating with a plasma cleaner, pouring about 5g of SU8-3050 photoresist, and homogenizing with a spin-coater (spin-coating parameters are 1000rpm,20s;20000rpm,360s;1000rpm,20 s). Heating at 65 ℃ for 1min, transferring to 95 ℃ for 20min to volatilize solvent in the photoresist, cooling, exposing in a photoetching machine (the exposure energy is 130mJ/cm 2), performing post-exposure baking (heating at 65 ℃ for 1min, transferring to 95 ℃ for 6 min), cooling, and washing out uncrosslinked photoresist by using a developing solution to obtain the chip mould meeting the design requirements;
s2-2: mixing dimethyl siloxane (DMS) and an initiator thereof at a ratio of 10:1 (w: w), vacuumizing to remove bubbles, and pouring into the mold;
S2-3: after removing bubbles invisible to the naked eye by vacuumizing again, heating at 95 ℃ for 30min for curing, and taking the cured PDMS out of the mold to obtain the first layer 110.
S3: the method for assembling the second layer 120 and the first layer 110 of the chip 100 includes:
the second layer 120 is bonded to the first layer 110 by oxygen plasma bonding, with the intermediate detection region 124 of the second layer 120 aligned with the array of microwells 111 of the first layer 110.
S4: the chip 100 is bonded to the slide by oxygen plasma bonding.
S5: preparation of a magnet support:
drawing a magnet support design drawing by adopting AutoCAD software, cutting a polymethyl methacrylate (PMMA) plate with the thickness of 2mm into a required shape according to the design drawing by a laser cutting machine, finally assembling the PMMA slice into a required support by using methylene dichloride, and fixing the magnet on the support.
Example 2
The microsphere loading method adopts the chip prepared in the embodiment 1, and comprises the following steps:
Step 1: 5. Mu.L of magnetic microspheres (DynabeadsTMM-270Strepitavidin, invitrogen) having a concentration of 1.2X10 5/. Mu.L (the number of microspheres is 6X 10 5, corresponding to about 2.7 times the number of microwells) were injected from the inlet 121 of the chip 100, and the middle 4mm microwell array 111 was filled with microspheres;
Step 2: standing for 20min, and allowing the microspheres to enter the micropores by gravity;
step 3: washing the microspheres without holes on the chip twice at the speed of 0.1 mu L/min for 60min each time;
Step 4: and placing the chip loaded with the microspheres on a magnet bracket, so that the microfluidic chip device is used for subsequent analysis.
Example 3
The microsphere loading method adopts the chip prepared in the embodiment 1, and comprises the following steps:
Step 1: 5. Mu.L of magnetic microspheres (DynabeadsTMM-270Strepitavidin, invitrogen) with the concentration of 4.8X10 4/mu.L (the number of the microspheres is 2.4X10 5 and is equivalent to 1.08 times of the number of micropores) are injected from a chip inlet, and the microspheres are distributed in a 4mm micropore array 111 in the middle;
Step 2: standing for 20min, and allowing the microspheres to enter the micropores by gravity;
step 3: washing the microspheres without holes on the chip twice at the speed of 0.1 mu L/min for 60min each time;
Step 4: and placing the chip loaded with the microspheres on a magnet bracket, so that the microfluidic chip device is used for subsequent analysis.
Comparative example 1
This comparative example was substantially the same as example 2 except that the concentration of the microsphere solution was 2.4X10 4/μl (1.2X10 5 microspheres, equivalent to about 0.54 times the number of microwells).
Comparative example 2
This comparative example was substantially the same as example 2 except that the concentration of the microsphere solution was 2.4X10 5/μl (1.2X10 6 microspheres, equivalent to about 5.4 times the number of microwells).
Experimental example 1
For the microfluidic chip device prepared in example 1, fluid flow in the microfluidic chip device was tested with dye as the flow medium. Fig. 6 is a physical diagram of a microfluidic chip device provided by the invention. Fig. 6 (a) is a front view of the chip after the second layer 120 and the first layer 110 of the chip are bonded to the slide 200. After the dye of fig. 6 (b) is injected into the chip, the flow channel is shown open, and the middle circular detection area 124 may be filled with solution. Fig. 6 (c) is a front view of the chip placed on the magnet holder. Fig. 6 (d) is a front view of the dye-infused chip placed on the magnet holder. Fig. 6 (e) is a side view of the chip placed on the magnet holder. Fig. 6 (f) is a side view of the dye-infused chip placed on the magnet holder.
Experimental example 2
Microspheres with different concentrations or numbers are added into the chip, and the microsphere access rates of the micropore array are different. For the microsphere loading methods of examples 2-3 and comparative examples 1-2, the results were observed using a microscopic field pattern on the chip before the chip was placed on the magnet holder in step 4.
As shown in (c) and (d) of fig. 7, example 2 was injected with 5 μl of a magnetic microsphere solution having a concentration of 1.2x10 5/μl (the number of microspheres is 6 x 10 5, corresponding to about 2.7 times the number of microwells), the non-perforated microspheres were easily washed away (step 3), and the microsphere perforation rate was as high as 99.8%.
As shown in FIGS. 7 (e) and (f), example 3 was injected with 5. Mu.L of a magnetic microsphere solution having a concentration of 4.8X10 4/. Mu.L (the number of microspheres is 2.4X10 5, which corresponds to 1.08 times the number of microwells), and the non-perforated microspheres were easily washed away (step 3), and the microsphere perforation rate was 92%.
As shown in FIGS. 7 (g) and (h), comparative example 1 was injected with 5. Mu.L of a magnetic microsphere solution having a concentration of 2.4X10 4/. Mu.L (1.2X10. 10 5 microspheres, equivalent to about 0.54 times the number of microwells), the number of microspheres was too small, and the microsphere access rate was only 74.8%.
As shown in FIGS. 7 (a) and (b), comparative example 2 was injected with 5. Mu.L of a magnetic microsphere solution having a concentration of 2.4X10 5/. Mu.L (1.2X10 6 microspheres, equivalent to about 5.4 times the number of microwells), and the microspheres without holes were difficult to wash out due to the excessive number of microspheres, and easily caused flow channel blockage.
Any numerical value recited in this disclosure includes all values incremented by one unit from the lowest value to the highest value if there is only a two unit interval between any lowest value and any highest value. For example, if the amount of a component, or a process variable such as temperature, pressure, time, etc., is stated to be 50-90, it is meant in this specification that values such as 51-89, 52-88 … …, and 69-71, and 70-71 are specifically recited. For non-integer values, 0.1, 0.01, 0.001 or 0.0001 units may be considered as appropriate. This is only a few examples of the specific designations. In a similar manner, all possible combinations of values between the lowest value and the highest value enumerated are to be considered to be disclosed.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims (10)

