WO2025209378A1 - Improved biochip for spatial transcriptomics analysis - Google Patents

Abstract

Provided in the present invention are a chip for analyzing spatial transcriptomics information of a biological sample, and a method for analyzing spatial transcriptomics information of a biological tissue sample by using the chip. A surface of the chip has a modification layer for binding to nucleic acid molecules, and probes forming an array, wherein the distance between lattice points in the array is substantially zero. Further provided in the present invention is a method for preparing the chip. By means of the chip and the analysis method provided in the present invention, more effective spatial omics information of cells of a tissue sample can be obtained.

Description

Improved biochips for spatial transcriptomic analysis

This application claims priority to the following Chinese patent application: Application number 202410381431.0, filed on March 30, 2024, entitled “Improved biochip for spatial transcriptomics analysis,” the entire contents of which are incorporated by reference into this application.

Technical Field

The present invention relates to the fields of biology and medical devices. Specifically, the present invention relates to a method for preparing a chip for analyzing nucleic acid information of cells in a biological sample and the application of the prepared chip, wherein the chip is suitable for analyzing spatial transcriptomic information of a biological tissue sample.

Background Art

Recent developments in gene expression analysis have made it possible to assess the spatial transcriptome of tissues using microarrays or RNA sequencing. However, known methods and chips for spatial transcriptome analysis still have some unresolved issues, such as the large spacing between each array point of the chip probe array, which greatly affects the information obtained.

Summary of the Invention

The present invention provides an improved biochip suitable for analyzing nucleic acid information of cells in biological samples, particularly suitable for analyzing spatial transcriptomic information of biological tissue samples. The present invention also provides a method for preparing the above-mentioned biochip.

Specifically, the present invention provides an improved method for preparing a biochip having an array, comprising the following steps:

A. Using a microfluidic device having multiple parallel microfluidic channels to transport and immobilize a first set of barcode nucleic acids on a chip surface, forming multiple first-direction barcode strips in a first direction, wherein the first set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip having a different first-direction barcode nucleic acid immobilized thereon;

B. Using the microfluidic device having multiple parallel microfluidic channels, applying and affixing a second set of barcode nucleic acids to the chip surface along the first direction at positions adjacent to the multiple first-direction barcode strips to form multiple second first-direction barcode strips, wherein the second set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip affixed with a different first-direction barcode nucleic acid, and each first-direction barcode strip affixed with a different barcode sequence;

C. Using the microfluidic device having multiple parallel microfluidic channels, applying and fixing a third set of barcode nucleic acids to the multiple first-direction barcode strips on the chip surface along a second direction perpendicular to the first direction to form multiple first- and second-direction barcode strips, wherein the third set of barcode nucleic acids includes multiple second-direction barcode nucleic acids having different barcode sequences, each second-direction barcode strip having one second-direction barcode nucleic acid, and each second-direction barcode strip having a different barcode sequence;

D. Optionally, using the microfluidic device having multiple parallel microfluidic channels, a fourth set of barcode nucleic acids is applied and fixed to the chip surface along the second direction at positions adjacent to the multiple first and second directional barcode strips to form multiple second directional barcode strips, wherein the fourth set of barcode nucleic acids includes multiple second directional barcode nucleic acids having different barcode sequences, each second directional barcode strip is fixed with a different second directional barcode nucleic acid, and each second directional barcode strip has a different barcode sequence.

On the chip surface where the multiple first-direction barcode strips intersect the multiple second-direction barcode strips, the second-direction barcode nucleic acids are connected to the first-direction barcode nucleic acids to form probes. The probes constitute array points, and each array point has a probe with a different sequence.

In one aspect of the method of the present invention, the spacing between the first first-direction barcode band and the adjacent second first-direction barcode band is substantially zero. In one aspect of the method of the present invention, the spacing between the first second-direction barcode band and the adjacent second second-direction barcode band is substantially zero. In one aspect of the present invention, the spacing is less than about 2.0 μm, preferably less than about 1.5 μm, and most preferably less than about 1.0 μm, for example, about zero.

In one aspect of the present invention, the method provides one or more alignment marks on the chip for aligning the microfluidic device with the barcoded nucleic acid when applying the barcoded nucleic acid using the microfluidic device having multiple parallel microfluidic channels, for example, by aligning the microfluidic device with corresponding alignment marks. In another aspect of the present invention, multiple alignment marks are provided on the chip, preferably more than 3 or 4, for example, 4-8.

In another aspect of the present invention, the method uses an alignment platform to control the alignment and movement of the microfluidic device and/or chip having multiple parallel microfluidic channels. In one aspect of the present invention, the alignment platform includes a device for fixing and/or moving the chip, a device for fixing and/or moving the microfluidic device, and a device for observing alignment marks on the chip and/or microfluidic channel. In one embodiment, the device for observing alignment marks on the chip and/or microfluidic channel is a microscope. More preferably, the device for observing alignment marks on the chip and/or microfluidic channel includes more than one microscope for simultaneously observing more than one alignment mark.

In one aspect of the present invention, the method further comprises fixing the chip surface linker nucleic acid on the chip surface, for example, on the surface of the entire chip. In the method, the chip surface linker nucleic acid can be connected to the first direction barcode nucleic acid. In one aspect of the method, the 3' end of the chip surface linker nucleic acid has a connection fragment for connecting to the first direction barcode nucleic acid through a single-stranded linker nucleic acid. In one aspect of the method, the 5' end of the chip surface linker nucleic acid has a group for connecting to the chip surface. Optionally, the 5' end portion of the chip surface linker nucleic acid has a primer fragment for an amplification reaction.

In one aspect of the present invention, in the method, the first-direction barcode nucleic acid comprises a first barcode segment. In another aspect of the present invention, the first-direction barcode nucleic acid further comprises a primer segment for amplification reaction at the 5' end.

In one embodiment of the present invention, in the method, the first-direction barcode nucleic acid has a group at the 5' end for connecting to the chip surface.

In one embodiment of the present invention, in the method, the first-direction barcode nucleic acid has a segment at the 5' end for connecting to the chip surface linker nucleic acid.

In one aspect of the present invention, the second-direction barcode nucleic acid includes a capture fragment at the 3' end for identifying and binding to the target nucleic acid in the biological sample (for example, a fragment for identifying and binding to mRNA or cDNA, such as a poly-T sequence) and a second barcode fragment.

In one aspect of the present invention, in the method, the second-direction barcode nucleic acid further has a unique molecular identifier (UMI).

In one aspect of the present invention, in the method, the 3' end of the first-direction barcode nucleic acid has a first connecting fragment for connecting to the second-direction barcode nucleic acid via a single-stranded connecting nucleic acid, and the 5' end of the second-direction barcode nucleic acid has a second connecting fragment for connecting to the first-direction barcode nucleic acid via the single-stranded connecting nucleic acid, and the first connecting fragment and the second connecting fragment are reverse complementary to the sequences at both ends of the single-stranded linker nucleic acid, respectively.

In one aspect of the present invention, the probe formed in the method includes a capture segment at its 3' end for recognizing and binding a target nucleic acid in a biological sample, as well as a first barcode segment and a second barcode segment. Preferably, the probe also includes a primer segment at its 5' end for amplification.

In one embodiment of the present invention, the sequence of each barcode segment of the first-direction barcode nucleic acid is specified, and/or the sequence of each barcode segment of the second-direction barcode nucleic acid is specified. In one embodiment of the present invention, the sequence of the first-direction barcode segment and the second-direction barcode segment of the probe are specified.

In one aspect of the present invention, the concentration of the nucleic acid applied through the microfluidic device having a plurality of parallel microfluidic channels in the method is about 0.1-100 uM, such as about 1-20 uM.

In one aspect of the present invention, in the method, the nucleic acid (e.g., the chip surface linker nucleic acid or the first-direction barcode nucleic acid) is immobilized on the chip surface by chemical bonding. The chemical bonding method is, for example, any one of chemical bonds involving amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, sulfhydryl groups, and silane groups, such as amino-aldehyde reactions. The surface of the chip can be coated with active groups such as amino groups, aldehyde groups, epoxy groups, isothiocyanate groups, sulfhydryl groups, and silane groups through surface chemical reactions. One end of the nucleic acid connected to the chip surface (usually the 5' end) has a group that forms a chemical bond with the coated active group.

In one aspect of the present invention, the first group of barcode nucleic acids can be immobilized on the chip surface by chemical bonding.

In another aspect of the present invention, the chip surface linker nucleic acid is fixed to the chip surface by chemical bonding, and then the first group of barcode nucleic acids is connected to the chip surface linker nucleic acid. One end (usually the 5' end) of the chip surface linker nucleic acid connected to the chip surface has a group that forms a chemical bond with the coated active group. In another embodiment of the present invention, the 3' end of the chip surface linker nucleic acid has a connecting segment for connecting to the first barcode nucleic acid through a single-stranded connecting nucleic acid. In another embodiment of the present invention, the 5' end of the first barcode nucleic acid has a connecting segment for connecting to the chip surface linker nucleic acid through a single-stranded connecting nucleic acid, the 3' end of the chip surface linker nucleic acid has a connecting segment for connecting to the first barcode nucleic acid through the single-stranded connecting nucleic acid, and the connecting segment at the 3' end of the chip surface linker nucleic acid and the connecting segment at the 5' end of the first barcode nucleic acid are respectively reverse complementary to the sequences at both ends of the single-stranded connecting nucleic acid. Furthermore, the 3' end of the first barcode nucleic acid has a linker segment for connecting to the second barcode nucleic acid via a single-stranded linker nucleic acid, and the 5' end of the second barcode nucleic acid has a linker segment for connecting to the first barcode nucleic acid via the single-stranded linker nucleic acid. The linker segment at the 3' end of the first barcode nucleic acid and the linker segment at the 5' end of the second barcode nucleic acid are respectively reverse complementary to the sequences at both ends of the single-stranded linker nucleic acid.

