CN114231607A - Microarray chip and preparation method and application thereof - Google Patents
Microarray chip and preparation method and application thereof Download PDFInfo
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- CN114231607A CN114231607A CN202111251159.7A CN202111251159A CN114231607A CN 114231607 A CN114231607 A CN 114231607A CN 202111251159 A CN202111251159 A CN 202111251159A CN 114231607 A CN114231607 A CN 114231607A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Abstract
The invention relates to a microarray chip and a preparation method and application thereof, the microarray chip comprises a substrate and a nucleic acid molecule identifier microarray connected with the upper surface of the substrate, the microarray is quadrilateral, the microarray chip realizes high-density modification of the nucleic acid molecule identifier microarray under the condition of extremely small substrate area, simultaneously uses extremely trace nucleic acid molecule identifiers, realizes the preparation of the chip with extremely low reagent cost, has simple operation, is suitable for large-scale industrial manufacture, has wide application field, can be used for the detection of biomolecules such as genes, proteins and the like, and improves the detection resolution.
Description
Technical Field
The invention belongs to the technical field of biochips and detection thereof, and particularly relates to a microarray chip, a preparation method of the microarray chip and application of the microarray chip.
Background
At present, the formation of nucleic acid molecule microarrays is mainly an in situ synthesis method and a spotting method. The in-situ synthesis method is characterized in that a photomask is used for controlling reaction positions, so that nucleotide molecules are connected to the surface of a carrier according to sequences, and due to low synthesis efficiency and complex process, the flux is low and the application is limited; the spotting method is a method of forming a large number of microarrays in an ordered arrangement by modifying nucleic acid molecules on the surface of a support (e.g., a substrate) with high precision by microprinting using an automated microspotting apparatus. The method has simple process, easy acquisition of analytical equipment and flexible design, and is widely used for scientific research and practical work.
However, the microprinting technology also has some considerable short plates: firstly, the microarray of nucleic acid molecules produced by an automatic microspotting device has an array resolution of usually more than 50 microns, and the low resolution limits the application of the microarray formed by the spotting method in some fields, such as single cell analysis or space group analysis; secondly, this method requires the use of a large number of supports for carrying microarrays at one time after the start of the spotting device, resulting in an increase in potential cost and waste of resources; meanwhile, the sample application device can also have the phenomenon of missing printing, namely, the required biological molecules are not printed at some point positions; finally, such automated microspotting devices are expensive, limiting the widespread adoption of this technology.
The defects and limitations of the conventional technologies need to be optimized and solved, which is a common appeal and general consensus in the industry. Therefore, it is necessary and urgent to develop a device or apparatus having high resolution, high sample capture rate, high throughput, economy, ease of operation, and general applicability.
Disclosure of Invention
The invention aims to provide a microarray chip which comprises a substrate, wherein the surface of the substrate is modified with a nucleic acid molecule identifier microarray, and the microarray is quadrilateral. The microarray chip realizes high-density modification of the microarray with the nucleic acid molecule identifier under the condition of extremely small substrate area, simultaneously uses extremely small amount of nucleic acid molecule identifiers, realizes the preparation of the chip with extremely low reagent cost, has simple operation, wide application field, can be used for the detection of biomolecules such as genes, proteins and the like, and improves the detection resolution.
Alternatively, the substrate material is not limited to a common glass substrate, but may be quartz, single crystal silicon, polylysine coating, nitrocellulose, polystyrene, Cyclic Olefin Copolymers (COCs), Cyclic Olefin Polymers (COPs), polypropylene, polyethylene and polycarbonate, Polydimethylsiloxane (PDMS), or other known materials in the art, or any combination thereof.
Optionally, the length of the substrate is 1-100cm, preferably 7.5 cm; the width can be 1-100cm, preferably 2.5 cm; the thickness may be 0.1-10mm, preferably 1 mm.
Alternatively, the substrate may be pretreated to create quadrilateral pores in the substrate surface using any method known in the art, such as laser etching. Wherein the width of each micropore can be 0.1nm-1000 μm, preferably 10-50 μm; the depth of each micropore may be from 0.1nm to 1000 μm, preferably from 10 to 50 μm; the center-to-center spacing of adjacent micropores may be in the range of 0.2nm to 1000. mu.m, preferably 10 to 50 μm. The area of each micropore may be 0.1nm2-10cm2Preferably 100 μm2-2500μm2。
Wherein the functionalized pretreatment of the substrate facilitates printing or modification of the nucleic acid molecule identifier. This step can be accomplished by any method known in the art, such as coating the polymer onto the substrate surface, activating chemical groups within the polymer, incorporating reactive or activatable functional groups into the polymer structure.
Among other things, any precursor known in the art that is any reactive or capable of being activated to form a precursor having reactive functional groups can be modified on the substrate surface using suitable methods, such as carboxylic acid groups activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), aldehyde groups, epoxy groups, 1, 4-Phenylisothiocyanatocyanate (PDITC) groups, streptavidin groups, and the like.
Wherein the nucleic acid molecule identifier microarray has a quadrilateral shape, which may be a regular square, or a quadrilateral shape having an irregular shape, and the angle of each corner inside the microarray may be 0 to 180 degrees, and in a preferred embodiment, the microarray has a square shape.
Wherein, the length of the nucleic acid molecule identifier microarray can be 0.1nm-1000 μm, preferably 10-50 μm; the width can be 0.1nm-1000 μm, preferably 10-50 μm; the area can be 0.1nm2-10cm2Preferably 100 μm2-2500μm2。
Wherein the center-to-center distance between adjacent microarrays can be 0.1nm to 1000 μm, preferably 10 to 50 μm.
Wherein, the surface of the substrate contains at least 1 microarray, for example, the surface of the substrate contains at least 1 microarray, and may contain 10, 100, 1000, 10000, 100000 microarrays, and in a preferred embodiment, the surface of the substrate contains 4900 microarrays.