1. A microfluidic chip device comprising a chip, the chip comprising:
a first layer, wherein a micropore array is arranged in the middle of the upper part of the first layer, 200000-300000 micropores are arranged in the micropore array, and the top of each micropore is opened; and
The second layer is arranged above the first layer and is connected with the first layer in a bonding way, the lower part of the second layer is provided with a runner, two ends of the runner are provided with an inlet and an outlet, the middle part of the runner is provided with a detection area, the inlet, the outlet and the detection area are communicated with the runner, and the bottom of the detection area is opened and covers the micropore array.
2. The device of claim 1, wherein the microfluidic chip device further comprises a slide disposed below the first layer, the slide being bonded to the first layer.
3. The device of claim 1, wherein the microfluidic chip device further comprises a magnet holder disposed below the chip and removably coupled to the chip.
4. The device of claim 1, wherein the diameter of the inlet and the outlet is 1-3mm, the length of the flow channel on either side of the detection zone is 4-6mm, the width and height of the flow channel are each 100-300 microns, the diameter of the detection zone is 4-6mm, the height of the detection zone is 100-300 microns, and the diameter of the microwell array is 3-5mm;
The diameter of the micropores is 4-6 microns, the depth of the micropores is 3-5 microns, and the distance between every two adjacent micropores is 2-4 microns.
5. A method of manufacturing a microfluidic chip device according to any one of claims 1 to 4, comprising the steps of:
S1: preparing a second layer;
s2: preparing a first layer; and
S3: bonding the second layer to the first layer by oxygen plasma bonding, the detection region of the second layer being aligned with the array of microwells of the first layer, to produce the chip.
6. The method according to claim 5, wherein step S1 comprises the steps of:
S1-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching;
S1-2: uniformly mixing dimethyl siloxane and an initiator in a mass ratio of (9-11) 1, vacuumizing to remove bubbles, and pouring into the die prepared in the step S1-1; and
S1-3: and vacuumizing again to remove bubbles invisible to naked eyes, heating and curing, taking out cured polydimethylsiloxane, and punching at an inlet and an outlet respectively to obtain the second layer.
7. The method according to claim 5, wherein step S2 comprises the steps of:
S2-1: etching the monocrystalline silicon wafer with SU8-3050 photoresist under the protection of a mask plate, and preparing a die for reverse die by wet photoetching;
s2-2: uniformly mixing dimethyl siloxane and an initiator in a mass ratio of (9-11) 1, vacuumizing to remove bubbles, and pouring into the die prepared in the step S2-1; and
S2-3: and vacuumizing again to remove bubbles invisible to naked eyes, heating and curing, and taking out the cured polydimethylsiloxane to obtain the first layer.
8. The method of claim 5, further comprising the step of:
s4: the chip was bonded to the slide with oxygen plasma bonding.
9. A method of loading microspheres using the microfluidic chip device of any one of claims 1-4 or the microfluidic chip device produced by the method of any one of claims 5-8, comprising the steps of:
Step 1: injecting a solution containing magnetic microspheres from the inlet of the chip, wherein the magnetic microspheres are distributed in the micropore array area;
Step 2: after standing, the magnetic microspheres enter micropores by gravity; and
Step 3: washing the magnetic microsphere without holes on the chip at a speed of 0.05-0.2 mu L/min for 30-120min.
10. The method of claim 9, wherein the microsphere loading method further comprises step 4: placing the chip obtained in the step 3 and loaded with the magnetic microspheres on the magnet support, and enabling the magnetic microspheres to be in magnetic attraction connection with the magnet support, so that the microfluidic chip device is used for subsequent analysis; and/or
Wherein the concentration of the magnetic microspheres in the magnetic microsphere-containing solution is 4.8X10 4/μl to 1.2X10 5/μl, and the total number of the magnetic microspheres in the magnetic microsphere-containing solution is 1-3 times the number of the micropores.
CN202410109276.7A 2024-01-26 2024-01-26 Microfluidic chip device, preparation method and microsphere loading method Pending CN117920368A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410109276.7A CN117920368A (en) 2024-01-26 2024-01-26 Microfluidic chip device, preparation method and microsphere loading method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410109276.7A CN117920368A (en) 2024-01-26 2024-01-26 Microfluidic chip device, preparation method and microsphere loading method