In one aspect of the present invention, in the method, the height of each microfluidic channel of the microfluidic channel device having a plurality of microfluidic channels arranged in parallel is about 5-100 μm, preferably about 25-75 μm.

In one aspect of the present invention, the width of each of the microchannels arranged in parallel in the method is about 10-100 μm, preferably about 15-50 μm, and most preferably about 20-30 μm.

In one aspect of the present invention, the probe density of the array of the chip prepared by the method is about 10 ³ -10 ⁴ /μm ² .

In one aspect of the present invention, the array of the chip prepared by the method has a probe uniformity deviation of less than 20%, preferably less than 10%, and more preferably less than 5%.

The present invention also provides an improved chip for analyzing nucleic acid information of biological samples.

In one aspect of the present invention, the improved chip for analyzing nucleic acid information of biological samples is prepared by the method provided by the present invention.

In one aspect of the present invention, a chip for analyzing nucleic acid information from a biological sample has an array of probes on its surface. The probe array comprises orthogonal rows and columns, and each probe at each point in the array has a different sequence that can be used to represent the spatial position of the probes. The chip is characterized in that the spacing between adjacent probe array points in the chip is substantially zero. In one aspect of the present invention, the spacing between adjacent array points is less than about 2.0 μm, preferably less than about 1.0 μm, and most preferably about 0, i.e., there is no space between adjacent array points.

In one aspect of the present invention, the probes include a first barcode and a second barcode. In another aspect of the present invention, each row of probes in the probe array has the same first barcode and each column of probes has the same second barcode; each row of probes has a different second barcode and each column of probes has a different first barcode.

In yet another aspect of the present invention, the sequence of each probe in the probe array includes a primer fragment at the 5' end for an amplification reaction. In yet another aspect of the present invention, the sequence of each probe in the probe array also includes a unique molecular identifier (UMI).

In one aspect of the present invention, the surface of the chip comprises a modified layer for binding to nucleic acid molecules and arrayed probes, the modified layer and the probes being chemically bonded. The sequence of nucleic acids at the array sites on the chip, from the 5' end to the 3' end, comprises a first barcode, a second barcode, and a capture segment for identifying and binding target nucleic acids in a biological sample.

In yet another aspect of the present invention, the chip for analyzing nucleic acid information from a biological sample comprises a chip surface linker nucleic acid across its entire surface; the probe is bound to the chip surface by linking to the chip surface linker nucleic acid. The sequence of nucleic acids at the array sites in the chip, from the 5' end to the 3' end, comprises the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture fragment for identifying and binding target nucleic acids in the biological sample. In another aspect of the present invention, the chip surface linker nucleic acid is fixed to the chip surface via chemical bonding.

In one aspect of the present invention, the probes of the array of the chip have a uniformity deviation of less than 20%, preferably less than 10%, and more preferably less than 5%.

In one aspect of the present invention, the size uniformity deviation of the array points of the chip is less than 10%, preferably less than 5%, and more preferably less than 2%.

In one aspect of the present invention, the width of each array point in the chip is about 10-100 μm, preferably about 15-50 μm, most preferably about 20-30 μm, for example about 25 μm or about 10 μm.

In one aspect of the present invention, the width of the chip is about 0.5-50 mm, preferably about 1-40 mm, and most preferably about 5-20 mm. "Width" generally refers to the dimension of the chip in the direction of its maximum width. For example, "a 40 mm chip" may include a chip that is 40 mm x 40 mm or 40 mm x 20 mm, etc.

The present invention also provides a method for analyzing spatial transcriptomic information of a biological tissue sample, the method comprising contacting the probe array of the chip of the present invention with the tissue sample. "Tissue samples" suitable for the present invention include tissue obtained from a subject, fixed, sliced, and mounted on a planar surface.

The method of the present invention involves contacting a tissue section with a probe array on a chip. The probes on the array can recognize and bind to nucleic acids, particularly mRNA, in cells within the tissue. Subsequent analysis includes reverse transcription and amplification, and can be performed using high-throughput next-generation sequencing (NGS) or sequencing-by-synthesis (SBS), and can be further analyzed using big data and artificial intelligence (AI).

The chip prepared by the present invention can be used to analyze intracellular molecules in tissue samples, especially thin tissue sections, including analysis of nucleic acids and proteins, such as by PCR, mass spectrometry, next-generation sequencing, or ELISA, to obtain their expression and spatial information.

In one aspect of the present invention, the biological sample is a tissue sample from a subject, such as a surgically removed tissue sample, preferably a thin tissue section obtained by microtomy. In one aspect of the present invention, the tissue sample is fixed and embedded (e.g., embedded in paraffin wax), and attached to a support such as a slide.

In one aspect of the present invention, the tissue slices can be subjected to morphological analysis and/or histological analysis, and the histological analysis is performed by H&E staining, IHC staining, ISH staining, and FISH staining.

In one aspect of the present invention, the analysis of the one or more biomolecules is performed by PCR, mass spectrometry, next generation sequencing, ELISA, big data and artificial intelligence (AI).

In one aspect of the present invention, the subject is selected from animals, farm animals, pets, and human subjects.

In one aspect of the present invention, the analyte further comprises one or more of a non-human cell, a human cell, a non-natural protein, a nucleic acid, a small molecule, a dye, a virus, a bacterium, a parasite, a protozoa, or a chemical substance. Small molecules include haptens, peptide tags, protein tags, fluorescent tags, nucleic acid tags, and combinations thereof.

In one aspect of the present invention, the chip can be used to analyze quantitative and/or qualitative data of markers in a sample. The markers include DNA, proteins, RNA, lipids, organelles, metabolites, or cells.

The chip of the present invention can be used to analyze tissue samples. The tissue sample includes a sample selected from the group consisting of one or more pre-malignant or malignant cells, cells from solid tumors, soft tissue tumors or metastases, tissue or cells from surgical margins, histologically normal tissue, one or more circulating tumor cells (CTCs), normal adjacent tissue (NAT), a blood sample from the same subject suffering from a tumor or at risk of suffering from a tumor, or a FFPE sample.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are some embodiments of the present invention.

FIG1 is a schematic flow chart of the basic process steps of the method for preparing a chip provided by the present invention.

FIG. 2 is an exemplary embodiment of a device having multiple microfluidic channels arranged in parallel used in the method of the present invention.

FIG3 is a schematic flow chart of an exemplary embodiment of a method for preparing a chip provided by the present invention.

FIG4 is an exemplary diagram of the microfluidic channel arrangement used in the method for preparing a biochip provided by the present invention.

FIG5 is a diagram illustrating the configuration of a system for calibrating and controlling the movement of a microfluidic channel used in the method for preparing a biochip provided by the present invention.

6A-6D are respectively exemplary views of steps A, B, C and D in the method for preparing a biochip provided by the present invention, showing the operation of aligning and positioning the microfluidic device and the chip to transport and fix the barcode nucleic acid, as well as the observation of the effect detection.

FIG7 is a HE staining image of mouse brain olfactory bulb tissue.

Figure 8 shows a UMI heatmap and numerical distribution plot obtained by spatial group analysis of mouse brain olfactory bulb tissue using the biochip provided by the present invention. The violin plot displays the distribution of UMI count data, reflecting the number of transcripts detected in each single cell (nUMI). The shape and width of the plot indicate the density distribution of the data; a wider width indicates a greater number of cells within that range. The dots represent the specific UMI counts for each cell, providing expression information for each cell. The figure on the right is a spatial UMI count distribution plot, showing the UMI count levels at various spatial locations on the tissue section. Each dot represents a spatial location, and the color bar, ranging from blue (low UMI count) to red (high UMI count), reflects the difference in the number of UMIs captured at that location. This plot reveals the abundance and heterogeneity of gene expression within different spatial regions, providing information about tissue structure and function.

Figure 9 is a Gene heat map and numerical distribution diagram obtained by performing spatial group analysis on mouse brain olfactory bulb tissue using the biochip provided by the present invention. The violin plot shows the distribution of the number of genes (nGene) detected in the sample, the vertical axis represents the number of nGene, the horizontal axis represents the sample, and the shape and width of the violin plot show the density distribution of the number of genes detected. The larger the width, the more cells are within the range, and the black dots in the figure represent the actual nGene count values of each cell. The Gene heat map can compare the differences in the number of genes detected between different groups and evaluate the quality of the data and the gene expression diversity of the sample. The figure on the right is a visualization of spatial transcriptomics data, showing the number of genes detected at various positions on the tissue section, each point represents a spatial position, and the color bar shows the difference in the number of nGenes from blue (fewer genes detected) to red (more genes detected). The figure intuitively presents the gene diversity and abundance of different spatial regions in the tissue, revealing the spatial heterogeneity of gene expression within the tissue.

Figure 10 is a UMAP clustering diagram obtained by performing spatial group analysis on mouse brain olfactory bulb tissue using the biochip provided by the present invention. Each point on the left represents a spatial location, and the color represents a different gene expression pattern or cell population. The UMAP algorithm displays data in a low-dimensional space, retaining its global and local structure, and is used to observe and identify differences in gene expression patterns, as well as how similar spatial expression groups cluster in the dimensionality reduction diagram. The spatial transcriptomic expression diagram on the right shows the gene expression at each spatial location in the tissue section, with each point representing a spatial location. The color and shape of the point correspond to the results in the UMAP diagram on the left, indicating the cell type or state at that location.

