CN116425109A - Pressure oscillation loading method of microsphere chip - Google Patents
Pressure oscillation loading method of microsphere chip Download PDFInfo
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- CN116425109A CN116425109A CN202310623757.5A CN202310623757A CN116425109A CN 116425109 A CN116425109 A CN 116425109A CN 202310623757 A CN202310623757 A CN 202310623757A CN 116425109 A CN116425109 A CN 116425109A
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- 239000004005 microsphere Substances 0.000 title claims abstract description 132
- 238000011068 loading method Methods 0.000 title claims abstract description 53
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- 229920002223 polystyrene Polymers 0.000 claims abstract description 70
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 55
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 55
- 239000010703 silicon Substances 0.000 claims abstract description 55
- 239000000758 substrate Substances 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000005530 etching Methods 0.000 claims abstract description 6
- 238000003825 pressing Methods 0.000 claims abstract description 5
- 108091034117 Oligonucleotide Proteins 0.000 claims description 14
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- 125000003277 amino group Chemical group 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 7
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
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- 229910021641 deionized water Inorganic materials 0.000 description 4
- 238000001000 micrograph Methods 0.000 description 4
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
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- QXJJQWWVWRCVQT-UHFFFAOYSA-K calcium;sodium;phosphate Chemical compound [Na+].[Ca+2].[O-]P([O-])([O-])=O QXJJQWWVWRCVQT-UHFFFAOYSA-K 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
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- 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
- B81C1/00341—Processes for manufacturing microsystems not provided for in groups B81C1/00023 - B81C1/00261
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B81—MICROSTRUCTURAL TECHNOLOGY
- B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
- B81B7/00—Microstructural systems; Auxiliary parts of microstructural devices or systems
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- Engineering & Computer Science (AREA)
- Manufacturing & Machinery (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Computer Hardware Design (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a pressure oscillation loading method of microsphere chips, which comprises the following steps: etching the silicon substrate to obtain a silicon substrate array with holes with diameters matched with the microspheres to be loaded; and adding a monodisperse polystyrene microsphere solution onto the silicon substrate array, applying pressure thereon while oscillating to load polystyrene microspheres into wells of the silicon substrate array. By the method, the microsphere inlet rate reaches more than 95 percent.
Description
Technical Field
The invention relates to the field of biological chips, in particular to a pressure oscillation loading method of a microsphere chip.
Background
The high-density gene chip is the most promising technology product in the current gene analysis and detection, and has excellent application prospect. Along with the continuous enhancement of life health consciousness of people in the world, related gene detection and disease screening industries are rapidly developed, and the high-density gene chip is widely used in various scenes including genetic disease detection, animal and plant breeding, new drug research and development, personalized medicine and the like at present by virtue of the advantages of high detection convenience, high throughput, rapid analysis, high sensitivity and the like.
The fixing mode of the material in the preparation of the gene chip mainly comprises an in-situ synthesis method, a sample application method, a microsphere loading method and the like, wherein the microsphere loading method is the method with the lowest cost and highest efficiency for preparing the high-density gene detection chip. The common preparation method is to use a silicon substrate as a matrix, and hundreds of thousands, millions or even hundreds of millions of small holes are formed on the surface of the silicon substrate, each small hole is used for fixing one microsphere, and the surface of the microsphere is covalently coupled with a nucleic acid probe for detection.
The loading of the microspheres on the silicon substrate is a key step in the preparation of the gene chip. Hundreds of thousands of microspheres modified with different nucleic acid probes are mixed uniformly in proportion and then randomly loaded in small holes of a silicon substrate. How to provide the loading rate of microspheres and to be suitable for mass production is one of the major difficulties in the manufacturing process of biochips. In addition, the loading of the microspheres is often accompanied by overlapping of multiple layers of microspheres, increasing the washing difficulty and the recovery cost of the microspheres, so avoiding multiple layers of overlapping is also one of the effective ways to increase the production efficiency of the biochip.
Disclosure of Invention
The invention aims to provide a pressure oscillation loading method of microsphere chips, which aims to solve the defects that in the prior art, microsphere loading is unstable, multilayer overlapping is easy to form and large-scale production is not suitable.