The principle of attaching the nucleic acid molecule identifier to the upper surface of the substrate according to the present invention may be any known principle in the art, including but not limited to physical, chemical, biological modification, such as electrostatic binding, carboxyamide reaction, biotin-streptavidin reaction, photocatalysis, radical polymerization, and the like.
Alternatively, the term "linked" as used herein includes both direct and indirect attachment, for example, attachment of a substrate surface to a nucleic acid molecule identifier, either directly by covalent attachment of a moiety therebetween or indirectly by attachment of an intermediate moiety or carrier.
The formation of the high-density quadrilateral nucleic acid molecule identifier microarray according to the present invention can be accomplished by any known method in the art, including but not limited to spotting, inverted attachment printing, microchip method, etc.
In order to make the understanding of the substrate having a high-density quadrilateral nucleic acid molecule identifier microarray according to the present invention more clear to those skilled in the art, the following detailed description will be made with reference to the accompanying drawings.
Correspondingly, the invention provides a manufacturing method of the high-density quadrilateral nucleic acid molecule identifier microarray substrate, which comprises the following steps:
specifically, microchannels arranged in parallel are attached to a substrate surface (fig. 1), and different first groups of nucleic acid molecule identifiers are respectively introduced into the microchannels by means of a microfluidic chip technology, so that the microchannels are connected to the substrate surface and unconnected nucleic acid molecule identifiers are removed. Then, the micro-channels are attached to the surface of the substrate again in a direction different from the direction of the above-mentioned channels, and a second set of different nucleic acid molecule identifiers is introduced into the micro-channels, and a unique nucleic acid molecule identifier is generated on the surface of the substrate through reaction (FIG. 2).
Optionally, the number of the pore channels in the parallel arrangement of the microchannels is at least 1 and more. For example, the parallel microchannels may comprise at least 10, at least 100, at least 1000, or at least 10000 microchannels arranged in parallel.
Optionally, the width of the pore channel in the parallel arrangement micro-channel is in the range of 0.1nm to 1000 μm. For example, the width of the parallel microchannels may be 1, 10, 100, 1000. mu.m.
Optionally, the width of the space between the channels in the parallel arrangement micro-channels is in the range of 0.1nm to 1000 μm. For example, the width of the channel pitch may be 1, 10, 100, 1000 μm.
Optionally, the liquid inlet arrangement of the parallel arrangement of microchannels comprises each channel connected to a separate liquid inlet, a plurality of channels connected to the same liquid inlet, and a channel connected to a plurality of liquid inlets.
Optionally, the liquid outlet arrangement of the parallel arrangement of microchannels comprises each channel connected to a separate liquid outlet, and also comprises a plurality of channels connected to the same liquid outlet or one channel connected to a plurality of liquid outlets.
Wherein "different" in the first and second sets of nucleic acid molecule identifiers means that the identifiers are different from each other with respect to other nucleic acid molecule identifiers of the invention attached to the surface of the substrate and other nucleic acid sequences of the invention.
Wherein said first set of nucleic acid molecule identifiers comprises: a universal region, a spatial coordinate sequence I and a nucleic acid molecule identifier hybridization region; the second set of nucleic acid molecule identifiers comprises: a nucleic acid molecule identifier hybridization area, a second space coordinate sequence, a molecule label and a nucleic acid molecule identifier capture area precursor; reacting to produce the unique nucleic acid molecule identifier comprises: universal region, spatial coordinate sequence one, nucleic acid molecule identifier hybridization region, spatial coordinate sequence two, molecular label, nucleic acid molecule identifier capture region (figure 2). The term "unique" refers to a nucleic acid molecule that is different from other identifiers of nucleic acid molecules attached to the surface of the substrate and related to subsequent applications and related to the present invention.
Wherein the universal region may comprise an active reaction site and a primer binding site, the active reaction site being available for attachment to the substrate surface, may comprise any reactive substance capable of binding to the substrate, and may also comprise a precursor capable of being activated to form a reactive site. Primer binding sites refer to nucleic acid sequences that can be used to bind universal primers for reactions such as amplification of nucleic acid molecule identifiers.
Alternatively, the primer binding site may be 1-1000 nucleotides in length.
Wherein the first spatial coordinate sequence serves to locate the nucleic acid molecule identifier to determine its position on the substrate surface, thereby providing information on the spatial location of the nucleic acid molecule captured by the nucleic acid molecule identifier capture zone (e.g., determining the spatial location of the captured nucleic acid molecule in the tissue sample).
Alternatively, the length of the first spatial coordinate sequence may be 1-1000 nucleotides.
Wherein the second spatial coordinate sequence is used to locate the nucleic acid molecule identifier to determine its position on the substrate surface, thereby providing information on the spatial position of the nucleic acid molecule captured by the nucleic acid molecule identifier capture zone (e.g., determining the spatial position of the captured nucleic acid molecule in the tissue sample).
Alternatively, the length of the second spatial coordinate sequence can be 1-1000 nucleotides.
Alternatively, the spatial coordinate sequences of the identifiers of the nucleic acid molecules introduced into each of the parallel-arranged microchannels are different from each other and can be distinguished from each other.
Wherein, complementary hybridization can be generated between the first and second sets of nucleic acid molecule identifier hybridization regions according to the Watson-Crick base complementary pairing principle known in the art.
Alternatively, the sequence length of the nucleic acid molecule identifier hybridizing region may be 1-1000 nucleotides.
Wherein the molecular tag can provide a tag for the nucleic acid captured by the unique nucleic acid molecular identifier, or provide a name of the nucleic acid, different unique nucleic acid molecular identifiers should comprise different molecular tags, i.e., the sequence order of the nucleic acid sequences of the molecular tags is unique for distinguishing the nucleic acid (e.g., mRNA) hybridized with different nucleic acid molecular identifiers.
Alternatively, the molecular tag may be 1-1000 nucleotides in length.
Wherein the nucleic acid molecule identifier capture zone precursor may comprise a nucleic acid sequence, for example a nucleic acid sequence consisting of a poly-A sequence, used to form the nucleic acid molecule identifier capture zone. Alternatively, the nucleic acid molecule identifier capture zone precursor may be 1-1000 nucleotides in length.