Figure 11 shows a t-SNE cluster diagram obtained by spatial group analysis of mouse olfactory bulb tissue using the biochip provided by the present invention. Each dot represents a spatial location, and different colors indicate different gene expression patterns or cell populations, revealing subtle differences between local clusters or cell populations in spatial data. The right panel displays gene expression at various spatial locations in the tissue section. The color and shape of the dots correspond to the results in the t-SNE diagram on the left, indicating the cell type or state at that location, as well as the spatial distribution and heterogeneity of gene expression within the tissue.

Figure 12 is a gene distribution heat map obtained by performing spatial group analysis on mouse brain olfactory bulb tissue using the biochip provided by the present invention, wherein A-D are the distribution heat maps of Cck (Cholecystokinin), Fabp7 (Fatty Acid Binding Protein 7), mt-Cytb (Mitochondrial Cytochrome b) and Plp1 (Proteolipid Protein 1), respectively.

DETAILED DESCRIPTION

To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

DETAILED DESCRIPTION

To make the objectives, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

Example 1

The present invention provides an improved biochip suitable for analyzing nucleic acid information of cells in biological samples and a method for preparing the chip. The chip provided by the present invention is suitable for analyzing spatial transcriptomic information of biological tissue samples.

Figure 1 is a flow chart of an exemplary method of the basic preparation process of a biochip with an array provided by the present invention. The surface of the chip provided by the present invention has probes forming an array, and the probe array includes orthogonal rows and columns. The probes at each array point in the array have different sequences, which can be used to reflect the spatial position of the probes. In one aspect of the present invention, the surface of the chip has a modification layer for binding to nucleic acid molecules and probes forming an array, and the modification layer and the probes are connected by chemical bonds or the like. In one embodiment, the chip for analyzing the nucleic acid information of a biological sample has a chip surface linker nucleic acid on its entire surface, and the probes are connected to the chip surface linker nucleic acid.

WO2023116938A1 discloses an exemplary method for preparing a chip for analyzing nucleic acid information of a biological sample, as employed by the applicant of the present application. As shown in FIG1 , the method mainly comprises the following steps:

Step 1. Provide a chip. Optionally, in order to fix the chip surface linker nucleic acid on the chip surface in the next step, the chip surface is coated with active groups such as amino, aldehyde, epoxy, isothiocyanate, thiol, silane, etc. through surface chemical reaction.

Step 2. Immobilize the chip surface linker nucleic acid on the chip surface, for example, on the surface of the entire chip; the chip surface linker nucleic acid may include a linker fragment at the 3' end for connecting to the first barcode nucleic acid, and a primer fragment at the 5' end for subsequent amplification reaction.

Step 3. Applying a first set of barcode nucleic acids to the chip surface through a plurality of parallel microfluidic channels, and forming a plurality of first barcode bands in a first direction under conditions that allow a ligation reaction between the linker nucleic acid and the first barcode nucleic acid on the chip surface, wherein the first set of barcode nucleic acids includes a plurality of first barcode nucleic acids having different barcode sequences, each first barcode band having a fixed first barcode nucleic acid, and the first barcode nucleic acids fixed to each first barcode band having a different barcode sequence.

Figure 2 shows an exemplary embodiment in which multiple barcode nucleic acids are applied to a chip surface via multiple parallel microchannels, undergo ligation reactions with surface linker nucleic acids on the chip surface, and are immobilized on the chip surface. The lower portion of the left image of Figure 2 shows a chip. The middle portion of the left image of Figure 2 shows a microfluidic device having multiple parallel microchannels (microchannel 1 to microchannel n). The side of the microchannel in contact with the chip surface, i.e., the bottom portion of the microchannel shown, allows the passage (permeability) of a solution or nucleic acids in the solution. For example, the side of the microchannel in contact with the chip surface lacks a microchannel wall. The microfluidic device is applied to the chip surface along a first direction, and then a designated solution, such as a solution containing barcode nucleic acids, is introduced into the microchannels. The upper portion of the left image of Figure 2 shows an exemplary device for facilitating the introduction of the solution, such as a vacuum suction device utilizing negative pressure, which can be positioned at the outlet of the microchannel. The right image of Figure 2 shows the addition of barcode nucleic acids containing different barcode sequences (barcode nucleic acids 1-n in the figure) into each microchannel through an inlet. In one aspect of the present invention, the barcode sequence of the barcode nucleic acid introduced into each microfluidic channel has a known or designated nucleotide sequence.

In one embodiment, the microfluidic device comprises parallel microfluidic channels, for example, approximately 20-100 microfluidic channels, which can be set based on the number of probe array points on the chip. In one aspect of the present invention, the width of each of the parallel microfluidic channels (i.e., the width of the barcode strip formed, i.e., the width of the chip probe array points) is approximately 10-200 μm, preferably approximately 15-150 μm, and most preferably approximately 25-100 μm.

It is beneficial that each array point of the probe array on the chip has the same width and length, that is, the detection area covered and the shape are the same. In the above-mentioned method for preparing the chip, the width of each barcode band formed by the nucleic acid transported and fixed by the microfluidic channel is the same. To achieve this purpose, in one aspect of the present invention, the width of each flow channel of the microfluidic device is the same (the flow channel width is the width of the barcode band formed). In another aspect, the spacing between each flow channel is the same and the same as the flow channel width.

As shown in Figure 1, the 5' end of the first barcode nucleic acid has a connecting segment for connecting to the chip surface linker nucleic acid through a single-stranded connecting nucleic acid (first linker); the connecting segment at the 3' end of the chip surface linker nucleic acid and the connecting segment at the 5' end of the first barcode nucleic acid are respectively reverse complementary to the sequences at both ends of the first linker.

Step 4. Remove the microfluidic channel in step 3, and apply a second set of barcode nucleic acids along a second direction (usually perpendicular to the first direction) to the plurality of first barcode strips on the surface of the chip in the first direction through another set of multiple parallel microfluidic channels to form a plurality of second barcode strips, wherein the second set of barcode nucleic acids includes a plurality of second barcode nucleic acids with different barcode sequences, each second barcode strip has one second barcode nucleic acid, and the second barcode nucleic acids fixed on each second barcode strip have different barcode sequences.

The exemplary second barcode nucleic acid shown includes a poly-T sequence at the 3' end that recognizes and binds to mRNA, a unique molecular identifier (UMI), and a second barcode segment.

In the illustrated example, the 3' end of the first barcode nucleic acid has a first linker fragment for ligating to the second barcode nucleic acid via a single-stranded linker nucleic acid (second linker). The 5' end of the second barcode nucleic acid has a second linker fragment for ligating to the first barcode nucleic acid via the second linker. The first linker fragment and the second linker fragment are reverse complementary to the sequences at both ends of the second linker nucleic acid. Under conditions that allow ligation between the first and second barcode nucleic acids, the second barcode nucleic acid is ligated to the first barcode nucleic acid on the chip surface where the multiple first barcode bands intersect the multiple second barcode bands to form a probe.

Step 5. Remove the microfluidic channel from step 4 to obtain a biochip having a probe array on its surface. Each array point on the probe array corresponds to a location where the plurality of first barcode bands intersect with the plurality of second barcode bands. Each array point has a probe molecule comprising a first barcode sequence and a second barcode sequence. The combination of the first barcode sequence and the second barcode sequence included in each probe molecule at each array point is different. Thus, the spatial position of the probe molecule in the array on the chip surface can be determined based on the first barcode sequence and the second barcode sequence at each array point.

The chip used as a base in step 1 generally refers to a solid substrate on which chemical, biological, biophysical or biochemical processes can be performed.

The chip can be made of any suitable material, and exemplary types of chip materials include glass, modified glass, functionalized glass, inorganic glass, microspheres (including inert and/or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, optical fibers or fiber bundles, various polymers other than the materials exemplified above, and porous microtiter plates. Specific types of exemplary plastics include acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon ^™ . Specific types of exemplary silica-based materials include various forms of silicon and modified silicon. The chip surface can be modified to accommodate the attachment of the target biopolymer by various methods known to those skilled in the art.

The array on the surface of the prepared chip has probes (or capture probes). The probe refers to a single-stranded nucleotide molecule that can recognize and bind to a target nucleic acid in a gene-specific or target-specific manner, such as a nucleic acid from a tissue sample, and has a specific nucleotide sequence, that is, a nucleotide sequence that can selectively anneal to a targeted nucleic acid, usually a complementary nucleotide sequence. Examples of analytes in tissue samples include genomic DNA, methylated DNA, specific methylated DNA sequences, messenger RNA (mRNA), poly A mRNA, mitochondrial DNA, viral RNA, microRNA, in situ synthesized PCR products, RNA/DNA hybrids, lipids, carbohydrates, and proteins. The capture probe can be a gene-specific capture probe that hybridizes, for example, with a specifically targeted mRNA or cDNA in the sample.

The probe has a barcode sequence for use in subsequent high-throughput next-generation sequencing (NGS) or synthesis sequencing (SBS) for analysis, such as in the sequencing analysis of large throughput. In these sequencings, a barcode sequence is used to mark and identify the source of the nucleic acid of the nucleic acid sequence obtained by sequencing. Barcode molecules are used to barcode nucleic acid molecules (e.g., RNA molecules) from biological particles (e.g., cells) to generate sequencing libraries, which are then sequenced to produce multiple sequencing reads. Some or all of the multiple sequencing reads include barcode sequences. In these sequencing applications, cell nucleic acids are typically amplified until the barcoded overlapping fragments in the object constitute at least 1X coverage, at least 2X, at least 3X, at least 4X, at least 5X, at least 10X, at least 20X, at least 40X or higher coverage of a specific portion or all of the cell genome. Once barcoded fragments are produced, they can be directly sequenced on a suitable sequencing system, such as an Illumina system. The presence of the same barcode on multiple sequences can provide information about the origin of the sequence.

