CN119120194A

CN119120194A - A spatial omics detection tool and preparation method thereof and spatial omics detection method

Info

Publication number: CN119120194A
Application number: CN202411293203.4A
Authority: CN
Inventors: 罗源; 赵建龙; 王大印
Original assignee: Shanghai Institute of Microsystem and Information Technology of CAS
Current assignee: Shanghai Institute of Microsystem and Information Technology of CAS
Priority date: 2024-09-14
Filing date: 2024-09-14
Publication date: 2024-12-13

Abstract

The invention provides a space histology detection tool, a preparation method thereof and a space histology detection method, wherein detection site electrodes which are arranged in an array in the detection tool are communicated with a liquid exchange micro-channel; the control system controls the electrifying state of the detection site electrode by using a site electrifying structure, and the microfluidic liquid changing system, the microelectrode array and the site electrifying structure are MEMS structures. The invention adopts an MEMS structure to obtain a space histology detection tool, improves the space resolution precision of space histology detection to a sub-single cell level, reduces the space histology detection cost and the detection complexity, simultaneously integrates a microelectrode array and a site electrification structure MEMS through a field effect tube and a capacitor, reduces the number of bonding pads, reduces the packaging difficulty, further improves the space resolution precision of space histology detection, electrically connects a detection site electrode and an external bonding pad from the bottom, further improves the space resolution precision of space histology detection, and finally sets a detachable probe card as a packaging structure to improve the replacement efficiency of the space histology detection tool.

Description

Space histology detection tool, preparation method thereof and space histology detection method

Technical Field

The invention belongs to the technical field of biology and the technical field of semiconductor integrated circuit manufacturing, and particularly relates to a space histology detection tool, a preparation method thereof and a space histology detection method.

Background

The spatial transcriptome, genomics and other spatial histology technologies can implement the detection of non-target genes (mutation information) and expression characteristics thereof on single-cell and even subcellular resolution, and accurately acquire the spatial distribution characteristics of gene expression on a molecular level so as to accurately describe heterogeneity of cell composition, spatial cell interaction, molecular interaction and the like, thereby analyzing the differences of sensitivity, side effects and the like of individual drugs on higher precision and more data dimension. In recent years, the application of space histology technology in basic life science research and major disease research of immunity, development, nerves and brain science has achieved numerous weight level achievements, and has shown great development potential of space histology technologies such as space transcriptome and genomics.

The acquisition of spatial location information is the core of spatial histology techniques. In the prior art, the spatial information acquisition modes of the spatial transcriptome and genomics technology can be divided into modes of micro-cutting, imaging, spatial bar code and the like. The method for acquiring spatial information through imaging comprises in situ hybridization (such as seqFISH, merFISH and the like) and in situ sequencing (STARMap, barseq and the like), wherein the two methods acquire sequence and position information through in situ complementary hybridization between a fluorescent probe and a gene or transcript and acquire images, but because of spectral bandwidth limitation, a plurality of hybridization operations are needed, the operation is relatively complex, the image data volume is huge, the efficiency is low, and an expensive high-resolution microscope and molecular probes are needed, compared with the spatial barcode technology which does not rely on imaging any more, the gene/transcript is captured through a nucleotide coding array, and the sequence and position information are acquired through the combination of the NGS sequencing technology, so that the equipment cost can be reduced, and the wide attention of the industry is drawn.

Spatial bar code technology by inkjet printingThe spatial bar code array is formed by methods such as a microbead array (HDST, slide-seq), a microfluidic auxiliary (DBiT-seq, decoder-seq) and the like, the spatial resolution precision is reduced from 55 mu m to be close to a single cell level, but the spatial resolution precision of the spatial histology analysis is limited by the structural density limitation of structures such as ink jet, microbeads and the like. The resolution of the spatial histology analysis was advanced to the submicron level by the Stereo-seq reported by the national institute of large genes in 2022, however, both the equipment cost and the operational complexity are increased.

Accordingly, there is a need for a structure or method that can improve the spatial resolution accuracy of a spatial histology analysis while reducing equipment costs and improving operational efficiency.

It should be noted that the foregoing description of the background art is only for the purpose of providing a clear and complete description of the technical solutions of the present application and is thus convenient for a person skilled in the art to understand, and it should not be construed that the above technical solutions are known to the person skilled in the art merely because these solutions are described in the background art section of the present application.

Disclosure of Invention

In view of the above drawbacks of the prior art, an object of the present invention is to provide a space histology detection tool, a method for preparing the same and a space histology detection method for solving the problems in the prior art.