The reaction system for generating a unique nucleic acid molecule identifier indicative of spatial coordinates may comprise any components that allow extension or amplification of a nucleic acid sequence, such as nucleotides, amplification enzymes (e.g., DNA polymerase) or ligases (e.g., T4-DNA ligase), buffers, ultra pure water, etc.
Optionally, a prehybridization step may be included prior to performing the nucleic acid sequence extension or amplification reaction, wherein the first and second nucleic acid molecule identifiers are subjected to complementary hybridization by a nucleic acid molecule identifier hybridization region according to the Watson-Crick base complementary pairing rules, which facilitates the generation of unique nucleic acid molecule identifiers.
Wherein the nucleic acid molecule identifier capture region may be used to capture nucleic acid sequences, optionally the capture domain may comprise a random sequence which may be used in combination with a poly-T oligonucleotide sequence (or poly-T analogue or the like) to improve capture efficiency.
Alternatively, the nucleic acid molecule identifier capture region may be a poly-T oligonucleotide sequence or a sequence functionally or structurally similar to a poly-T oligonucleotide sequence (e.g., a poly-U or deoxythymidine analog) that can bind to a nucleic acid (e.g., mRNA) carrying a poly-A sequence.
Alternatively, the sequence length of the nucleic acid molecule identifier capture region may be 1-1000 nucleotides. .
Wherein the arrangement of the functional regions of the nucleic acid molecule identifier and the unique nucleic acid molecule identifier includes but is not limited to the order or position listed in the present invention, and one or more of the functional sequences of the nucleic acid molecule identifiers can be arranged in any suitable order.
Alternatively, after the generation of the quadrangular nucleic acid molecule identifier microarray, a washing step may be performed, which removes substances other than the quadrangular nucleic acid molecule identifier microarray from the surface of the substrate. Preferably, the treatment may be performed using a buffer containing a surfactant, a salt, or the like.
Alternatively, the quadrilateral nucleic acid molecule identifier microarray may be characterized by any method known in the art, for example, using fluorescently labeled tag sequences or sequencing analysis, or the like.
Accordingly, the present invention provides a specific application of the substrate having the high-density quadrilateral nucleic acid molecule identifier microarray: it is used for spatial transcriptomics studies of tissue samples.
Specifically, a tissue sample frozen section is attached to the surface of a substrate, so that the sample section covers the surface of the high-density quadrilateral nucleic acid molecule identifier microarray, and then HE staining and imaging are performed on the tissue sample. And then digesting the frozen section, capturing the mRNA sequence of the tissue by the nucleic acid molecule identifier microarray through a nucleic acid molecule identifier capturing area, synthesizing a first strand and a second strand of cDNA, amplifying the cDNA, establishing a nucleic acid library and analyzing spatial transcriptomics, corresponding the transcriptomics information of the analyzed sample to the corresponding spatial position according to the spatial coordinate sequence information, and fitting the spatial transcriptomics information with the tissue sample image to obtain the spatial transcriptomics information of the tissue sample.
Alternatively, to facilitate analysis of the spatial location of the tissue sample corresponding to the unique nucleic acid molecule identifier having a spatial coordinate sequence, the tissue sample may be imaged using any method known in the art, such as light, dark field imaging, confocal imaging, and the like.
Alternatively, to facilitate analysis of the spatial location of the tissue sample corresponding to the unique nucleic acid molecule identifier having a spatial coordinate sequence, the orientation of the tissue sample can be indicated using any method known in the art, such as etching a specific print on the surface of the substrate.
Alternatively, the sample may be digested in any manner known to those skilled in the art such that the mRNA sequences within it may be captured by the unique nucleic acid molecule identifier, e.g., chemical digestion, enzymatic digestion, etc.
Alternatively, first strand cDNA synthesis can be performed using any method known in the art, such as reverse transcription, i.e., by reverse transcription of the reaction fluid to produce captured copies of mRNA from the tissue sample, which copies of cDNA are reflective of the transcriptome information contained in the tissue sample. The unique nucleic acid molecule identifier of the present invention can itself serve as a priming sequence for a reverse transcription reaction.
Alternatively, after the cDNA copies are generated, the captured mRNA sequences may be removed and this step may be accomplished by any method known in the art, such as chemical, physical, biological, and the like.
Alternatively, after the cDNA copy is generated, the tissue section from the substrate surface may be removed, and this step may be performed using any method known in the art, such as enzymatic degradation.
Alternatively, second strand cDNA synthesis may be performed using any method known in the art, such as isothermal nucleic acid amplification, in which first strand copies of tissue sample-derived cDNA from the captured components are generated by isothermal amplification of a reaction fluid, wherein the generated first strand copies of cDNA reflect transcriptome information contained in the tissue sample and facilitate exponential increase in copy number.
Alternatively, a universal primer, a specific primer (e.g., a template switch primer), or a random primer may be used in the reaction to generate the second strand of cDNA, thereby producing a full-length or random-length nucleic acid fragment product that may correspond to information on the sequence of the captured mRNA.
Alternatively, cDNA amplification can be carried out using any method known in the art, such as the polymerase chain reaction, using the cDNA copies described above as templates, using DNA polymerase enzymes, the required primers and corresponding buffer systems. The amount of cDNA generated by this step should be sufficient for the nucleic acid library building step.
Alternatively, nucleic acid library building may be performed using any method known in the art, for example by polymerase chain reaction, following introduction of the Illumina tag sequence into the cDNA amplification product.
Alternatively, prior to the construction of the nucleic acid library, the cDNA amplification products may be subjected to fragmentation, sequence repair, sequencing adaptor ligation, etc., which facilitates library construction and sequencing analysis. This step can be performed using any method known in the art, such as physical, chemical, or biological methods, among others.
Alternatively, fragment length screening of nucleic acid sequences can be performed before and after the nucleic acid library is established, and this step can be performed using any method known in the art, such as nucleic acid sequence length analysis and the like.