The invention aims to provide a pressure oscillation loading method of a microsphere chip, which comprises the following steps:
1) Etching the silicon substrate to obtain a silicon substrate array with holes with shapes and diameters matched with the microspheres to be loaded; and
2) Adding a monodisperse polystyrene microsphere solution onto the silicon substrate array, applying pressure thereon while oscillating to load polystyrene microspheres into wells of the silicon substrate array; and
3) And cleaning the silicon substrate array, and then performing ultrasonic cleaning to obtain the microsphere chip.
In a specific embodiment, the polystyrene microsphere is a polystyrene microsphere that is covalently bound to an oligonucleotide chain.
In one embodiment, the monodisperse polystyrene microsphere solution is dropped onto the silicon substrate array and then pressure is applied to the polystyrene microsphere solution while shaking, thereby loading the polystyrene microspheres into the wells of the silicon substrate array.
In one embodiment, the method of pressure oscillation loading of microsphere chips is performed in a loading mold comprising a body and a plurality of chip grooves disposed on the body.
In a specific embodiment, the polystyrene microsphere solution is simultaneously oscillated by applying pressure to the polystyrene microsphere solution by: and covering a cover glass on the silicon substrate array in which the monodisperse polystyrene microsphere solution is dripped, then paving a layer of high-precision glass balls on the cover glass, covering a glass slide on the chip loading mould, and then sealing, wherein the glass slide faces upwards for oscillating loading.
In a specific embodiment, the surface modified carboxyl polystyrene microsphere is activated by EDC and NHS to obtain an activated polystyrene microsphere; the activated polystyrene microsphere is incubated with an oligonucleotide chain with an amino group modified at the 5' end, and the polystyrene microsphere with the oligonucleotide chain covalently bonded is obtained.
In a specific embodiment, the oscillating power of the oscillation is 60W.
In a specific embodiment, the polystyrene microspheres are subjected to ultrasonic dispersion in ultrapure water to obtain a monodisperse polystyrene microsphere solution having a concentration of 1 mg/mL-5 mg/mL.
In a specific embodiment, the power of the ultrasonic cleaning is 40W, and the cleaning time is 2 min for 3 times.
It is another object of the present invention to provide a microsphere chip prepared by the above method.
The pressure oscillation loading method of the microsphere chip has higher microsphere inlet rate which reaches more than 95.55%, the loaded microsphere is very stable, and the ultrasonic cleaning is allowed for a plurality of times, so that the multilayer overlapping adhesion of the microsphere is reduced, and the microsphere is not easy to fall off in the subsequent experimental step.
Drawings
FIG. 1 is a block diagram of a loading die used in the present invention; fig. a is a front view, and fig. B is a side view.
Fig. 2 shows a physical view of a loading die used in the present invention.
Fig. 3 shows a physical diagram of a pressure oscillation loading device used in the present invention.
Fig. 4 shows a micrograph of the product prepared according to example 1 of the present invention.
Fig. 5 shows a micrograph of the product prepared in example 2 of the present invention after high intensity ultrasound.
Fig. 6 shows a micrograph of the product prepared in example 3 of the present invention after incubation with high intensity ultrasound and complementary fluorescent probes.
Figure 7 shows a comparison of access rates for different loading modes of example 4 of the present invention.
Detailed Description
For a better understanding of the invention, its full technical route, and its advantages, reference should be made to the following detailed description of the invention taken in conjunction with the accompanying drawings. The embodiments described below are only some of the embodiments of the present invention and do not include all of the embodiments. Based on the embodiments of the present invention, other embodiments that can be obtained by a person skilled in the art without any inventive effort are within the scope of the present invention.
In the examples described below, unless otherwise indicated, conventional methods used are recognized or commonly employed by those skilled in the art, and instruments, reagents, consumables, etc. used in the examples of the present invention are available through normal commercial routes.