In order to achieve the aim, the invention provides a space histology detection tool which comprises a microfluidic liquid exchange system, a microelectrode array, a site electrification structure and a control system, wherein the microfluidic liquid exchange system is connected with the microelectrode array;

The microfluidic liquid exchange system comprises a liquid exchange micro-channel with controllable on-off state, wherein the micro-electrode array comprises n detection site electrodes arranged in an array, and n is an integer greater than or equal to 1; the liquid exchange micro-channel is communicated with each detection site electrode so as to introduce label raw materials into the detection site electrodes from the liquid exchange micro-channel, the detection site electrodes are used for electrochemically synthesizing label nucleic acid sequences for capturing DNA and preparing libraries in space histology after being electrified;

the site electrification structure is electrically connected with each detection site electrode, the control system is electrically connected with the site electrification structure, and the control system controls the electrifying state of each detection site electrode through the site electrification structure;

the microfluidic liquid exchange system, the microelectrode array and the site electrification structure are MEMS structures.

Optionally, the space histology detection tool comprises n site electrification structures, each site electrification structure comprises a connecting wire and an external bonding pad which are electrically connected, the external bonding pad is electrically connected with the control system, each detection site electrode is correspondingly and electrically connected with the connecting wire of one site electrification structure, and the control system controls the electrifying state of each detection site electrode through the connecting wire and the external bonding pad.

The micro-electrode array and the site power-on structure are MEMS integrated circuits, wherein the site power-on structure comprises n capacitors, n identical field effect transistors which are arranged in an a-row b-column array, a-row control lines, b-column control lines and n external bonding pads, and a and b are integers more than or equal to 1;

each grid electrode in each row of field effect tubes is commonly connected with a row control line, each source electrode in each column of field effect tubes is commonly connected with a column control line, and one end, which is not connected with each field effect tube, of each row control line and each column control line is electrically connected with 1 external bonding pad;

each capacitor is connected with 1 corresponding field effect tube and 1 detection site electrode, each capacitor comprises a first electrode, a second electrode and an insulating medium, the first electrode is electrically connected with the corresponding detection site electrode, the second electrode is electrically connected with the drain electrode of the corresponding field effect tube, the insulating medium is positioned between the first electrode and the second electrode, and the control system controls the energizing state of the corresponding detection site electrode by controlling the input electric signals of a row control line and a column control line of the field effect tube.

Optionally, the first electrode is a polysilicon region at a preset position around the drain electrode correspondingly connected to the capacitor, the second electrode is the drain electrode correspondingly connected to the capacitor, and the insulating medium is a silicon dioxide layer located between the first electrode and the second electrode.

Optionally, the field effect transistor is an NMOS transistor.

Optionally, the space histology detection tool further comprises a probe card, wherein the probe card is used for packaging the microelectrode array and the site power-on structure, the probe card comprises n probes and a PCB circuit, one end of each probe is electrically connected with the external bonding pad corresponding to one detection site electrode, the other end of each probe is electrically connected with the external bonding pad through the PCB circuit to lead out the electrical connection of the external bonding pad to a packaging pin on the outer surface, and the control system is electrically connected with the packaging pin to control the power-on state of the detection site electrode.

Optionally, n is greater than or equal to 5000.

Optionally, the distance between two adjacent detection site electrodes is less than or equal to 10 micrometers, or the distance between two adjacent detection site electrodes is less than or equal to 5 micrometers.

The invention also provides a space histology detection method, which is carried out by adopting any one of the space histology detection tools, and comprises the following steps:

controlling a liquid exchange micro-channel in a micro-fluidic liquid exchange system to introduce label raw materials to a detection site electrode in a microelectrode array, and controlling the detection site electrode which is introduced with the label raw materials to be electrified through a control system so as to electrochemically synthesize a label nucleic acid sequence from the label raw materials of the detection site electrode;

The control system controls the detection site electrode to be powered off, and the tissue slice to be detected is transferred to the detection site electrode after cell membrane perforation is carried out on the tissue slice to be detected;

Performing DNA unbinding and DNA cutting on the tissue slice to be detected transferred to the detection site electrode to obtain a plurality of DNA fragments, and capturing each DNA fragment obtained after cutting by the label nucleic acid sequence in the corresponding detection site electrode;

Library preparation is carried out on the tag nucleic acid sequence of the DNA fragment captured by the detection site electrode so as to enable the DNA fragment captured by the tag nucleic acid sequence to replicate the tag structure of the corresponding tag nucleic acid sequence, and the DNA fragment with the tag structure is collected and processed into a structure capable of sequencing, so that transcripts are obtained;

And carrying out high-throughput sequencing on the DNA fragments with the tag structures in transcripts obtained by library preparation and collection treatment to obtain the spatial position information of the DNA fragments in the tissue section to be detected.