Optionally, before and after the nucleic acid library is established, appropriate nucleic acid purification methods can be used to remove potentially introduced interferents, such as non-target nucleic acid sequences, nucleotides, salts, etc., which facilitates the reliability of the assay results. This step may be performed using any method known in the art, such as magnetic bead separation.
Alternatively, the target nucleic acid sequence may be analyzed using any method known in the art, such as first generation sequencing, second generation sequencing, third generation sequencing, and the like. Generally, such methods are sequence-specific methods, e.g., the methods may employ a sequence analysis method of the amplification reaction type using primers for the sequence being analyzed, which amplification reaction may be a linear or non-linear reaction, e.g., Polymerase Chain Reaction (PCR), isothermal amplification reaction (e.g., RPA), and the like.
Optionally, the analyzing step should comprise analysis of the unique nucleic acid molecule identifier, whereby spatial localization of the analyzed sequence and captured nucleic acid sequence information can be obtained.
The above is only a typical application of the microarray substrate with high density quadrilateral nucleic acid molecule identifiers of the present invention, and the application range of the substrate of the present invention includes the capture of any nucleic acid, protein, polysaccharide molecule in tissue samples, and also includes the application of the method to any nucleic acid, protein, polysaccharide molecule in cells, such as tRNA, rRNA, and viral RNA.
Alternatively, the tissue sample in the application of the substrate of the invention may be a tissue sample of any organism or spatial structure of an organism, e.g. plant, animal, fungus.
Alternatively, the tissue sample in the application of the substrate of the present invention may be any type or kind of tissue sample, for example, a dead or living tissue sample, a fresh tissue may be used as the tissue sample of the present invention. The tissue samples of the present invention also include any treated or untreated tissue samples and are not limited to the illustrated embodiments, such as fixed, unfixed, ambient, paraffin tissue samples.
Alternatively, the application of the substrate of the invention can be any kind of biological omics testing and analysis without being limited to transcriptomics, such as epigenomics, proteomics, metabolomics, etc.
Drawings
FIG. 1 is a diagram showing microchannels used for preparing a high-density quadrilateral nucleic acid molecule identifier microarray substrate, wherein the height of the microchannel is 20 μm, the width of the microchannel is 10 μm, and the center-to-center distance of the microchannel is 30 μm;
FIG. 2 is a schematic diagram of the preparation of a microarray substrate for unique nucleic acid molecule identifiers, showing the complementary sequences thereof;
FIG. 3 is a search of the activation conditions for attaching a first set of nucleic acid molecule identifiers to the surface of a magnetic bead substrate;
FIG. 4 is a plot of the ligation time between the surface of the magnetic bead matrix and the first set of nucleic acid molecule identifiers;
FIG. 5 is a search of reaction conditions for generating unique nucleic acid molecule identifiers on the surface of a magnetic bead matrix;
FIG. 6 is a substrate with a high-density quadrilateral oligonucleotide microarray, in FIG. 6, all unique nucleic acid molecule identifiers are subjected to liquid modification by twice placement in the lateral and longitudinal directions of microchannels, a first group and a second group of nucleic acid molecule identifiers are respectively introduced to the surface of the substrate, and then the high-density quadrilateral oligonucleotide microarray is obtained on the surface of the substrate through extension reaction and multistep cleaning, wherein the 3' end of the unique nucleic acid molecule identifier is a poly-T sequence, the quality detection is performed by using a fluorescence-labeled poly-A probe and gene sequencing, and all microchannel cross sites are modified with unique nucleic acid molecule identifiers carrying spatial coordinate sequences;
FIG. 7 shows the result of staining mouse brain tissue sections;
FIG. 8 is a graph showing the concentration measurement of the amplified cDNA;
FIG. 9 shows the quality control of the cDNA amplification product, as shown in the example of FIG. 9, the reverse transcription, cDNA synthesis and amplification are performed on the mRNA captured by the microarray, the products are enriched and collected, and the length of the cDNA is measured, and the detection result shows that a cDNA sequence of 300 and 1000bp is generated, which is consistent with the expected result;
FIG. 10 is a nucleic acid library concentration determination;
FIG. 11 shows quality control of nucleic acid library. The length of the nucleic acid library sequence derived from the tissue sample is determined, and the detection result shows that the nucleic acid sequence with the length of 300-700bp is generated at a sufficient concentration and is consistent with the expected result;
FIG. 12 is a library data quality analysis.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparation of microchannels
Placing a glass plate with a uniform chromium film and a photoresist layer under a mask plate with pore channels arranged in parallel, exposing for 10s by an ultraviolet lamp, and then placing a substrate in a developing solution to soak for 30s to obtain the photoresist glass surface with a micro-pore channel pattern with a chromium layer; soaking in chromium etching solution for 5min, and placing the surface in glass etching solution (mass ratio HF: HNO)3：NH4F：H2Soaking in 25:23.5:9.35:450) for 40min to obtain a glass pore channel mold with the morphology structure of the micro-pore channel pattern; uniformly mixing Polydimethylsiloxane (PDMS) prepolymer and a curing agent according to a mass ratio of 10:1, vacuum degassing for 30min, pouring the mixture onto the surface of a glass mold, placing the glass mold in an oven at a temperature of 60 ℃, curing for 10h, and uncovering the glass mold to obtain PDMS microfluidic channels which are arranged in parallel, wherein the height of each channel is 20 micrometers, the width of each channel is 10 micrometers, and the center distance of each channel is 30 micrometers (shown in figure 1).
Example 2: preparation of unique nucleic acid molecule identifier on magnetic bead substrate surface
In order to search the reaction conditions for generating a unique nucleic acid molecule identifier, this example performed preliminary experiments of reaction conditions using carboxyl magnetic beads (Spherotech) having a size of 30 μm, in which the exposure time of fluorescence was 1s each.