In one aspect of the present invention, a pressure oscillation loading method for a microsphere chip is provided, including:
1) Etching the silicon substrate to obtain a silicon substrate array with holes with shapes and diameters matched with the microspheres to be loaded;
2) Adding a monodisperse polystyrene microsphere solution onto the silicon substrate array, applying pressure thereon while oscillating to load polystyrene microspheres into wells of the silicon substrate array; and
3) And cleaning the silicon substrate array, and then performing ultrasonic cleaning to obtain the microsphere chip.
In one embodiment, the silicon substrate is previously subjected to a cleaning and baking process to remove water. In one embodiment, the silicon substrate array having holes with a shape and diameter matching those of the polystyrene microspheres is a silicon substrate array having a diameter similar to that of the polystyrene microspheres. The shape and size of the hole are not particularly limited, and the shape and size of the hole are matched with those of the microsphere. For example, the cross section of the hole may be any pattern such as a circle or an ellipse, but is not limited thereto. For example, when the diameter of the microsphere is 4.5 μm to 5.5 μm, the diameter of the hole may be 5 μm to 6 μm (the difference between the diameter of the hole and the diameter of the microsphere is not more than 0.5 μm), and the depth of the hole may be 2.5 μm to 3.5 μm, so that the phenomenon that the loading of the microsphere is unstable and the microsphere is easily removed from the hole due to too shallow depth is avoided. Too deep hole depth is avoided, and too small exposed area of the microsphere surface in the hole is avoided, so that subsequent experimental operations such as liquid incubation and the like are not facilitated. Of course, the depth of the hole is not limited thereto, and may be adjusted accordingly according to the diameter of the microsphere.
In one embodiment, the polystyrene microspheres are ultrasonically dispersed in ultrapure water to prepare a monodisperse polystyrene microsphere solution having a concentration of 1 mg/mL-5 mg/mL.
In one embodiment, the pressure oscillation loading method of the microsphere chip is performed in a loading mold comprising a main body and a plurality of chip grooves provided on the main body. FIG. 1 is a block diagram of a loading die used in the present invention; FIG. 2 shows a physical view of a loading die used in the present invention; fig. 3 shows a physical diagram of a pressure oscillation loading device used in the present invention.
In one embodiment, a monodisperse polystyrene microsphere solution is dropped onto a silicon substrate array and then pressure is applied to the polystyrene microsphere solution while shaking, thereby loading polystyrene microspheres into wells of the silicon substrate array.
In one embodiment, pressure is applied to the polystyrene microsphere solution while shaking by: after a cover glass is covered on a silicon substrate array to which a monodisperse polystyrene microsphere solution is dripped, a layer of high-precision glass balls is paved on the cover glass, the cover glass is covered on a loading mould, then the loading mould is sealed, and the loading mould is oscillated and loaded with the glass slide face upwards. In one embodiment, the surface of the silicon substrate array is washed with deionized water after loading, and ultrasonic washing is performed 3 times to complete the loading of the polystyrene microspheres.
In one embodiment, the silicon substrate array has a thickness of 0.6 to mm and the apertures have a depth of 2.5 μm to 3.5 μm. In one embodiment, the body of the loading die has a length of 75 mm, a width of 25 mm, a thickness of 5 mm, and a plurality of (e.g., 10) chip recesses with a depth of 3 mm disposed therebetween. In one embodiment, the amount of polystyrene microsphere solution dropped on the chip is 100-150 μl. In one embodiment, the cover slip has a length of 9 mm, a width of 9 mm, a thickness of 0.2 mm, a glass material, and smooth sides. In one embodiment, the high precision glass spheres have a diameter of 1 mm and a smooth surface. In one embodiment, the slide has a length of 75 mm, a width of 25 mm, a thickness of 1 mm, a glass material, and smooth sides.
In one embodiment, the oscillating power is 60W and the loading time is 30 minutes.
In one embodiment, the power of the ultrasonic cleaning is 40W and the cleaning is 3 times, each time for 2 minutes.
In one embodiment, the polystyrene microsphere is a polystyrene microsphere that is covalently bound to an oligonucleotide chain. In one embodiment, the surface modified carboxyl polystyrene microsphere is activated by EDC and NHS to obtain an activated polystyrene microsphere, and the activated polystyrene microsphere is incubated with the 5' end modified amino-oligonucleotide chain to obtain a polystyrene microsphere covalently bound to the oligonucleotide chain.
