CN113324813A - Biological probe sample application method and process based on high-density dot matrix - Google Patents
Biological probe sample application method and process based on high-density dot matrix Download PDFInfo
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- CN113324813A CN113324813A CN202110583096.9A CN202110583096A CN113324813A CN 113324813 A CN113324813 A CN 113324813A CN 202110583096 A CN202110583096 A CN 202110583096A CN 113324813 A CN113324813 A CN 113324813A
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- 239000000523 sample Substances 0.000 title claims abstract description 108
- 239000011159 matrix material Substances 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 39
- 230000008569 process Effects 0.000 title claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 119
- 238000005507 spraying Methods 0.000 claims abstract description 86
- 239000006260 foam Substances 0.000 claims abstract description 83
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 239000007921 spray Substances 0.000 claims abstract description 28
- 238000004140 cleaning Methods 0.000 claims abstract description 18
- 238000007789 sealing Methods 0.000 claims abstract description 11
- 239000011248 coating agent Substances 0.000 claims abstract description 10
- 238000000576 coating method Methods 0.000 claims abstract description 10
- 239000011148 porous material Substances 0.000 claims abstract description 9
- 238000005070 sampling Methods 0.000 claims abstract description 6
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 4
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 239000000919 ceramic Substances 0.000 claims description 10
- 238000012544 monitoring process Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 4
- 239000011534 wash buffer Substances 0.000 claims description 4
- 239000004593 Epoxy Substances 0.000 claims description 3
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- HFGPZNIAWCZYJU-UHFFFAOYSA-N lead zirconate titanate Chemical group [O-2].[O-2].[O-2].[O-2].[O-2].[Ti+4].[Zr+4].[Pb+2] HFGPZNIAWCZYJU-UHFFFAOYSA-N 0.000 claims description 3
- 229910052451 lead zirconate titanate Inorganic materials 0.000 claims description 3
- 230000009871 nonspecific binding Effects 0.000 claims description 3
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims description 3
- 229910000077 silane Inorganic materials 0.000 claims description 3
- 239000000463 material Substances 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000004043 dyeing Methods 0.000 abstract description 3
- 239000011493 spray foam Substances 0.000 abstract description 3
- 238000000018 DNA microarray Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 2
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- 239000012634 fragment Substances 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
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- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
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- 238000002558 medical inspection Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
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- 238000001291 vacuum drying Methods 0.000 description 1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
Abstract
The invention discloses a biological probe sample application method and a biological probe sample application process based on a high-density dot matrix, which specifically comprise the following steps: preparing a sample, distributing the sample into a pore plate, and placing the pore plate on a sample table; selecting a coating substrate, and sequentially placing the coating substrate on a substrate table; inputting a foam spraying instruction into a control device of the non-contact foam spraying device, sucking the liquid sample in the orifice plate by the non-contact foam spraying device, and moving the liquid sample to the position above the substrate table; carrying out non-contact spray on the position of 80 mu m above the substrate, and moving the substrate according to the instruction to realize the dot matrix layout shape instruction; after the same sample is sprayed with foam, cleaning the non-contact type foam spraying device, and repeating the foam spraying task until the set substrates completely complete the lattice layout; after the lattice layout is finished, drying and sealing all the substrates; and finally, sampling the substrate for fluorescence detection. The method adopts non-contact type spray foam, effectively improves the sample application efficiency, greatly avoids the problem of reagent mixed dyeing, and greatly improves the sample application density.
Description
Technical Field
The invention relates to the technical field of life science, in particular to a biological probe sample application method and a biological probe sample application process based on a high-density dot matrix.
Background
Biochip technology has its origin in nucleic acid molecule hybridization. The biochip is generally referred to as a micro-array hybridization type chip (microarray) in which biological information molecules (such as gene fragments, DNA fragments or polypeptides, proteins, sugar molecules, tissues, etc.) are immobilized on a mutual support medium at a high density, and the sequence and position of each molecule in the array are known and are a predetermined sequence array. The biochip (biochip or bioarray) integrates biochemical analysis process on the chip surface according to the principle of specific interaction between biological molecules, thereby realizing high-flux rapid detection of DNA, RNA, polypeptide, protein and other biological components. Nowadays, biochips play an important role in the fields of medical inspection and biological research.