A10 mu l volume of carboxyl magnetic beads is washed 3 times by 2- (N-morpholine ethanesulfonic acid) buffer solution with pH5.0, then the magnetic beads are activated by EDC and MES solution mixtures with the ratio of 1:1, 3:2 and 9:1 respectively to connect a first group of nucleic acid molecule identifiers, quality inspection is carried out by using fluorescent sequences capable of hybridizing with the first group of nucleic acid molecule identifiers, the connection amount of the first group of nucleic acid molecule identifiers is characterized by the fluorescence intensity, and a negative sequence is added into a control group, so that the connection amount of the first group of nucleic acid molecule identifiers is the highest when the ratio of the two groups is 1:1 (figure 3). Then, the ratio is adopted to search the connection time of the first group of nucleic acid molecule identifiers, the reaction time is set to be 30min, 2h, 4h and 12h respectively, and a negative sequence is added into a control group, so that the result shows that after the reaction time is more than 2h, the performance of each group is close to and better than that of an experimental group (figure 4) with the reaction time being less than 2h, and the reaction time is adopted in the next pre-experiment.
After ligation of the first set of nucleic acid molecule identifiers under optimal conditions, a second set of nucleic acid molecule identifiers is added and gradient annealing is performed: cooling to 20 ℃ at 95 ℃, cooling to 0.1 ℃ per second, and then adding a unique nucleic acid molecular identifier to generate a reaction system: klenow polymerase large fragment (NEB) and corresponding buffer, dNTP, ddH2O, set up different enzyme gradients: 0.5. mu.l, 2.5. mu.l, 5. mu.l, reaction conditions: eluting the magnetic beads by 8M urea after the reaction is finished at 25 ℃, 30min, 75 ℃ and 10min, and then utilizing the fluorescence-labeled nucleic acid sequence A20(the nucleic acid sequence information is shown in Table 3), and the results show that the brightness of the three experimental groups is close to that of the control group added with the negative sequence (figure 5), so that the subsequent experiments are carried out by adopting a reaction system with the enzyme amount of 0.5 mu l.
Table 1: partial first nucleic acid molecule identifier sequence information
| Sequence name | Sequence information 5 '→ 3' |
| P1-1 | NH2-CTACACGACGCTCTTCCGATCTACCTTGCGACTGGCCTGC |
| P1-2 | NH2-CTACACGACGCTCTTCCGATCTCAGGTGTGACTGGCCTGC |
| P1-3 | NH2-CTACACGACGCTCTTCCGATCTAGGAATTAGACTGGCCTGC |
| P1-4 | NH2-CTACACGACGCTCTTCCGATCTGCGATACAACTGGCCTGC |
| P1-5 | NH2-CTACACGACGCTCTTCCGATCTACCGATAGACTGGCCTGC |
| P1-6 | NH2-CTACACGACGCTCTTCCGATCTCCACTGGAACTGGCCTGC |
| P1-7 | NH2-CTACACGACGCTCTTCCGATCTAGACATGCACTGGCCTGC |
| P1-8 | NH2-CTACACGACGCTCTTCCGATCTAGAGAAGTACTGGCCTGC |
| P1-9 | NH2-CTACACGACGCTCTTCCGATCTACGGATATACTGGCCTGC |
| P1-10 | NH2-CTACACGACGCTCTTCCGATCTAGTATCGTACTGGCCTGC |
Table 2: partial second nucleic acid molecule identifier sequence information
| Sequence name | Sequence information 5 '→ 3' |
| P2-1 | NBAAAAAAAAAAAAAAAAAANNNNNNNNCTCAGACAGCAGGCCAGT |
| P2-2 | NBAAAAAAAAAAAAAAAAAANNNNNNNNAAGTTACGGCAGGCCAGT |
| P2-3 | NBAAAAAAAAAAAAAAAAAANNNNNNNNAGCTATGTGCAGGCCAGT |
| P2-4 | NBAAAAAAAAAAAAAAAAAANNNNNNNNACAGTAACGCAGGCCAGT |
| P2-5 | NBAAAAAAAAAAAAAAAAAANNNNNNNNAGGTCGCTGCAGGCCAGT |
| P2-6 | NBAAAAAAAAAAAAAAAAAANNNNNNNNACAACTAGGCAGGCCAGT |
| P2-7 | NBAAAAAAAAAAAAAAAAAANNNNNNNNGGCAAGCAGCAGGCCAGT |
| P2-8 | NBAAAAAAAAAAAAAAAAAANNNNNNNNCAGCACTCGCAGGCCAGT |
| P2-9 | NBAAAAAAAAAAAAAAAAAANNNNNNNNCAAGACTAGCAGGCCAGT |
| P2-10 | NBAAAAAAAAAAAAAAAAAANNNNNNNNCTAAGCTCGCAGGCCAGT |
TABLE 3 example partial correlation sequence
Example 3: preparation of microarray substrate with high-density quadrilateral nucleic acid molecule identifier
Attaching 70 micro-channels which are arranged in parallel to the surface of a substrate coated with N-hydroxysuccinimide groups (NHS), respectively introducing different first group of nucleic acid molecule identifiers (the first group of nucleic acid molecule identifiers comprises a universal region, a spatial coordinate sequence I and a nucleic acid molecule identifier hybridization region from 5 'end to 3' end, wherein part of nucleic acid molecule identifier information is shown in table 1), connecting the first group of nucleic acid molecule identifiers with the surface of the substrate, then introducing a substrate cleaning solution (Tris-HCl, pH8.0 and 0.05% Tween-20) into the channels, and washing away redundant nucleic acid molecule identifiers. Attaching the micro-channel to the surface of the substrate again in a direction perpendicular to the channels, wherein the intersection point of the two channels is positioned in the center of the substrate, respectively introducing different second group of nucleic acid molecule identifiers (the nucleic acid molecule identifier capture region, the molecule tag, the second space coordinate sequence and the nucleic acid molecule identifier hybridization region are formed by the 5 'end to the 3' end, part of the nucleic acid molecule identifier information is shown in table 2), enabling the second group of nucleic acid molecule identifiers to generate complementary hybridization with the first group of nucleic acid molecule identifiers according to the Watson-Crick base complementary pairing principle through the nucleic acid molecule identifier hybridization region, and washing off the unhybridized second group of nucleic acid molecule identifiers. Introducing an extension reaction solution (Klenow polymerase large fragment and an adapter buffer solution thereof, dNTP and the like) into the micro-channel, incubating for 1h at 37 ℃, then performing enzyme inactivation and washing the surface of the substrate to generate a unique nucleic acid molecule identifier carrying a space coordinate, wherein the unique nucleic acid molecule identifier consists of a 5 'end to a 3' end: the kit comprises a general area, a space coordinate sequence I, a nucleic acid molecule identifier hybridization area, a space coordinate sequence II, a molecule label and a nucleic acid molecule identifier capture area.