In another aspect of the invention, there is provided a microsphere chip prepared by the preparation method as described above.
The following provides a detailed description of embodiments of the present invention with reference to examples.
Example 1
Preparation of polystyrene microspheres covalently bound to oligonucleotide chains
a. Activation of polystyrene microsphere
Polystyrene microsphere with surface modified carboxyl with particle size of 3-5.5 μm is prepared into suspension with 4-morpholinoethanesulfonic acid buffer solution (MES, pH=6) to prepare 1 mg/mL-5 mg/mL, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution prepared at present are added to make the EDC and NHS concentration in the system 25 mg/mL, and the reaction is carried out at room temperature with shaking for 1 h.
b. Covalent binding of polystyrene microspheres to oligonucleotide chains
10 nmol of dry powder (sequence of oligonucleotide chain: 5'-AGATCGTACGCTATCAGTGCCACGCCTTGTTCCTTCCTATGTTGTTGTCTTGCTTAGAAACCACCGAGGAAGT-3') of 5' -end modified amino oligonucleotide chain with a length of 73 mer to be covalently bound is dissolved by MES buffer solution, and mixed with the obtained activated polystyrene microsphere suspension, wherein the polystyrene microsphere content is 1 mg-5 mg, after the reaction is carried out by shaking at room temperature for 4 h, PBST is centrifugally washed three times (5000 rpm,5 min) and resuspended by ultrapure water, and the mixture is stored for standby.
Pressure oscillation loading method of microsphere chip
1. Etching the silicon substrate to obtain a silicon substrate array of holes having diameters matching those of the microspheres to be loaded:
the silicon substrate is subjected to cleaning and baking treatment in advance to remove water, photoresist is uniformly spin-coated on the surface of the silicon substrate to form a film with uniform thickness, the photoresist is baked and then subjected to ultraviolet exposure through a chromium mask 365 and nm, an alkali washing development is carried out, a glue film of the exposed part is removed, and the photoresist is removed after ICP etching, so that a silicon substrate array with holes with diameters approximately equal to those of polystyrene microspheres is obtained;
2. a solution of monodisperse polystyrene microspheres is added to the silicon substrate array, whereupon pressure is applied while oscillating to load polystyrene microspheres into the wells of the silicon substrate array.
Injecting polystyrene microsphere with covalent bond oligonucleotide chain into ultrapure water, preparing monodisperse polystyrene microsphere solution with concentration of 5 mg/mL by ultrasonic dispersion, placing a silicon substrate array into a loading mould, uniformly dripping 125 mu L of polystyrene microsphere solution onto the silicon substrate array, covering a cover glass on a chip, uniformly spreading a layer of high-precision glass sphere with particle size of 1 mm on the cover glass, covering the cover glass on the mould, winding and sealing the cover glass by a sealing film, fixing the cover glass on a vortex oscillator with the front face upwards, carrying out oscillation loading (60W, 30 min), cleaning the surface of the silicon substrate by deionized water after loading, carrying out short-time ultrasonic cleaning (40W, 2 min) for 3 times, and counting the microsphere access rate by autofluorescence (ultraviolet fluorescence channel) of the polystyrene microsphere under a microscope.
The microsphere chip obtained by the method has the advantage that the rate of the holes of the microspheres in the chip reaches 95.55 percent. The micrograph of the microsphere chip is shown in FIG. 4.
High-intensity ultrasonic repeated cleaning
And 5 times of high-intensity ultrasonic cleaning (100W, 5 min) are carried out on the microsphere chip, and the microsphere access rate is counted through autofluorescence (ultraviolet fluorescence channel) of the polystyrene microsphere under a microscope.
The microsphere chip is subjected to 5 times of high-intensity ultrasonic cleaning, the rate of holes of microspheres in the chip reaches 86.57%, and a microscopic image is shown in fig. 5.