The invention also provides a preparation method of the space histology detection tool, which is used for preparing the space histology detection tool, and comprises the following steps:

providing a substrate with a first oxide layer on the upper surface;

a first metal layer is arranged on the first oxide layer;

patterning the first metal layer;

covering a second oxide layer on the upper surface of the gap between the patterned first metal layers;

Patterning the second oxide layer over the patterned first metal layer to reveal a portion of the first metal layer under; setting a second metal layer for a gap between the patterned second oxide layers, wherein the second metal layer and the first metal layer below form corresponding electric connection;

A third metal layer is arranged on the surface of the second metal layer, and the third metal layer covers the second metal layer and the second oxide layer, the surface of which is exposed;

The third metal layer is patterned to obtain a microelectrode array and an external bonding pad, wherein the microelectrode array is electrically connected with the second metal layer, the microelectrode array comprises n detection site electrodes which are arranged in an array, each detection site electrode is electrically connected with the first metal layer and a corresponding external bonding pad through the second metal layer below, the detection site electrodes are used for carrying out electrochemical synthesis on label raw materials which are introduced into the detection site electrodes after the external bonding pad is electrified, and the label nucleic acid sequences are used for capturing DNA and preparing libraries in space histology.

As described above, the space histology detection tool and the preparation method and the space histology detection method thereof have the following beneficial effects:

According to the invention, the detection site electrode capable of performing DNA capture by electrochemically synthesizing the tag nucleic acid sequence is obtained by adopting the MEMS device, so that the density of the detection site electrode is greatly improved, the spatial resolution precision of the space histology detection is reduced to a sub-single cell level, the space histology analysis of a tissue slice with a larger area can be adapted, the equipment cost for performing the space histology detection is greatly reduced, and the efficiency of the space histology detection is improved;

According to the invention, the microelectrode array and the site electrification structure are prepared through the integration of the structures of the field effect transistor and the capacitor by the MEMS integrated circuit, so that the number of required conductive pads is reduced, the packaging difficulty is reduced, and the further improvement of the density of the detection site electrode is facilitated;

According to the invention, the detection site electrode is electrically connected with the external bonding pad from the bottom metal, so that the density of the detection site electrode is further improved, and the resolution of space histology detection is improved;

according to the invention, the detachable probe card is arranged as the packaging structure, so that the replacement efficiency of the space group chemical detection tool is improved.

Drawings

Fig. 1 is a schematic view showing a partial exploded view of a three-dimensional structure of a space group chemical examination tool in example 1 of the present invention.

Fig. 2 is a schematic view showing a partial exploded view of a spatial histology inspection tool in a spatial group according to embodiment 2 of the present invention.

FIG. 3 is a schematic structural diagram showing the structure of the site-specific electrification of the space-histology detection tool in example 2 of the present invention.

Fig. 4 is a schematic diagram showing the electrical connection structure of a single fet of the spatial histology inspection tool according to embodiment 2 of the present invention.

FIG. 5 is a schematic side sectional view showing the structure of the semiconductor structure of the site-applied electrical structure of the space-histology inspection tool in example 2 of the present invention.

FIG. 6 shows a schematic structural diagram of the synthetic tag nucleic acid sequence in step A1 of the method for detecting space histology in example 3 of the present invention.

FIG. 7 is a schematic diagram showing the structure of a substrate provided in step B1 of the method for preparing a space histology inspection tool in example 4 of the present invention.

Fig. 8 is a schematic structural diagram showing the first metal layer disposed in step B2 of the method for preparing a space histology detection tool in embodiment 4 of the present invention.

Fig. 9 is a schematic structural diagram showing a first metal layer patterned in step B3 of the method for preparing a space histology detection tool in embodiment 4 of the present invention.

Fig. 10 is a schematic structural diagram showing the process for preparing a space histology detection tool according to embodiment 4 of the present invention, wherein the process is performed in step B4 by coating the second oxide layer.

Fig. 11 is a schematic structural diagram showing the arrangement of the second metal layer in the step B5 of the preparation method of the space histology detection tool in embodiment 4 of the present invention.

Fig. 12 is a schematic structural diagram showing the arrangement of the third metal layer in the step B6 of the preparation method of the space histology detection tool in embodiment 4 of the present invention.

Fig. 13 is a schematic structural diagram showing a method for preparing a space histology detection tool according to embodiment 4 of the present invention, wherein the third metal layer is patterned in step B7.