In the present invention, the prepared probe contains two barcode sequences. The two barcode sequences can help determine the position of the probe in the array on the chip surface (determine the position of the X dimension and the Y dimension respectively), and thus also have the function of a position tag. The barcode sequence on the probe can correspond to the array points in the array on the chip, and can also indicate the position of the cells on the tissue it recognizes, including single cells, in the tissue sample. Examples of other molecules that can be coupled to the nucleic acid tag include antibodies, antigen-binding domains, proteins, peptides, receptors, haptens, etc.

The probe also includes one or more unique molecular identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid fragment or two or more non-contiguous nucleic acid fragments that serve as a label or identifier for a specific analyte or a capture probe that binds a specific analyte. A UMI is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample.

Nucleic acids, such as chip surface linker nucleic acids, can be fixed to the chip using various methods known in the art. The fixation of nucleic acids refers to direct or indirect attachment to the chip through covalent or non-covalent bonds. In one aspect of the present invention, fixation refers to keeping the nucleic acid stationary or attached to the chip during reactions such as nucleic acid amplification and/or sequencing. In one aspect of the present invention, fixation can also refer to the nucleic acid stationary or attached to the chip being able to detach from the chip surface under specified conditions during subsequent reactions such as nucleic acid amplification and/or sequencing. Exemplary non-covalent attachments include, but are not limited to, non-specific interactions (such as hydrogen bonding, ionic bonding, van der Waals interactions, etc.) or specific interactions (such as affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). The chip surface linker nucleic acid can also be fixed to the chip surface by physical adsorption, such as by hydrophobic interactions, electrostatic attraction, etc.

In steps 3 and 4 of this exemplary method, the linker nucleic acid on the chip surface and the first barcode nucleic acid, as well as the first barcode nucleic acid and the second barcode nucleic acid, can be linked by various methods known in the art. For example, by complementing each other with sequences at different ends of another single-stranded nucleic acid fragment (the linker nucleic acid), the three nucleic acid fragments (the first barcode nucleic acid, the second barcode nucleic acid, and the linker nucleic acid) can be formed under conditions suitable for a ligation reaction to achieve the purpose of linking.

In this example method, the chip surface linker nucleic acid has a primer fragment at the 5' end for subsequent amplification reaction, such as a universal primer sequence used in known sequencing methods.

In this example method, the chip surface linker nucleic acid has a group or sequence at its 5' end for attachment to the chip surface. For example, if the chip surface is modified with an aldehyde group, the chip surface linker nucleic acid has an amino group at its 5' end. In another aspect of the present invention, the chip surface linker nucleic acid has a partner that can form a specific interaction with a factor modified on the chip, for example, the factor and partner are antibody-epitope, avidin-biotin, streptavidin-biotin, or lectin-carbohydrate, respectively.

In this example method, the first barcode nucleic acid includes a first barcode fragment.

In this exemplary method, the second barcode nucleic acid comprises a second barcode segment. In one aspect of the present invention, the second barcode nucleic acid has a capture segment at its 3' end for recognizing and binding a target in a biological sample, such as a segment that recognizes and binds to mRNA or cDNA, such as a segment that recognizes a poly-T sequence of mRNA.

In this example method, the 3' end of the first barcode nucleic acid has a first linker fragment for ligating to the second barcode nucleic acid. In another aspect of the present invention, the 5' end of the second barcode nucleic acid has a second linker fragment for ligating to the first barcode nucleic acid. In yet another aspect of the present invention, the first linker fragment and the second linker fragment are each complementary to one end of a linker nucleic acid. Under ligatable conditions (e.g., in the presence of T4 ligase), the first linker fragment and the second linker fragment combine with the linker nucleic acid to achieve ligation of the first barcode nucleic acid to the second barcode nucleic acid.

In the present invention, an improved chip for analyzing nucleic acid information of biological samples and a method for preparing the chip are provided. The surface of the improved chip for analyzing nucleic acid information of biological samples has probes forming an array, and the probe array includes orthogonal rows and columns. The probes at each array point of the array have different barcode sequences, which can be used to reflect the spatial position of the probes, and is characterized in that the spacing between adjacent array points of the probe array in the chip is substantially zero. In another aspect of the present invention, the spacing between adjacent array points is less than about 2.0 μm, preferably less than about 1.0 μm, and most preferably is about 0, that is, there is no gap between adjacent array points. The absence of gaps between array points on the chip is extremely beneficial for analyzing the spatial transcriptomic information of biological tissue samples, thereby achieving continuous and gapless gene capture areas and full tissue coverage, without loss of transcripts in some areas, improving the continuity and accuracy of gene capture data, and improving spatial fidelity.

FIG3 is a schematic flow chart of an exemplary embodiment of a method for preparing an improved chip provided by the present invention.

The method for preparing an improved biochip having an array of the present invention comprises the following steps:

A. Using a microfluidic device having multiple parallel microfluidic channels to transport and immobilize a first set of barcode nucleic acids on a chip surface to form multiple first-direction barcode strips, wherein the first set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip having a different first-direction barcode nucleic acid immobilized thereon;

B. Using another microfluidic device having multiple parallel microfluidic channels, transporting a second set of barcode nucleic acids in the first direction at positions adjacent to the multiple first-direction barcode strips and immobilizing them on the chip surface to form multiple second first-direction barcode strips, wherein the second set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip immobilized with a different first-direction barcode nucleic acid, and each first-direction barcode strip immobilized with a different barcode sequence;

C. applying a third set of barcode nucleic acids to the plurality of first-direction barcode bands on the chip surface along a second direction perpendicular to the first direction using the microfluidic device having the plurality of parallel microfluidic channels to form a plurality of first- and second-direction barcode bands, wherein the third set of barcode nucleic acids includes a plurality of second-direction barcode nucleic acids having different barcode sequences, each second-direction barcode band having a second-direction barcode nucleic acid, and each second-direction barcode band having a different barcode sequence;

D. Optionally, using the microfluidic device having multiple parallel microfluidic channels, affix a fourth set of barcode nucleic acids in the second direction on the chip surface at positions adjacent to the multiple first and second-direction barcode bands to form multiple second-direction barcode bands, wherein the fourth set of barcode nucleic acids includes multiple second-direction barcode nucleic acids having different barcode sequences, each second-direction barcode band having a different second-direction barcode nucleic acid affixed thereto, and each second-direction barcode band having a different barcode sequence affixed thereto;

On the chip surface where the plurality of first-direction barcode strips intersect the plurality of second-direction barcode strips, the second-direction barcode nucleic acid is linked to the first-direction barcode nucleic acid to form probes. The probes constitute array points, and each array point has a probe with a different sequence.

In the present invention, the width of each microchannel of the parallel microchannels (i.e., the width of the barcode strip formed, i.e., the width of the chip probe array) is about 10-200 μm, preferably about 15-150 μm, and most preferably about 25-100 μm.

In the present invention, it is advantageous for each probe array site on the chip to have the same width and length, that is, the same coverage area and shape. In the aforementioned method of preparing a chip with substantially zero spacing between adjacent sites, the barcode strips formed by nucleic acids transported and immobilized via the microfluidic channel have the same width.

To achieve this objective, in one aspect of the present invention, the microfluidic device used in the method has multiple parallel microfluidic channels, each of which has the same channel width (i.e., the width of the barcode strips formed), and the spacing between the channels is the same and the same as the channel width. Thus, in the multiple first-direction barcode strips formed in step A and the multiple first-second-direction barcode strips formed in step C, the width of the spacing between adjacent barcode strips is the same as the width of the barcode strips. Furthermore, in steps B and D, a microfluidic device having the same channel width and spacing between the channels as in steps A and C is also used to form multiple second first-direction barcode strips or second second-direction barcode strips adjacent to the multiple first first-direction barcode strips, thereby forming first-direction barcode strips or second-direction barcode strips with no gaps or substantially no gaps between adjacent barcode strips.

In yet another aspect of the present invention, in steps A and C of the method, the microfluidic device having multiple parallel microfluidic channels has the same channel width, and the spacing between the channels is the same as the channel width. In steps B and D, the microfluidic device has a channel width greater than the channel width of the microfluidic device in steps A or C (the spacing between the channels of the microfluidic device remains the same as that of the microfluidic device in steps A and C, i.e., the channel width and the total length of the channel walls are the same). In other words, the barcode strips formed in steps B and D are wider than the barcode strips formed in steps A and C. Thus, when forming multiple second first-direction barcode bands or second second-direction barcode bands adjacent to the multiple first first-direction barcode bands or first second-direction barcode bands in steps B and D, the "redundant" portion of the barcode bands formed by the microfluidic channel used in steps B and D relative to the barcode bands formed by the microfluidic channel used in steps A or C can compensate for gaps between the barcode bands caused by microfluidic channel positioning errors. The inventors of the present application unexpectedly discovered that the overlap of different barcode nucleic acids in adjacent barcode bands caused by the flow channel width of the microfluidic device used in steps B and D being greater than the flow channel width of the microfluidic device used in steps A or C does not result in significant (statistically significant) probe misalignment (i.e., the presence of probes with nucleic acid sequences at adjacent sites at the array site) in adjacent array sites of the prepared chip, and therefore does not significantly reduce the uniformity deviation of the probes in the array sites of the chip. Without being limited by theory, the inventors believe that this is because, during the steps of adding and/or ligating nucleic acid fragments to form probes at each array site, the groups or nucleic acids used to immobilize or ligate the nucleic acid fragments at the overlapped locations have been completely or nearly completely consumed by the nucleic acid fragments already immobilized or ligated in the previous step. For example, in step B, even if the microfluidic channel overlaps with the first-dimensional barcode strip formed in step A, the groups or chip surface linker nucleic acids available for binding to the second set of barcode nucleic acids transported at the overlapped locations are already occupied by the first set of barcode nucleic acids in step A. Therefore, there is no or nearly no second set of barcode nucleic acids available for immobilization at the overlapped locations. For another example, in step D, even if the microfluidic channel overlaps with the second-dimensional barcode strip formed in step C, the first barcode nucleic acids available for binding to the fourth set of barcode nucleic acids transported at the overlapped locations are already occupied by the third set of barcode nucleic acids in step C. Therefore, there is no or nearly no fourth set of barcode nucleic acids available for immobilization at the overlapped locations.