In the production work, the ideal biochip should have the following characteristics:
1. the fluorescence background signal of the chip substrate is low; 2. the probe has high specificity and can accurately mark the spatial information of a target gene; 3. the probe and the substrate are connected and fixed with high stability and are not easy to fall off in the hybridization, washing and post-treatment processes.
In addition, the spotting process for immobilizing the probes on the substrate also has some influence on the results of the assay. The traditional contact type sample application process not only needs repeated sampling, wastes raw materials and reduces efficiency, but also needs frequent cleaning of sample application heads when sample application is carried out on different types of samples, and the possibility of mixed dyeing of reagents is difficult to avoid completely.
Disclosure of Invention
The invention aims to provide a biological probe spotting method and a biological probe spotting process based on a high-density dot matrix, which aim to solve the problems in the background art. In order to achieve the purpose, the invention provides the following technical scheme: a biological probe spotting method and a biological probe spotting process based on a high-density dot matrix specifically comprise the following steps:
2, selecting different coated substrates according to requirements, sequentially placing the coated substrates on a substrate table in sequence, and numbering the positions of the coated substrates according to a numbering mode;
and 3, inputting a foam spraying instruction in a control device of the non-contact type foam spraying device, wherein the foam spraying instruction comprises the following steps: the number of substrates, the number of droplets sprayed with foam each time, dot spacing, repeated spraying times, dot matrix layout shape, dot matrix area and spraying mode;
and 9, performing sampling fluorescence detection on the cleaned substrate, and verifying the usability of the substrate by using a substrate scanner.
Preferably, the non-contact type foam spraying device in the step 3 comprises a piezoelectric ceramic nozzle and a flash monitoring camera, each foam spraying module of the non-contact type foam spraying device has 8 channels, the piezoelectric ceramic nozzle is mainly made of lead zirconate titanate (PZT) and is communicated with the channels of the foam spraying modules, and the flash monitoring camera can monitor the liquid drop foam spraying speed, the quantity of liquid drops, the size of a dot matrix and the distance between the dot matrixes in real time and feed back the speed, the quantity, the size and the distance to the control device.
Preferably, the coated substrate in step 2 comprises an epoxy silane coated substrate and an N-hydroxysuccinimide ester coated substrate.
Preferably, the liquid solvent in step 1 is printbuffer, and the printbuffer is selected based on optimal microfluidic properties of the droplet and the efficiency of biochemical coupling of the sample to the substrate.
Preferably, the number of the substrates refers to the number of the substrates needing to be arranged with the dot matrix on the substrate table in one production process; the volume and the number of the liquid drops sprayed out by a single nozzle channel under the condition that the number of the sprayed liquid drops is the same position each time; the dot pitch includes a dot pitch (X) and a dot pitch (Y) which respectively represent an X pitch of the droplet dots on the substrate and a Y pitch of the droplet dots on the substrate; the repeated spraying times are the spraying times of the same sample; the dot matrix layout shape is a dot matrix distribution shape on the substrate; the lattice area is a side length parameter of the lattice layout; the spray mode is a single-channel mode, which means that 1 spray channel completes the whole instruction; the multi-channel mode refers to 8 channels spraying foam simultaneously.
Preferably, the coating substrate numbering in the step 2 is numerical along the X-axis direction, and the Y-axis direction is letter number.
The invention has the technical effects and advantages that: the method adopts a non-contact foam spraying technology, overcomes the defects that the needle head is in contact with the substrate in the contact foam spraying process, the interference of the ascending and descending processes of the spray head during working on the dot matrix is avoided, and the error rate is reduced; the non-contact type foam spraying effectively improves the sample application efficiency, greatly avoids the problem of reagent mixed dyeing, and greatly improves the sample application density.
Drawings
FIG. 1 is a schematic numbering scheme in example 1 of the present invention;
FIG. 2 is a schematic view showing spotting of substrates in example 2 of the present invention;
FIG. 3 is a fluorescence detection diagram of a high density array bio-substrate in example 2 of the present invention.