Publications (1)

Publication Number Publication Date
CN117920368A true CN117920368A (en) 2024-04-26

Family

ID=90757179

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410109276.7A Pending CN117920368A (en) 2024-01-26 2024-01-26 Microfluidic chip device, preparation method and microsphere loading method

Country Status (1)

Country Link
CN (1) CN117920368A (en)

Similar Documents

Publication Publication Date Title
US7638339B2 (en) Hydrodynamic focusing devices
AU2009329750C1 (en) Assay device and method for performing biological assays
US8034629B2 (en) High precision scanning of encoded hydrogel microparticles
CN101948741A (en) Microfluidic gene chip for nucleic acid sequencing
WO2013126556A1 (en) Ultrahigh throughput microinjection device
CN209974793U (en) Digital PCR device and centrifugal microfluidic chip
CN109453827A (en) The micro-fluidic chip of flow control is realized based on the microarray of lyophily and/or lyophoby
CN111254046B (en) Device and method for co-capturing single cell and single microsphere
CN217140437U (en) High-stability liquid drop sorting system and micro-fluidic chip comprising same
Wu et al. Multicompartmental hydrogel microspheres as a tool for multicomponent analysis
CN113083384A (en) Droplet type micro-fluidic chip suitable for nucleic acid hybridization detection and preparation method thereof
CN117920368A (en) Microfluidic chip device, preparation method and microsphere loading method
CN104718153B (en) For the method producing structurized microcarrier
CN113713868B (en) Micro-flow control chip and manufacturing method thereof
CN216149778U (en) Micro-fluidic chip
KR101106022B1 (en) Cell-based chemotaxis kit and fabricating method thereof
CN115463626A (en) Hydrophilic-hydrophobic patterned substrate circulation microchannel reactor and preparation method thereof
CN115970775B (en) Centrifugal-driven micro-fluidic chip, preparation method and application
Yang et al. Development of a 3d-tesla micromixer for bio-applications
Wang et al. One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes
Chen et al. Particle focusing mechanisms in λ‐DNA solution flowing in a straight microchannel
CN117732525B (en) Manufacturing method of microfluidic chip
McCoy et al. High-Throughput, Portable Microfluidic Aptamer Assay
Höjer Characterization of Agarose Microdroplet Properties for Acoustic Trapping of Encapsulated Bacteria and On-chip DNA Extraction
Reynolds et al. Droplet microfluidics

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