Binding complementary fluorescent probes
The microsphere chip is incubated with a complementary fluorescent probe (5 ' -end modified FAM group, sequence such as 5'-GTGGCACTGATAGCGTACGATCT-3') of the oligonucleotide strand (room temperature, 30 min), and the complementary efficiency of the probe is counted by autofluorescence (ultraviolet fluorescence channel) of the polystyrene microsphere and green fluorescence of the FAM fluorescent probe under a microscope.
The microsphere chip can still be combined with a complementary fluorescent probe with high efficiency after 5 times of high-intensity ultrasonic cleaning, the combination efficiency reaches 98.85%, and the microscopic diagram is shown in fig. 6.
Comparative example
Common loading method 1: dropping the monodisperse polystyrene microsphere solution on the silicon substrate array to ensure that the silicon substrate array is covered by the solution, and after the solution volatilizes, cleaning the surface of the silicon substrate by deionized water, and completing loading;
common loading method 2: dropping the monodisperse polystyrene microsphere solution on one side of the silicon substrate array, scraping the silicon substrate array from the side to the opposite side for multiple times by using a cover glass, rotating the silicon substrate array by 90 degrees clockwise, repeating the scraping operation, continuing rotating until the scraping operation is completed in all directions of the silicon substrate array, cleaning the surface of the silicon substrate by using deionized water, and finishing loading.
The microsphere chips of example 1, the microsphere chips loaded by the general loading method 1 and the microsphere chips loaded by the general loading method 2 were subjected to 5 times of high-intensity ultrasonic cleaning (100W, 5 min), and the porosity was compared after each cleaning. The results show that the microspheres loaded on the microsphere chip are more stable and have smaller change of the hole rate by the invention (figure 7).
While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (10)
1. A pressure oscillation loading method of a microsphere chip comprises the following steps:
1) Etching the silicon substrate to obtain a silicon substrate array with holes with shapes and diameters matched with the microspheres to be loaded;
2) Adding a monodisperse polystyrene microsphere solution onto the silicon substrate array, applying pressure to the polystyrene microsphere solution while oscillating to load polystyrene microspheres into wells of the silicon substrate array; and
3) And cleaning the silicon substrate array, and then performing ultrasonic cleaning to obtain the microsphere chip.
2. The method of claim 1, wherein the polystyrene microsphere is a polystyrene microsphere that is covalently bound to an oligonucleotide chain.
3. The method of claim 1, wherein the monodisperse polystyrene microsphere solution is dripped onto the silicon substrate array and then pressure is applied to the polystyrene microsphere solution while shaking, thereby loading the polystyrene microspheres into the wells of the silicon substrate array.
4. The method of claim 1, wherein the pressure oscillation loading method of the microsphere chips is performed in a loading mold comprising a main body and a plurality of chip grooves provided on the main body.
5. The method of claim 4, wherein pressure is applied to the polystyrene microsphere solution while oscillating by: and covering a cover glass on the silicon substrate array in which the monodisperse polystyrene microsphere solution is dripped, then paving a layer of high-precision glass balls on the cover glass, covering a glass slide on the chip loading mould, and then sealing, wherein the glass slide faces upwards for oscillating loading.
6. The method according to claim 2, wherein the surface-modified carboxyl-modified polystyrene microspheres are activated by EDC and NHS to obtain activated polystyrene microspheres; the activated polystyrene microsphere is incubated with an oligonucleotide chain with an amino group modified at the 5' end, and the polystyrene microsphere with the oligonucleotide chain covalently bonded is obtained.
7. The method of claim 1, wherein the oscillating power of the oscillation is 60W.
8. The method according to claim 1, wherein the polystyrene microspheres are subjected to ultrasonic dispersion in ultrapure water to obtain a monodisperse polystyrene microsphere solution having a concentration of 1 mg/mL to 5 mg/mL.
9. The method of claim 1, wherein the power of the ultrasonic cleaning is 40W and the cleaning is 3 times, each time for 2 minutes.
10. A microsphere chip prepared by the method of any one of claims 1-9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117070602A (en) * | 2023-10-16 | 2023-11-17 | 之江实验室 | Preparation method of gene chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117070602A (en) * | 2023-10-16 | 2023-11-17 | 之江实验室 | Preparation method of gene chip |
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