In one embodiment of the present invention, in steps B and D, the channel width of the microfluidic device used is approximately 5.0 μm (i.e., approximately 2.5 μm on one side of the channel) larger than the channel width of the microfluidic device in step A or C, preferably approximately 4.0 μm (i.e., approximately 2.0 μm on one side of the channel), more preferably approximately 2.0 μm (i.e., approximately 1.0 μm on one side of the channel), for example, approximately 0.5 μm (i.e., approximately 0.25 μm on one side of the channel).

The inventors of the present application also unexpectedly discovered that in the above-mentioned method for preparing the improved chip of the present invention, although there is contact and extrusion between the flow channel wall of the microchannel and the barcode band formed in the previous step, the contact and extrusion do not have a significant effect on the nucleic acid bound to the formed barcode band, nor do they have a significant effect on the number and quality of effective probes on the chip finally formed.

In one aspect of the present invention, in the method, when preparing the chip, one or more alignment points are provided on the chip for calibration and positioning with alignment marks provided on the microfluidic device when applying the barcoded nucleic acid using the microfluidic device having multiple parallel microfluidic channels. Through positioning, particularly precise positioning, the microfluidic device can be used to accurately transport and fix the barcoded nucleic acid on the chip surface at a specified position, for example, to transport and form another nucleic acid barcode band in parallel adjacent to an already formed nucleic acid barcode band. In the present invention, the positioning error is typically less than 5.0 μm, preferably less than 2.0 μm, more preferably less than 1.0 μm, for example, approximately 0. Thus, when the second set of barcoded nucleic acids is formed into a second first-direction barcode band adjacent to the first first-direction barcode band, the spacing between them can be controlled to be less than 5.0 μm, preferably less than 2.0 μm, more preferably less than 1.0 μm, for example, approximately 0 μm. This means that there is no gap between the first first-direction barcode band and the adjacent second first-direction barcode band, thereby achieving a chip with no gaps between adjacent array sites of the probe array. In one aspect of the present invention, multiple alignment points are provided on the chip, preferably equal to or greater than 3, and more preferably equal to or greater than 4.

In one aspect of the present invention, the method employs an alignment platform system to control the alignment and movement of a microfluidic device and/or chip having multiple parallel microfluidic channels to transport and immobilize the barcoded nucleic acid on the chip surface. In one embodiment of the present invention, the alignment platform includes a device for securing and/or moving the chip, a device for securing and/or moving the microfluidic device, and a device for observing alignment marks on the chip and/or microfluidic channel. Preferably, the device for observing alignment marks on the chip and/or microfluidic channel is a microscope. More preferably, the device for observing alignment marks on the chip and/or microfluidic channel includes more than one microscope for simultaneously observing more than one alignment mark.

As shown in Figure 3, the method may further include immobilizing a chip surface linker nucleic acid on the chip surface, for example, across the entire chip surface; the chip surface linker nucleic acid is used to link to the first barcode nucleic acid. For example, the chip surface linker nucleic acid may include a 3'-terminal linker fragment for linking to the first barcode nucleic acid. Prior to this step, the chip surface may also be modified to allow the chip surface linker nucleic acid to be directly or indirectly attached to the chip via covalent or non-covalent bonds.

The present invention also provides a chip for analyzing nucleic acid information from biological samples. In one aspect of the present invention, the chip for analyzing nucleic acid information from biological samples is prepared by the aforementioned method. In one aspect of the present invention, the surface of the chip for analyzing nucleic acid information from biological samples comprises an array of probes, the probe array comprising orthogonal rows and columns. The spacing between adjacent array sites in the chip provided by the present invention is substantially zero. In another aspect of the present invention, the spacing between adjacent array sites is less than approximately 2.0 μm, preferably less than approximately 1.0 μm, and most preferably approximately zero, i.e., there are no gaps between adjacent array sites. The probes in each array site of the probe array of the chip of the present invention each have a different barcode sequence, which can be used to indicate the spatial position of the probes. In one aspect of the present invention, the probes comprise a first barcode and a second barcode. In another aspect of the present invention, the probes in each row of the probe array have the same first barcode and the probes in each column have the same second barcode; the second barcodes of the probes in each row are different, and the first barcodes of the probes in each column are different. In one aspect of the present invention, the chip for analyzing nucleic acid information from biological samples comprises a chip surface linker nucleic acid across its entire surface. In yet another aspect of the present invention, the 5' end of each probe in the probe array is the chip surface linker nucleic acid. In yet another aspect of the present invention, the sequence of each probe in the probe array, from the 5' end to the 3' end, includes the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture segment for identifying and binding target nucleic acids in a biological sample. In yet another aspect of the present invention, the sequence of each probe in the probe array includes a primer segment for amplification reaction at the 5' end. In yet another aspect of the present invention, the sequence of each probe in the probe array also includes a unique molecular identifier (UMI).

The chip prepared by the method provided by the present invention can be used to analyze intracellular molecules in tissue samples, especially thin tissue sections, including analysis of nucleic acids and proteins, such as by PCR, mass spectrometry, next-generation sequencing, or ELISA, to obtain their expression and spatial information.

The present invention also provides a method for analyzing spatial transcriptomic information of a biological tissue sample, the method comprising contacting the aforementioned array with a tissue sample. "Tissue samples" suitable for the present invention include tissue obtained from a subject, fixed, sliced, and mounted on a planar surface. The tissue sample can be a formalin-fixed paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or a frozen tissue sample, among others. The method of the present invention can be performed before or after staining the tissue sample. For example, after hematoxylin and eosin staining, the tissue sample can be spatially analyzed according to the methods provided herein. The method can include analyzing the histology of the sample (e.g., using hematoxylin and eosin staining) and then spatially analyzing the tissue. Formalin fixation and paraffin embedding (FFPE) of tissue sections typically involves fixing the tissue obtained from the subject in formaldehyde (e.g., 3%-5% formaldehyde in phosphate-buffered saline) or Bouin's solution, embedding in wax, cutting into thin slices, and then mounting on a planar surface, such as a microscope slide. The method of the present invention involves contacting the tissue slice with an array of probes on a chip, wherein the probes on the array can recognize and bind to nucleic acids, particularly mRNA, of cells in the tissue. Subsequent analysis includes reverse transcription and amplification, and can be performed by high-throughput next-generation sequencing (NGS) or synthesis sequencing (SBS).

"Sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. Sequencing can be performed using a variety of currently available systems, such as, but not limited to, sequencing systems from Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies. In some cases, the systems and methods provided herein can be used in conjunction with proteomic information.

In some embodiments, nucleic acids in tissue sections (e.g., formalin-fixed paraffin-embedded (FFPE) tissue sections) are transferred to an array and captured on the array by hybridization with a capture probe. In some embodiments, the capture probe can be a universal capture probe that hybridizes, for example, with an adapter region in a nucleic acid sequencing library, or a poly-A tail of an mRNA. In some embodiments, the capture probe can be a gene-specific capture probe that hybridizes, for example, with a specifically targeted mRNA or cDNA in a sample.

In some embodiments, the nucleic acid in the tissue section (e.g., FFPE section) is transferred to the array and captured on the array by single-stranded connection with universal adapter oligonucleotides. In other embodiments, the nucleic acid on the chip can be transferred to the tissue section (e.g., FFPE section). The probe bound on the chip can be made to fall off in the solution and enter the cell on the tissue in contact with it by methods known in the art. For example, a photodegradable joint can be added to the junction of the nucleic acid probe and the chip, or the nucleic acid probe can be bound to the chip by a pH-sensitive joint, and then the nucleic acid probe is separated from the chip by changing the pH value of the solution.

The chip of the present invention and its use can determine the spatial position of the probe molecule in the array on the chip surface through the first barcode sequence and the second barcode sequence of each array point, thereby obtaining the position information of the cell containing the nucleic acid molecule in the tissue.

In one aspect of the present invention, the method further comprises subjecting the tissue thin sections to morphological analysis and/or histological analysis, wherein the histological analysis is performed by H&E staining, IHC staining, ISH staining, and FISH staining.

In one aspect of the invention, the method comprises one or more of a non-human cell, a human cell, a non-natural protein, a nucleic acid, a small molecule, a dye, a virus, a bacterium, a parasite, a protozoa, or a chemical substance.

In one aspect of the present invention, the small molecule in the method comprises a hapten, a peptide tag, a protein tag, a fluorescent tag, a nucleic acid tag, or a combination thereof.

In one aspect of the invention, the method comprises generating quantitative and/or qualitative data of the marker.

In one aspect of the present invention, in the method, the marker comprises DNA, protein, RNA, lipid, organelle, metabolite, or cell.

In one aspect of the invention, the method wherein the marker comprises genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids and/or organelles.

In one aspect of the invention, the method comprises an altered nucleotide sequence encoding an altered amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, a copy number change, an expression level change, a protein level change, a protein activity change, or a methylation status change in cancer tissue or cancer cells compared to normal healthy tissue or cells.