Dripping a fluorescence-labeled nucleic acid sequence A on the surface of a substrate20And after incubation for 1h, cleaning, and then representing by adopting a fluorescence microscope, wherein the result shows that the surface of the substrate is modified with a nucleic acid molecule identifier dot matrix (figure 6), the experiment is simultaneously provided with a negative control area, and a fluorescence sequence without a captured area is introduced into alternate pore channels, so that the result shows that a fluorescence image is formed only at the position of the pore channel introduced with the captured area, and the nucleic acid molecule identifier is amplified and sequenced at the same time, thereby proving that the unique nucleic acid molecule identifier has correct composition and contains the space coordinate sequence, the molecule mark and the capture domain.
The size of the nucleic acid molecule identifier quadrilateral microarray for modification is 10 mu m, which is obviously smaller than that of a circular lattice generated by the existing ink-jet printing modification method, and the center distance between the microarrays is 30 mu m, which is far higher than the density of the microarrays generated by the modification method in the prior art. Therefore, 4900 high-density arrays and different modifications of the nucleic acid molecule identifier microarray with space coordinates are realized by adopting 140 nucleic acid molecule identifiers, and the reagent cost required by full-length synthesis of sequences in the ink-jet printing modification method in the prior art is greatly reduced.
Example 4: spatial transcriptomics studies using quadrilateral high density nucleic acid molecular identifier arrays
The size of the quadrilateral nucleic acid molecule identifier microarray manufactured by the invention reaches the single cell resolution, and the embodiment combines the nucleic acid molecule identifier microarray with a space coordinate, so that the analysis of the single cell transcriptome on the space can be realized.
The freshly taken mouse brain tissue was embedded and trimmed to size by OCT (mixture of polyethylene glycol and polyvinyl alcohol), and then a frozen tissue section with a thickness of 10 μm was cut with a cryomicrotome (Leika) and attached to the surface of the high-density quadrilateral nucleic acid molecule identifier microarray. The substrate with the tissue sample attached thereto was placed on a preheated PCR instrument, incubated at 37 ℃ for 1min, the sample was fixed with pre-cooled methanol at-20 ℃, the sliced sample was incubated with isopropanol at room temperature for 1min and air-dried, then the tissue slice was stained with hematoxylin staining solution, rewet staining solution and eosin staining solution, and photographed using a bright field microscope, and the exposure time and the photographing range were adjusted so that the boundaries of the tissue slice and the array were visible (fig. 7).
The substrate was clamped using a frame and tissue digest (0.1% pepsin, 0.1N hydrochloric acid) was added dropwise into the frame to bring it into full contact with the tissue section and incubated at 37 ℃ for 30min, then the digest was removed and sodium citrate buffer was added for washing and the first strand synthesis reaction was performed in the frame (the reaction system contained reverse transcriptase and corresponding buffer system, template switching primer, etc., and the template switching primer information is shown in table 3), and after incubation at 42 ℃ for 90min, the liquid was removed. Adding KOH solution (100mM) dropwise into the frame, incubating at room temperature for 3min, removing the KOH solution, adding Tris-hydroxymethyl-aminomethane buffer (10mM Tris-HCl, pH 8.5), standing for 10s, removing the KOH solution, performing second strand synthesis reaction (containing Bst 2.0 polymerase and corresponding buffer system and corresponding primer, and primer sequence information shown in Table 3) in the frame, incubating at 65 ℃ for 30min, removing the reaction solution, adding Tris-hydroxymethyl-aminomethane buffer (10mM Tris-HCl, pH 8.5), standing for 10s, removing the KOH solution (100mM), incubating at room temperature for 3min, transferring the solution to an EP tube without nuclease contamination, and adding Tris-hydroxymethyl-aminomethane buffer (1M Tris-HCl, pH 7.2). Putting the reaction product on ice, adding cDNA amplification reaction solution (containing high-fidelity DNA polymerase and a corresponding buffer system thereof, dNTP and an amplification primer, and primer sequences are shown in Table 3), fully mixing, and performing polymerase chain reaction under the following conditions:
step 1: heating at 98 deg.C for 3 min;
step 2: 15s at 98 ℃;
and step 3: 63 ℃ for 20 s;
and 4, step 4: 60s at 72 ℃;
and 5: go to step 2 for a total of 21 cycles;
step 6: 60s at 72 ℃;
and 7: and stopping at 4 ℃.
The product was purified according to the SPRI-select nucleic acid fragment selection kit (Beckman Coulter) instructions, cDNA concentration was determined using NanoDrop, and cDNA fragment size distribution was determined using an automated electrophoresis system (Agilent technologies). The cDNA amplification product concentration and the purification results are shown in FIGS. 8 and 9, and the detection results show that the cDNA length is distributed between 300 and 1000bp, which is consistent with the expected results.