Detailed Description
In the description of the present invention, it should be noted that unless otherwise specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly and may be, for example, fixedly connected, detachably connected, integrally connected, mechanically connected, or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements.
Example 1
A biological probe spotting method and a biological probe spotting process based on a high-density dot matrix specifically comprise the following steps:
and 9, performing sampling fluorescence detection on the cleaned substrate, and verifying the usability of the substrate by using a substrate scanner.
Example 2
A production process of a DNA high-density dot matrix biochip comprises the following steps: oligo probes with 5 '-biotin modification, 3' -Cy5 modification were dissolved in printbuffer and dispensed into 96-well plates as needed, 10. mu.L of sample was added to each well; placing the 96-well plate filled with the sample on a sample table; sequentially placing 100N-hydroxysuccinimide ester coating chips on a substrate table; starting a central control computer of the non-contact type foam spraying device, opening corresponding software on the central control computer, and inputting a production instruction, wherein the instruction is as follows: the number of substrates: 100, number of foam droplets per spray (volume/number of droplets): 40pL/1, dot spacing (X): 150 μm, dot pitch (Y): 150 μm, repeated spray times: 16, lattice layout shape: rectangle, lattice area: 10.3mm × 10.0mm, spray pattern: 8; electrifying the non-contact type foam spraying device, adjusting the foam spraying environment, and starting the non-contact type foam spraying device when the foam spraying environment reaches 50-60% RH and the temperature is 18 ℃; the non-contact type spray device receives an input production instruction, returns to a zero position (initial sample loading position), and sucks an oligo sample in a 96-well plate by utilizing capillary action; after the sample is sucked, the PZT spray head of the non-contact type foam spraying device is transferred to the position A1-A4 of the substrate table by the height of 80 microns under the control of the portal frame; starting voltage pulse, carrying out spray on an 8-channel PZT spray head, wherein the spray amount of liquid drops each time is 40pL, the distance between the spray heads is set to be 1mm, (taking 1mm as an example, the spray amount is not limited to 1mm, and can be set to be 0.5-2mm), sequentially carrying out sample application on 100 substrates A1-A4, cleaning residual liquid of the PZT spray head after sample application on the site A is finished, and absorbing a new sample again; according to the input production instruction, the non-contact type foam spraying device controls the spray head to accurately move 10 micrometers (taking 10 micrometers as an example, the spray head is not limited to 10 micrometers, and can be set to a certain distance between 10 micrometers and 100 micrometers) each time as shown in fig. 2, so that all sample application work is completed, and a high-speed stroboscopic monitoring camera on the non-contact type foam spraying device is used for carrying out real-time monitoring in the sample application process; after sample application is completed, the non-contact type foam spraying device is transferred to a cleaning position to remove residual samples and clean and dry the sample feeding channel; after the spotting of 100 high-density substrates is completed, fixing for at least 3 hours, then transferring to a vacuum drier, and vacuum-drying overnight at room temperature; the dried substrate is carefully and gently transferred to a cleaning device for sealing and cleaning; removing the non-hybridized and immobilized probes on the substrate using a washing buffer containing a surfactant; the cleaned substrate is subjected to sampling fluorescent inspection to verify the usability of the substrate; placing the extracted substrate on a substrate scanner, and exciting CY5 fluorescence to perform fluorescence scanning under the fluorescence channel as shown in FIG. 3; after the detection is qualified, the produced high-density lattice biological substrate can be put into use.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (6)
1. A biological probe sample application method and a process based on a high-density dot matrix are characterized in that: the method specifically comprises the following steps of,
step 1, dissolving a probe and a sample in a liquid solvent according to requirements, distributing the solution into a pore plate, and placing the pore plate on a sample table;
2, selecting different coated substrates according to requirements, sequentially placing the coated substrates on a substrate table in sequence, and numbering the positions of the coated substrates according to a numbering mode;
and 3, inputting a foam spraying instruction in a control device of the non-contact type foam spraying device, wherein the foam spraying instruction comprises the following steps: the number of substrates, the number of droplets sprayed with foam each time, dot spacing, repeated spraying times, dot matrix layout shape, dot matrix area and spraying mode;