In one aspect of the invention, the marker is measured in the method by single-cell sequencing, single-nucleus sequencing, flow cytometry, immunohistochemistry staining, hematoxylin and eosin staining, whole genome sequencing, high-throughput sequencing, mass spectrometry, DNA microarray, or a combination thereof.

In one aspect of the present invention, the method further comprises subjecting the tissue thin sections to morphological analysis and/or histological analysis, wherein the histological analysis is performed by H&E staining, IHC staining, ISH staining, and FISH staining.

In one aspect of the invention, the method wherein the one or more biomolecules are analyzed by PCR, mass spectrometry, next generation sequencing, or ELISA, or by artificial intelligence (AI) analysis, or by big data analysis.

Example 2 Preparation of Chip

Figure 3 is a schematic flow diagram of an exemplary embodiment of the method for preparing a biochip provided by the present invention. Figure 4 is an exemplary diagram of the microfluidic channel configuration used in the method for preparing a biochip provided by the present invention. Figure 5 is an exemplary diagram of the configuration of a system for calibrating and controlling the movement of the microfluidic channel used in the method for preparing a biochip provided by the present invention.

As shown in FIG3 , in the method provided by the present invention, a glass sheet is used as a chip substrate, and the chip surface is modified with active groups such as amino, aldehyde, epoxy, isothiocyanate, mercapto, and silane through surface chemical reactions.

In this embodiment, a commercially available optical epoxy-modified glass sheet ( Slide E) is the chip substrate.

A universal chip surface linker nucleic acid with a 5' amino-terminal modification and the following sequence was synthesized, where the underlined T base is FITC-modified:

5'amino-CTACACGACGC T CTTCCGATC-3'

Synthesize 100 5' end phosphorylated first direction barcode nucleic acids with the following sequences:

5' phosphorylated-CTCTTTCCC T AC12345678ACGACGCTCTTC-3'

Wherein, "12345678" represents a barcode segment having 8 nucleotides, wherein the sequence of the 8 nucleotides is known (specified). The sequences of the barcode segments (referred to as the first barcode) of the 100 first group of barcode nucleic acids are different, and the sequence of the first barcode of each first group of barcode nucleic acid is known (specified). The underlined T base is FITC-modified. In this embodiment, each step of adding the barcode segment in chip synthesis is observed or quality controlled by fluorescent modification of the barcode segment and detection of the generated fluorescent signal. In other embodiments, the barcode segment may not be fluorescently modified.

The 100 first-direction barcode nucleic acids were divided into two groups (50 in each), and named as the first group (first) first-direction barcode nucleic acids and the second group (second) first-direction barcode nucleic acids, respectively.

A second set of 100 barcoded nucleic acids with the following sequences were synthesized:

5' Phosphorylated-GAGTGATTGCT T GTGACGCCTT 87654321 NNNNNNNNNN TTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'

Here, "87654321" represents an 8-nucleotide barcode segment, wherein the sequence of the 8-nucleotide segment is known (assigned). The sequences of the barcode segments (referred to as the second barcode) of the 100 second-group barcode nucleic acids are different, and the sequence of the second barcode of each second-group barcode nucleic acid is known (assigned). The underlined T base is Cy3-modified.

The 100 second-direction barcode nucleic acids were divided into two groups (50 in each), and named as the first group (first) second-direction barcode nucleic acids and the second group (second) second-direction barcode nucleic acids, respectively.

A first linker nucleic acid having the following sequence was synthesized:

5’-AGGGAAAGAGAGATCGGAAG-3’

A second linker nucleic acid having the following sequence was synthesized:

5’-GCAATCACTCGAAGAGCGT-3’

The cell culture chamber is attached to the glass slide, and the chamber and the glass slide are pressed together using a frame to improve the sealing. Use a pipette to add 10-20uM universal chip surface linker nucleic acid (dissolved in 300mM sodium phosphate buffer pH8.5) into the chamber. After covering the bottom of the chamber, the glass slide is placed in a constant temperature mixer at 40°C and 800rpm for oscillation and mixing for 3 hours. After the reaction is completed, the modified glass slide is washed with 0.1% Triton X-100, 1mM HCl, and 100mM KCl in sequence, and then blocked with 0.1M Tris pH 9.0, 50mM ethanolamine, and 0.1% SDS at 50°C. After blocking, rinse the substrate with deionized water for 1 minute, and then blow dry the substrate with nitrogen. The FITC fluorescence signal of the universal nucleic acid is observed under a fluorescence microscope to confirm the completion of the reaction and the effect of the surface modification.

A device comprising parallel microchannels as shown in FIG2 was prepared by soft lithography using polydimethylsiloxane (PDMS), wherein the bottom of the microchannels was open and the height of the microchannels was about 70 μm.

FIG4 is an exemplary diagram of the microfluidic channel arrangement used in the method for preparing a biochip provided by the present invention. From top to bottom, the flow channel arrangement diagrams of the microfluidic channel devices used in steps A, B, C, and D in FIG3 are shown. The spacing between adjacent microfluidic channels in steps A, B, C, and D is 300 μm. In addition, the flow channel width of the microfluidic channel devices in steps A and C is 150 μm (equivalent to a distance of 150 μm between adjacent flow channels), and the flow channel width of the microfluidic channel devices in steps B and D is 154 μm (equivalent to a distance of 146 μm between adjacent flow channels: the spacing between adjacent microfluidic channels is 300 μm minus the flow channel width of 154 μm).

The PDMS microfluidic device is bonded to a glass chip slide to seal the channel. A clamping tool is used to press the top of the channel against the base glass slide to improve the seal. One end of the microfluidic channel serves as the solution inlet, and the other end is connected to a vacuum suction device via a port.

In one embodiment, the detailed operations of steps A, B, C, and D in FIG3 are as follows.

After attaching the first microfluidic device to the glass slide, a buffer solution is introduced into the flow channel to expel the gas within. Then, 10-20 μM of the first set of barcode nucleic acids is added to the flow channel: a first set of first-direction barcode nucleic acids (the first barcode nucleic acid in each flow channel has a different barcode sequence than the first barcode nucleic acids in other flow channels), a first linker nucleic acid, and T4 ligase are introduced into each flow channel. HF glass etchant is added to the control mark inlet of the microfluidic channel used to prepare alignment marks at the four corners of the glass slide. After the probe solution and glass etchant are added, negative pressure is applied to the corresponding flow channel outlet to drive the probe solution and glass etchant through the flow channel. After the flow channel is filled, the reaction is allowed to stand at 37°C for 30 minutes. After the reaction is completed, the substrate is rinsed with deionized water for 1 minute and then blown dry with nitrogen. The FITC fluorescence signal of the first barcode nucleic acid is observed using a fluorescence microscope to confirm the completion of the reaction and the connection reaction between the first set of barcode nucleic acids and the universal chip surface linker nucleic acid at the flow channel coverage area to form the first set of barcode bands.

Figure 5 is an exemplary diagram of the setup of a system for calibrating and controlling the movement of microfluidic channels used in the method for preparing a biochip provided by the present invention. The system includes a device for fixing and/or moving the chip, a device for fixing and/or moving the microfluidic device, and a device for observing the alignment marks on the chip and/or microfluidic channel. Specifically, a WH-AM-01 alignment platform (purchased from: Suzhou Wenhao Microfluidic Technology Co., Ltd.) was used to fit the second PDMS microfluidic channel device to the glass slide processed in the previous step through the alignment marks set on the chip, so that the flow channel is completely close to the flow channel formed by the glass slide processed in the previous step. A second group of first-direction barcode nucleic acid, a first linker nucleic acid, and T4 ligase are introduced into each flow channel, and negative pressure is applied at the corresponding flow channel outlet to drive the probe solution through the flow channel. After the flow channel is filled, the reaction is allowed to stand at 37°C for 30 minutes. After the reaction is completed, the substrate is rinsed with deionized water for 1 minute, and then the substrate is blown dry with nitrogen.

The WH-AM-01 alignment platform is operated according to the manufacturer's instructions. The basic steps include: connecting the USB cable of the microscope of the alignment platform to the computer, opening the camera software, and turning on the camera ruler function; placing the cleaned PDMS microfluidic device on the lower surface of the upper stage and the chip slide on the upper surface of the lower stage; adjusting the Z-axis knob of the upper stage until the PDMS microfluidic device and the chip slide are close but not touching; adjusting the two microscopes so that the alignment marks of the upper PDMS microfluidic device can be clearly found at the same time, and adjusting the microscope bracket X -Use the Y-axis knob to move the horizontal and vertical lines of the ruler to the alignment position of the upper PDMS microfluidic device; adjust the focus of the microscope until the alignment position of the lower chip slide can be clearly found, and adjust the X-Y-θ axis knob of the lower slide stage to move the alignment mark of the lower chip slide to the same ruler positioning line as the upper PDMS microfluidic device to complete the rough alignment; slowly twist the Z-axis knob of the upper stage to slowly move the upper PDMS microfluidic device downward until the PDMS microfluidic device is bonded to the glass slide; after the alignment and bonding are completed, remove the PDMS microfluidic device-glass slide assembly for subsequent experiments.

Using the WH-AM-01 alignment platform, align the third PDMS microfluidic device with the glass slide processed in the previous step along the direction perpendicular to the flow channels of the first and second PDMS microfluidic devices using the alignment marks. After the buffer solution is introduced into the flow channel to expel the gas in the flow channel, 10-20uM of the first set of second-direction barcode nucleic acids is introduced: one second-direction barcode nucleic acid (the second barcode nucleic acid of each flow channel has a barcode sequence different from the second barcode nucleic acid of other flow channels) and the second linker nucleic acid and T4 ligase are introduced into each flow channel. After filling the flow channel, the reaction is allowed to stand at 37°C for 30 minutes. After the reaction is completed, the substrate is rinsed with deionized water for 1 minute and then blown dry with nitrogen.