The purified cDNA amplification products were transferred to ice, processed according to the rapid DNA fragmentation/end repair/dA addition module kit instructions (tiangen biochemical technology), and the reaction products were subjected to SPRI-select fragment sorting. Taking part of products to perform oligonucleotide joint connection (the oligonucleotide joint information is shown in table 3) according to the specification of a quick connection module kit (Tiangen biochemical technology), performing SPRI-select fragment sorting on reaction products, taking the sorted products to perform nucleic acid library establishment reaction (a reaction system contains high-fidelity DNA polymerase and a buffer system thereof, dNTP and Illumina primers, and primer information is shown in table 3), and setting reaction conditions:
step 1: at 98 ℃ for 1 min;
step 2: 20s at 98 ℃;
and step 3: 67 ℃ for 30 s;
and 4, step 4: 72 ℃ for 20 s;
and 5: go to step 2 for a total of 12 cycles;
step 6: 60s at 72 ℃;
and 7: and stopping at 4 ℃.
The product was purified according to the SPRI-select nucleic acid fragment selection kit (Beckman Coulter) instructions, the concentration of the nucleic acid library was determined using NanoDrop, the size distribution of the nucleic acid library fragments was determined using an automated electrophoresis system (Agilent technologies), the concentration and distribution of the amplified nucleic acid library were shown in FIGS. 10 and 11, and the results of the detection indicated that the length distribution of the nucleic acid library was between 300 and 700bp, consistent with the expected results.
Novaseq is selected to sequence a nucleic acid library, the sequenced nucleic acid library is subjected to sequence analysis, the captured mRNA information is subjected to spatial positioning by combining the spatial coordinate sequence information of the nucleic acid molecule identifier, the expression information of the target gene at a specific position in the space is determined by matching the target gene, and the counting and quantification of the expression of the target gene are performed by counting the molecular markers corresponding to the target gene, so that the data error generated by the amplification deviation of the target gene can be reduced. And processing the sequencing data, and matching the unique nucleic acid molecule identifier microarray of each spatial point with the tissue slice staining image so as to visualize the sequencing data of the nucleic acid library at the spatial position of the tissue slice.
The sequencing result shows that the corresponding nucleic acid molecule identifier information can be found in the analysis result of the nucleic acid library, the sequence information of the spatial coordinate sequence and the molecular label is correct, and the data quality can meet the analysis requirement (figure 12). The result of spatial transcriptome sequencing data standardization shows that each quadrilateral nucleic acid molecule identifier microarray can capture more than 2000 genes, the quality of the captured genes is higher, and most sequencing data results of the same microarray show that the microarray is derived from the same cell, which shows that the substrate is adopted to realize spatial transcriptomics analysis under the single cell resolution. The results of the space lattice clustering and subgroup analysis and the lattice clustering Marker analysis show that the mouse brain tissue typical gene staining result is correct, and the common brain cell Marker is reflected in the data. Meanwhile, subdivision of anatomical regions, signal path analysis, differential gene screening and function analysis, dot matrix population gene enrichment analysis and cell communication analysis are carried out.
The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (37)
1. A microarray chip, comprising: the microarray comprises a substrate and a nucleic acid molecule identifier microarray connected with the upper surface of the substrate, wherein the shape of the microarray is a quadrangle.
2. The microarray chip of claim 1, wherein: the substrate is glass, or quartz, single crystal silicon, polylysine coatings, nitrocellulose, polystyrene, cyclic olefin copolymers, cyclic olefin polymers, polypropylene, polyethylene, and polycarbonate or polydimethylsiloxane, or any other material known in the art, or any combination thereof.
3. A microarray chip according to claims 1-2, characterized in that: the length of the substrate is 1-100 cm; the width is 1-100 cm; the thickness is 0.1-10 mm.
4. A microarray chip according to claims 1-3, characterized in that: the upper surface of the substrate is pre-treated with a functionalizing group that is a 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide activated carboxylic acid group, an aldehyde group, an epoxy group, a 1, 4-phenylisothiocyanate isocyanate group, a streptavidin group, or other groups known in the art or any combination thereof.
5. A microarray chip according to claims 1-4, characterized in that: the nucleic acid molecule identifier is achieved by coating the polymer onto the substrate surface, activating chemical groups within the polymer, incorporating reactive or activatable functional groups into the polymer structure, or other methods known in the art, or any combination thereof.
6. A microarray chip according to claims 1-5, characterized in that: the nucleic acid molecule identifier microarray is square or irregular quadrangle, and the angle of each corner inside the microarray is 0-180 degrees.
7. The microarray chip of any one of claims 1 to 6, wherein: the length of the nucleic acid molecule identifier microarray is 0.1nm-1000 mu m; the width is 0.1nm-1000 μm; the microarray of nucleic acid molecule identifier has an area of 0.1nm2-10cm2(ii) a The distance between centers of the adjacent quadrilateral nucleic acid molecule identifier microarrays is 0.1nm-1000 μm.
8. The microarray chip of any one of claims 1 to 7, wherein: the number of the nucleic acid molecule identifier microarrays on the upper surface of the substrate is at least 1.
9. A microarray chip according to claims 1-8, characterized in that: the nucleic acid molecule identifiers are attached to the upper surface of the substrate by electrostatic binding, a carboxyammonia reaction, a biotin-streptavidin reaction, photocatalysis, free radical polymerization, or other methods known in the art, or any combination thereof.
10. A method for preparing a microarray chip according to claims 1 to 9, wherein the nucleic acid molecule identifier microarray is attached to the functionalized upper surface of the substrate by microchip technology, comprising the steps of:
attaching micro-channels arranged in parallel to the upper surface of a substrate, respectively introducing different first groups of nucleic acid molecule identifiers into the micro-channels through a micro-fluidic chip technology, connecting the micro-channels to the upper surface of the substrate, and removing the unconnected nucleic acid molecule identifiers; then attaching the micro channels to the upper surface of the substrate again in a direction different from the pore channels, respectively introducing different second groups of nucleic acid molecule identifiers into the micro channels, and generating unique nucleic acid molecule identifiers on the upper surface of the substrate through reaction.
11. The method of claim 10, wherein: at least 1 microchannel is arranged in parallel.
12. The production method according to claims 10 to 11, characterized in that: the width range of the pore channel in the micro-channels arranged in parallel is 0.1nm-1000 μm.