step 4, the non-contact type foam spraying device obtains a specific instruction, moves to the sample platform, sucks the liquid sample in the pore plate according to the sample loading requirement, and then moves to the position above the substrate platform;
step 5, the foam spraying device is driven by a moving structure on a portal frame, moves according to the input dot spacing, moves to the position of a first substrate, performs non-contact foam spraying at the position of 80 mu m above the substrate, performs a foam spraying task at the same position according to the number command of foam spraying liquid drops each time, moves above the substrate according to the dot spacing command and the numbering mode of the position of the coated substrate, and finally realizes a dot matrix layout shape command;
step 6, after the same sample is sprayed with foam, the non-contact type foam spraying device moves to a cleaning device to clean the previous sample, a spray head of the non-contact type foam spraying device is cleaned and dried to prepare for sucking the next sample, and after the cleaning is finished, the non-contact type foam spraying device is driven by a moving device to move to a sample table to perform sample introduction work of the next sample to prepare for foam spraying of the next sample; repeating the foam spraying task until the set substrates complete the lattice layout, and completing one substrate production work;
step 7, after the dot matrix layout is finished, after the substrate is fixedly placed for a period of time, drying and sealing all the substrates in an ultra-clean vacuum environment, and in the sealing process, using BSA or casein to inhibit the non-specific binding of the substrates and the sample at the temperature of 60-65 ℃;
step 8, transferring the dried substrate to a cleaning device for sealing and cleaning, and removing probes on the non-hybridized and fixed substrate by using a washing buffer solution containing a surfactant;
and 9, performing sampling fluorescence detection on the cleaned substrate, and verifying the usability of the substrate by using a substrate scanner.
2. The method and process for spotting biological probes based on high-density dot matrix according to claim 1, wherein: the non-contact type foam spraying device in the step 3 comprises a piezoelectric ceramic sprayer and a flash monitoring camera, each foam spraying module of the non-contact type foam spraying device is 8 channels, the main material of the piezoelectric ceramic sprayer is lead zirconate titanate piezoelectric ceramic (PZT) and is communicated with the channels of the foam spraying modules, and the flash monitoring camera can monitor the foam spraying speed of liquid drops, the quantity of the liquid drops, the size of a dot matrix and the space of the dot matrix in real time and feed back the speed, the quantity, the size and the space of the dot matrix to the control device.
3. The method and process for spotting biological probes based on high-density dot matrix according to claim 1, wherein: the coated substrate in the step 2 comprises an epoxy silane coated substrate and an N-hydroxysuccinimide ester coated substrate.
4. The method and process for spotting biological probes based on high-density dot matrix according to claim 1, wherein: the liquid solvent in the step 1 is printbuffer, and the principle of the printbuffer is the optimal microfluidic property of the drop and the biochemical coupling efficiency of the sample and the substrate.
5. The method and process for spotting biological probes based on high-density dot matrix according to claim 2, wherein: the number of the substrates refers to the number of the substrates needing to be laid out with the dot matrix on the substrate table in one production process; the volume and the number of the liquid drops sprayed out by a single nozzle channel under the condition that the number of the sprayed liquid drops is the same position each time; the dot pitch includes a dot pitch (X) and a dot pitch (Y) which respectively represent an X pitch of the droplet dots on the substrate and a Y pitch of the droplet dots on the substrate; the repeated spraying times are the spraying times of the same sample; the dot matrix layout shape is a dot matrix distribution shape on the substrate; the lattice area is a side length parameter of the lattice layout; the spray mode is a single-channel mode, which means that 1 spray channel completes the whole instruction; the multi-channel mode refers to 8 channels spraying foam simultaneously.
6. The method and process for spotting biological probes based on high-density dot matrix according to claim 1, wherein: and the numbering mode of the coating substrate in the step 2 is that the number is a number along the X-axis direction, and the number is a letter number along the Y-axis direction.
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| CN202110583096.9A CN113324813A (en) | 2021-05-27 | 2021-05-27 | Biological probe sample application method and process based on high-density dot matrix |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110583096.9A CN113324813A (en) | 2021-05-27 | 2021-05-27 | Biological probe sample application method and process based on high-density dot matrix |
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