Using the WH-AM-01 alignment platform, align the fourth PDMS microfluidic device with the glass slide processed in the previous step using the alignment marks, ensuring that the channels are completely aligned with the channels formed on the glass slide processed in the previous step. After purging the channels with buffer, introduce 10-20 μM of a second set of second-direction barcode nucleic acids: one second-direction barcode nucleic acid (each channel's second barcode nucleic acid has a different barcode sequence than the other channels), a second linker nucleic acid, and T4 ligase into each channel. After the channels are filled, incubate at 37°C for 30 minutes.

After the ligation reaction is complete, the flow channel is rinsed with 1× PBS buffer and ultrapure water. The flow channel is then removed, and the substrate is rinsed with deionized water for 1 minute, followed by drying with nitrogen. The Cy3 fluorescence signal of the second barcode nucleic acid is observed using a fluorescence microscope to confirm the completion of the reaction and the ligation reaction between the second barcode nucleic acid and the first set of barcode nucleic acids at the flow channel intersections, forming a barcode array and completing chip fabrication.

The prepared chip is vacuum-sealed and stored in a dark place at room temperature or in a refrigerator at 4°C. The efficiency of each barcode nucleic acid ligation reaction and the intensity (density) and uniformity of the probes on the prepared chip are evaluated by observing the fluorescence signal on the prepared chip.

6A-6D are respectively views of the operations of aligning and positioning the microfluidic device and the chip to transport and fix the barcoded nucleic acid in steps A, B, C, and D in FIG. 3 , and observation views of the effect detection.

Figure 6A illustrates the operation and effect of step A in Figure 3, namely, forming the first first-direction barcode strip on the chip. As shown in the left image and the enlarged partial view in the upper right corner of Figure 6A, alignment marks at the four corners of the chip are used to position the microfluidic device. A first set of barcode nucleic acids is vertically introduced into the chip, where the entire surface of the chip is already immobilized with chip surface linker nucleic acids, forming the first set of barcode nucleic acid strips. The fluorescence image in the lower right corner of Figure 6A shows the formation of 40 vertical barcode strips on the chip, spaced apart by the same strip width. The fluorescence of the barcode nucleic acid strips immobilized on the chip by the chip surface linker nucleic acids dims, while the fluorescence at chip locations unbound by barcode nucleic acids (between the barcode nucleic acid strips and at the chip periphery) maintains its original intensity. Figure 6B illustrates the operation and effect of step B, namely, forming a second first-direction barcode strip adjacent to the first first-direction barcode strip on the chip. As shown in the left image and the enlarged partial image in the upper right corner of Figure 6B, alignment marks at the four corners of the chip were used to position the microfluidic device. A second set of first-direction barcode nucleic acids was added adjacent to the first first-direction barcode strip already formed on the chip surface to form a second first-direction barcode nucleic acid strip. The fluorescence image in the lower right corner of Figure 6B shows the formation of 80 vertical barcode strips with no gaps between them on the chip. The gaps between the barcode strips formed in step B are also darkened due to the addition and binding of the second set of first-direction barcode nucleic acids to the chip surface linker nucleic acids.

Figure 6C illustrates the operation and results of step C in Figure 3, namely, forming first and second directional barcode strips on the chip. As shown in the left image and the enlarged partial view in the upper right corner of Figure 6C, alignment marks at the four corners of the chip are used to position the microfluidic device. A third set of barcode nucleic acids is then introduced perpendicularly to the chip, where 80 vertical barcode strips with no gaps between them have already been formed. The fluorescence image in the lower right corner of Figure 6C shows the formation of 40 horizontal barcode strips on the chip, spaced apart by the same strip width. The second horizontal barcode nucleic acid, fixed to the chip by binding to the first barcode nucleic acid, displays a Cy3 fluorescent label, while locations on the chip where the second barcode nucleic acid is not bound exhibit no fluorescent signal.

Figure 6D illustrates the operation and results of step D, which involves forming a second second-directional barcode band adjacent to the first and second-directional barcode bands on the chip. As shown in the left image and the enlarged partial view in the upper right corner of Figure 6D , alignment marks at the four corners of the chip are used to position the microfluidic device. A second set of second-directional barcode nucleic acids is added adjacent to the first and second-directional barcode bands already formed on the chip surface to form a second second-directional barcode nucleic acid band. The fluorescence image in the lower right corner of Figure 6D shows the formation of 80 horizontal barcode bands with no gaps between them on the chip. The gaps between the barcode bands formed in step C also display Cy3 fluorescence labeling due to the addition and binding of the second barcode nucleic acid to the first-directional barcode nucleic acid. It is important to note that the darker stripes of fluorescence signal seen in the chip array in the image are not the gaps between the barcode bands, but rather the entire barcode band (due to differences in probe density caused by factors such as nucleic acid fragment ligation efficiency).

The efficiency of each barcode nucleic acid ligation reaction and the intensity (density) and uniformity of the probes on the prepared chip were evaluated by observing the fluorescent signals on the prepared chip.

Images and measurements were taken using a fluorescence microscope (Olympus IX53) and a CCD camera (Olympus DP74). Fluorescence intensity values and boundaries were extracted using ImageJ, and the intensity uniformity and actual width of the array spots of the prepared chip were calculated. The results showed that there were virtually no gaps between the spots in the array of the prepared chip, and the uniformity of the probe modification between adjacent spots in the same horizontal or vertical direction varied by less than approximately 3.0%.

In another exemplary method for preparing a biochip provided by the present invention, the spacing between adjacent microchannels in steps A, B, C and D is 300 μm, and the channel width is 150 μm (equivalent to a distance of 150 μm between adjacent channels). The chip is prepared using the same process as above. After testing, in some of the obtained chips, there is a gap of about 0.5 μm to about 1.0 μm between adjacent array points.

Example 3 Tissue sample preparation and staining

(1) Tissue OCT embedding

Take a fresh mouse olfactory bulb tissue sample and quickly rinse the tissue surface with pre-chilled PBS or saline to remove any residual liquid. Then, blot dry with clean absorbent paper. Place the tissue in an embedding chamber and add OCT embedding medium until the tissue is completely covered. Ensure there are no bubbles around the tissue and place the embedding chamber on dry ice until the OCT is completely frozen.

(2) Frozen sections

The freezing microtome temperature was set to -20°C for the chamber and -10°C for the specimen head. Before sectioning, the frozen tissue and substrate were placed in a -20°C freezing microtome chamber for equilibrium for at least 30 minutes, and then cryosectioned in the freezing microtome chamber with a thickness of 10 μm.

(3) Tissue fixation and HE staining

Attach the cut tissue sections to the barcode array-modified substrate prepared in Example 2 and incubate at 37°C for 1 minute. Completely immerse the tissue-attached substrate in pre-chilled methanol and fix at -20°C for 30 minutes. After fixation, remove the substrate, wipe dry the liquid on the back, and add 500 μl of isopropanol to the tissue sections. Incubate at room temperature for 1 minute. After 1 minute, remove the isopropanol, and allow the sections to air dry at room temperature for 5-10 minutes.

Add 1 ml of hematoxylin to evenly cover the tissue section on the substrate and incubate at room temperature for 7 minutes. Remove the hematoxylin reagent, rinse the substrate in RNase-free water, and let it dry. Add 1 ml of bluing solution and incubate at room temperature for 2 minutes. Remove the bluing solution, rinse the substrate in RNase-free water, and wipe the liquid on the back of the substrate. Add 1 ml of eosin mixture and incubate at room temperature for 1 minute.

After removing the eosin, the slides were rinsed in RNase-free water and allowed to dry until the tissue was opaque. After incubating the slides at 37°C for 5 minutes, brightfield imaging was performed. Figure 7 shows a histological HE staining of mouse brain olfactory bulb tissue.

(IV) Tissue Permeabilization

Assemble the fixture chamber onto the prepared tissue microarray, ensuring each tissue section is located within its corresponding chamber. Add 70 μl of permeabilization enzyme (0.1% pepsin diluted in 0.1 N HCl) to the chamber and permeabilize the tissue at 37°C. Remove the permeabilization enzyme and wash with 0.1× SSC.

Example 4: Reverse transcription reaction of tissue sample slices using a chip

Add 70 μl of reverse transcription mixture to the cleaned chamber in Example 3. The reverse transcription mixture includes: 1x first-strand buffer, 5 mM DTT, 500 μM dNTPs, 0.19 μg/μl BSA, 1% DMSO, 2.5 μM Template Switch Oligo, 20 U/μl Superscript III, and 2 U/μl RNase inhibitor.

After reverse transcription is complete, aspirate the reverse transcription mixture from the chamber. Add 70 μl of 0.08 M KOH to the chamber, incubate at room temperature for 5 minutes, and then rinse once with 100 μl of RNase-free water.

Add the cDNA second-strand synthesis reaction solution to the cleaned chamber. The second-strand synthesis reaction solution contains: 1x First Strand Buffer, 10U Klenow ^Exo- , and 2.5μM Second Strand Primer. Seal the chamber with tape and place it on a thermostat at approximately 37°C for 1 hour.