13. The production method according to claims 10 to 12, characterized in that: the width of the space between the pore channels in the parallel arrangement micro-pore channels is 0.1nm-1000 μm.
14. The production method according to claims 10 to 13, characterized in that: the liquid outlet or inlet arrangement in the parallel arrangement micro-channels comprises that each pore channel is respectively connected with an independent liquid outlet or inlet, also comprises a plurality of pore channels connected with the same liquid outlet or inlet, and also comprises a pore channel connected with a plurality of liquid outlets or inlets.
15. The method for preparing a polymer according to claims 10 to 14, wherein: the first and second sets of nucleic acid molecule identifiers are different from each other relative to other nucleic acid molecule identifiers and other nucleic acid sequences attached to the upper surface of the substrate.
16. The method for preparing a polymer according to claims 10 to 15, wherein: the first group of nucleic acid molecule identifiers comprises a universal region, a first spatial coordinate sequence and a nucleic acid molecule identifier hybridization region; the second set of nucleic acid molecule identifiers comprises a nucleic acid molecule identifier hybridization region, a spatial coordinate sequence two, a molecular tag, and a nucleic acid molecule identifier capture region precursor; the unique nucleic acid molecule identifier generated by the reaction comprises a universal region, a spatial coordinate sequence I, a nucleic acid molecule identifier hybridization region, a spatial coordinate sequence II, a molecular label and a nucleic acid molecule identifier capture region.
17. The production method according to claims 10 to 16, characterized in that: the universal region comprises active reaction sites for attachment to the substrate surface and primer binding sites, including any reactive species capable of binding to the substrate and also precursors capable of being activated to form reactive sites.
18. The method for preparing a polymer according to claims 10 to 17, wherein: the length of the primer binding site, the spatial coordinate sequence I, the spatial coordinate sequence II, the nucleic acid molecule identifier hybridization region, the molecular label, the nucleic acid molecule identifier capture region precursor and the nucleic acid molecule identifier capture region is 1-1000 nucleotides.
19. The method for preparing a polymer according to claims 10 to 18, wherein: the space coordinate sequence I or the space coordinate sequence II locates the nucleic acid molecule identifier.
20. The method for preparing a polymer according to claims 10 to 19, wherein: the spatial coordinate sequences of the nucleic acid molecule identifiers introduced into each of the parallel-arranged microchannels are different from each other and distinguishable from each other.
21. The production method according to claims 10 to 20, characterized in that: complementary hybridization occurs between the first and second sets of nucleic acid molecule identifier hybridizing regions according to the Watson-Crick base complementary pairing rules known in the art.
22. The production method according to claims 10 to 21, characterized in that: the molecular tag provides a tag for the nucleic acid captured by the unique nucleic acid molecule identifier, or provides a name for the nucleic acid, and different unique nucleic acid molecule identifiers should comprise different molecular tags.
23. The method for preparing a polymer according to claims 10 to 22, wherein: the nucleic acid molecule identifier capture zone precursor comprises a nucleic acid sequence for forming a nucleic acid molecule identifier capture zone.
24. The method of manufacturing according to claims 10-23, wherein: the reaction for generating a unique nucleic acid molecule identifier indicative of spatial coordinates comprises a reaction that can extend or amplify a nucleic acid sequence.
25. The method of manufacturing according to claims 10-24, wherein: the nucleic acid molecule identifier capture region is used to capture nucleic acid sequences.
26. The method of manufacturing according to claims 10-25, wherein: the arrangement of the functional regions of the nucleic acid molecule identifier and the unique nucleic acid molecule identifier includes, but is not limited to, the order or position recited in the present invention, and one or more of the functional sequences in the nucleic acid molecule identifiers described above may be arranged in any suitable order.
27. Use of the microarray chip according to claims 1-9 for spatial transcriptomics studies, characterized in that the steps are as follows: attaching the frozen tissue sample slice to the surface of a substrate, covering the frozen slice on the surface of the nucleic acid molecule identifier microarray, and then performing HE (high intensity electrophoresis) staining and imaging on the tissue sample; and then digesting the frozen section, capturing the mRNA sequence of the tissue by the nucleic acid molecule identifier microarray through a nucleic acid molecule identifier capturing area, synthesizing a first strand and a second strand of cDNA, amplifying the cDNA, establishing a nucleic acid library and analyzing spatial transcriptomics, corresponding the transcriptomics information of the analyzed sample to the corresponding spatial position according to the spatial coordinate sequence information, and fitting the spatial transcriptomics information with the tissue sample image to obtain the spatial transcriptomics information of the tissue sample.
28. The use according to claim 27, wherein: the tissue sample is imaged by light, dark field imaging, confocal imaging, or other methods known in the art, or any combination thereof.
29. The use according to claim 27, wherein: the tissue sample is digested by chemical digestion, enzymatic digestion, or other methods known in the art, or any combination thereof.
30. The use according to claim 27, wherein: the tissue self mRNA is hybridized to the nucleic acid molecule identifier capture region of the microarray by Watson-Crick base complementary pairing protospecies.
31. The use according to claim 27, wherein: the first strand of the cDNA is synthesized by reverse transcription or other methods known in the art, or any combination thereof.
32. The use according to claim 27, wherein: the second strand of cDNA is synthesized by isothermal amplification of nucleic acids or by methods known in the art, or any combination thereof.
33. The use according to claim 27, wherein: the means of cDNA amplification is polymerase chain reaction or methods known in the art or any combination thereof.
34. The use according to claim 27, wherein: the nucleic acid library is established by polymerase chain reaction or by methods known in the art or any combination thereof.
35. The use according to claim 27, wherein: the nucleic acid library is analyzed using first generation sequencing, second generation sequencing, third generation sequencing, or any combination thereof.
36. The use according to claim 27, wherein: the tissue sample species may be plant, animal, fungal, or any combination thereof.
37. The use according to claim 27, wherein: the tissue sample type may be dead body, biopsy tissue sample, fresh tissue, or any combination thereof.
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