After the reaction is completed, aspirate the second-strand synthesis reaction solution in the chamber and then add 100ul RNase-free water to wash once. Then add 35μl 0.08M KOH to the chamber and incubate at room temperature for 10 minutes. Prepare several new 1.5ml centrifuge tubes and add 10μl Tris (1M, pH 7.0) to them. Transfer the 35μl sample in the chamber to the corresponding centrifuge tube containing Tris and mix well to complete the second-strand preparation of cDNA.

cDNA amplification

Prepare a PCR amplification reaction mixture in a new 1.5ml microcentrifuge tube placed on ice. The PCR reaction mixture includes: 1× Kapa HiFi Hotstart ReadyMix, 0.8μM cDNA Forward Primer, 0.8μM cDNA Reverse Primer, 35μl cDNA template, in a total volume of 100μl. Amplify the cDNA using the following protocol:

After amplification, the amplified product was purified using 0.6× AMpure XP Beads. The purified product was used for library construction and sequencing.

Example 5 Library construction and sequencing

Fragmentation, end repair, and A addition

Place a new PCR tube on ice and prepare the fragmentation reaction solution. The fragmentation reaction solution should include: 5 μl FEA Buffer V2, 10 μl purified DNA from the previous step, 25 μl ddH2O, and 10 μl FEA Enzyme Mix V2, for a total volume of 50 μl. Mix thoroughly by pipetting or vortexing, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and run the following program:

This achieves the purpose of fragmenting the DNA while blunting the ends of the fragmented DNA, phosphorylating the 5' end and adding a dA tail to the 3' end.

Connector connection

Place a new PCR tube on ice and prepare the adapter ligation reaction solution. The adapter ligation reaction solution should include: 25μl Rapid Ligation Buffer V2, 50μl of the fragmented DNA purified in the previous step, 15μl ddH2O, 5μl Rapid DNA Ligase V2, 5μl of adapter (10pM), for a total volume of 100μl. Use a pipette or vortex to mix thoroughly, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and run the following program:
Heated cover 105℃ On
20℃ 15min
4℃ Hold

After the ligation reaction, 0.6×XP SPRIselect Beads were used to purify the ligation product.

Library amplification

Place a new PCR tube on ice and prepare the library amplification reaction solution. The library amplification reaction solution includes: 25 μl VAHTS HiFi Amplification Mix, 20 μl of the adapter-ligated DNA purified in the previous step, and 5 μl Index PCR Primer Mix (10 pM each), for a total volume of 50 μl. Use a pipette or vortex to mix thoroughly, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and run the following program:

After the amplification reaction, the amplified product was purified using 0.9× AMpure XP Beads.

Sequencing

The library was sequenced using PE150 using Illumina NovaSeq 6000.

Data processing and analysis

a) Use data processing software such as umitools (version: 1.1.2) to extract UMI and Barcode in Read1.

b) Use STAR (version 2.5.3a) and other data processing software to align read2 to the mouse reference genome, mm10 (GENCODE vM23/Ensembl 98).

featureCounts (Version 2.0.3)assign gene.

c) Use data processing software such as umitools (version: 1.1.2) to generate a barcode-gene expression matrix and obtain the "sequencing," "mapping," "spots," and other data in Table 1 below.

Table 1 Analysis results

d) Use Python (version: 3.10.8) to write a program to convert the barcode-gene expression matrix into a barcode spatial distribution map.

e) Use Adobe Illustrator to compare the barcode spatial distribution map with the HE image to obtain the barcode-HE image correspondence table.

f) Seurat (version: 4.3.0) software was used to process the barcode-gene expression matrix, barcode-HE map correspondence table, and HE map to output a UMI heat map and value distribution map (Figure 8), a gene heat map and value distribution map (Figure 9), a UMAP cluster map (Figure 10), a TSNE cluster map (Figure 11), and a gene distribution map (Figures 12A-D).

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (15)

A method for preparing a biochip having an array comprises the following steps: A. applying and immobilizing a first set of barcode nucleic acids onto a chip surface using a microfluidic device having multiple parallel microfluidic channels to form multiple first-direction barcode strips in a first direction, wherein the first set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip having a different first-direction barcode nucleic acid immobilized thereon; B. Using the microfluidic device having multiple parallel microfluidic channels, applying and affixing a second set of barcode nucleic acids to the chip surface along the first direction at positions adjacent to the multiple first-direction barcode strips to form multiple second first-direction barcode strips, wherein the second set of barcode nucleic acids includes multiple first-direction barcode nucleic acids having different barcode sequences, each first-direction barcode strip affixed with a different first-direction barcode nucleic acid, and each first-direction barcode strip affixed with a different barcode sequence; C. applying a third set of barcode nucleic acids to the plurality of first-direction barcode bands on the chip surface along a second direction perpendicular to the first direction using the microfluidic device having the plurality of parallel microfluidic channels to form a plurality of first- and second-direction barcode bands, wherein the third set of barcode nucleic acids includes a plurality of second-direction barcode nucleic acids having different barcode sequences, each second-direction barcode band having a second-direction barcode nucleic acid, and each second-direction barcode band having a different barcode sequence; D. Optionally, using the microfluidic device having multiple parallel microfluidic channels, affix a fourth set of barcode nucleic acids in the second direction on the chip surface at positions adjacent to the multiple first and second-direction barcode bands to form multiple second-direction barcode bands, wherein the fourth set of barcode nucleic acids includes multiple second-direction barcode nucleic acids having different barcode sequences, each second-direction barcode band having a different second-direction barcode nucleic acid affixed thereto, and each second-direction barcode band having a different barcode sequence affixed thereto; On the chip surface where the plurality of first-direction barcode strips intersect the plurality of second-direction barcode strips, the second-direction barcode nucleic acid is linked to the first-direction barcode nucleic acid to form probes. The probes constitute array points, and each array point has a probe with a different sequence. The method according to claim 1, wherein one or more alignment marks (preferably multiple alignment marks, for example, 2-4) are provided on the chip for calibrating and positioning the microfluidic device when applying the barcoded nucleic acid using the microfluidic device having multiple parallel microfluidic channels, for example, by calibrating and positioning with corresponding alignment marks provided on the microfluidic device. Preferably, an alignment platform system is used to control the alignment and movement of the microfluidic device and/or chip having a plurality of microfluidic channels arranged in parallel. The method according to claim 1, further comprising immobilizing the chip surface linker nucleic acid on the chip surface, for example, on the entire surface of the chip (the chip surface linker nucleic acid can be connected to the first direction barcode nucleic acid), Preferably, the 3' end of the chip surface linker nucleic acid has a connecting fragment for connecting to the first direction barcode nucleic acid through a single-stranded connecting nucleic acid. The method according to claim 1, wherein the first direction barcode nucleic acid comprises a first barcode fragment, and optionally, the 5' end portion of the first direction barcode nucleic acid further comprises a primer fragment for an amplification reaction. The method according to claim 1, wherein the second-direction barcode nucleic acid comprises a probe fragment at the 3' end for identifying and binding to a target nucleic acid in a biological sample (e.g., a fragment that recognizes and binds to mRNA or cDNA, such as a poly-T sequence) and a second barcode fragment. Optionally, the second-direction barcode nucleic acid further comprises a unique molecular identifier (UMI). The method according to any one of claims 1 to 5, wherein the spacing between the first first-direction barcode band and the adjacent second first-direction barcode band is substantially zero, Optionally, the spacing between the first second-direction barcode band and the adjacent second second-direction barcode band is substantially zero. The method of claim 6, wherein the pitch is less than about 2.0 μm, preferably less than about 1.5 μm, most preferably less than about 1.0 μm, for example, about 0. The method according to any one of claims 1 to 7, wherein the array spots of the prepared chip have a probe uniformity deviation of less than 20%, preferably less than 10%, and more preferably less than 5%. A chip for analyzing nucleic acid information of a biological sample, wherein the surface of the chip has probes forming an array, the probe array comprising orthogonal rows and columns, characterized in that the spacing between adjacent array points of the probe array of the chip is substantially zero, The probes at each array point in the array have different sequences, wherein the probes include a first barcode and a second barcode, the probes in each row of the probe array have the same first barcode and the probes in each column have the same second barcode; the second barcodes of the probes in each row are different and the first barcodes of the probes in each column are different, For example, the pitch between adjacent array points of the probe array is less than about 2.0 μm, preferably less than about 1.0 μm, and most preferably about 0. The chip according to claim 9, wherein the surface of the chip has a modified layer for binding to nucleic acid molecules, and the sequence of the nucleic acid at each array point in the probe array includes a first barcode, a second barcode, and a capture fragment for identifying and binding to the target nucleic acid in the biological sample from the 5' end to the 3' end. The chip according to claim 9 has a chip surface linker nucleic acid on its (entire) surface, and the sequence of the nucleic acid at each array point in the probe array includes the chip surface linker nucleic acid, a first barcode, a second barcode, and a capture fragment for identifying and binding the target nucleic acid in the biological sample from the 5' end to the 3' end. The chip according to claim 9, wherein the probe uniformity deviation of each of the array sites of the probe array is less than 20%, preferably less than 10%, and more preferably less than 5%, or, The size uniformity deviation of the probe array spots is less than 10%, preferably less than 5%, and more preferably less than 2%. The chip according to claim 9, wherein the width of each array site of the probe array is about 10-100 μm, preferably about 15-50 μm, most preferably about 20-30 μm, for example about 25 μm or about 10 μm. The chip according to any one of claims 9 to 13, prepared by the method according to any one of claims 1 to 10. A method for analyzing spatial transcriptomic information of a biological tissue sample using a chip according to any one of claims 9 to 14, the method comprising contacting the array of the chip with a tissue sample, wherein the probes on the array recognize and bind to nucleic acids of cells in the tissue, Preferably, the method is used to analyze intracellular molecules in tissue samples, especially thin tissue sections, including analysis of nucleic acids and proteins, for example, by PCR, mass spectrometry, next-generation sequencing, ELISA, or big data and artificial intelligence (AI) to obtain their expression and spatial information.