Description of element reference numerals

110. A microfluidic liquid exchange system; 111, liquid exchange micro channel, 112, label nucleic acid sequence, 113, label structure, 120, microelectrode array, 121, detection site electrode, 131, connection wire, 132, external bonding pad, 140, control system, 210, capacitor, 211, first electrode, 212, second electrode, 213, insulating medium, 214, silicon dioxide layer, 215, polysilicon region, 220, field effect transistor, 221, drain, 222, grid, 223, source, 224, channel region, 231, row control line, 232, column control line, 233, grounding line, 234, row addressing integrated circuit, 235, column addressing integrated circuit, 310, substrate, 311, first oxide layer, 312, second oxide layer, 321, first metal layer, 322, second metal layer, 323, third metal layer, 324, titanium layer, 325, platinum layer.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

As described in detail in the embodiments of the present invention, the schematic drawings showing the structure of the apparatus are not partially enlarged to general scale, and the schematic drawings are merely examples, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in actual fabrication.

For ease of description, spatially relative terms such as "under", "below", "beneath", "above", "upper" and the like may be used herein to describe one element or feature's relationship to another element or feature as illustrated in the figures. It will be understood that these spatially relative terms are intended to encompass other orientations of the device in use or operation in addition to the orientation depicted in the figures.

In the context of the present application, a structure described as a first feature being "on" a second feature may include embodiments where the first and second features are formed in direct contact, as well as embodiments where additional features are formed between the first and second features, such that the first and second features may not be in direct contact.

It should be noted that, the illustrations provided in the present embodiment merely illustrate the basic concept of the present invention by way of illustration, and only the components related to the present invention are shown in the drawings rather than the number, shape and size of the components in actual implementation, and the form, number and proportion of each component in actual implementation may be arbitrarily changed, and the layout of the components may be more complex.

Example 1:

the embodiment provides a space histology detection tool, as shown in fig. 1, which comprises a microfluidic liquid exchange system 110, a microelectrode array 120, a site electrification structure and a control system 140;

The microfluidic liquid exchange system 110 comprises a liquid exchange micro-channel 111 with controllable on-off state, wherein the micro-electrode array 120 comprises n detection site electrodes 121 which are arranged in an array, n is an integer greater than or equal to 1, the liquid exchange micro-channel 111 is communicated with each detection site electrode 121 so as to introduce label raw materials into the detection site electrodes 121 from the liquid exchange micro-channel 111, the detection site electrodes 121 are used for carrying out electrochemical synthesis on label nucleic acid sequences 112 (shown in fig. 6) of the label raw materials introduced into the detection site electrodes 121 after being electrified, and the label nucleic acid sequences 112 are used for capturing DNA and preparing libraries in space histology technology;

The site electrification structure is electrically connected with each detection site electrode 121, the control system 140 is electrically connected with the site electrification structure, and the control system 140 controls the electrifying state of each detection site electrode 121 through the site electrification structure;

the microfluidic liquid exchange system 110, the microelectrode array 120 and the site electrification structure are MEMS structures.

In the prior art, the spatial information acquisition modes of the spatial transcriptome and genomics technology can be divided into modes of micro-cutting, imaging, spatial bar code and the like. The method for acquiring spatial information through imaging comprises in situ hybridization (such as seqFISH, merFISH and the like) and in situ sequencing (STARMap, barseq and the like), wherein the two methods acquire sequence and position information through in situ complementary hybridization between a fluorescent probe and a gene or transcript and acquisition of images, but because of spectral bandwidth limitation, multiple hybridization operations are needed, the operation is relatively complex, the image data volume is huge, the efficiency is low, and an expensive high-resolution microscope and molecular probes are needed, compared with the spatial barcode technology which does not rely on imaging any more, but captures the gene/transcript through a nucleotide coding array and acquires the sequence and position information through the NGS sequencing technology, the equipment cost can be reduced. Current spatial bar code technology is through inkjet printingThe spatial bar code array is formed by methods such as a microbead array (HDST, slide-seq), a microfluidic auxiliary (DBiT-seq, decoder-seq) and the like, the spatial resolution precision is reduced from 55 mu m to be close to a single cell level, but the spatial resolution precision of the spatial histology analysis is limited by the structural density limitation of structures such as ink jet, microbeads and the like. The resolution of the spatial histology analysis was advanced to the submicron level by the Stereo-seq reported by the national institute of large genes in 2022, however, both the equipment cost and the operational complexity are increased. Meanwhile, since the prior art is generally used for detection by using conventional and conventional tools such as a microscope and a probe, the prior art does not use MEMS structure and process for space histology analysis and detection.

The invention adopts a cross-field technical scheme, breaks through the technical bias and blind spot of detection tools in the traditional space histology analysis, obtains the detection site electrode 121 capable of capturing DNA by electrochemically synthesizing the tag nucleic acid sequence 112 by adopting the MEMS device as the space histology detection tool, greatly improves the density of the detection site electrode 121 by utilizing the high integration degree which can be realized by utilizing the maturation process of the MEMS and is far superior to the site density of the space bar code formed by ink-jet, microbeads and the like in the prior art, reduces the space resolution precision of the space histology detection to the sub-single cell level, can adapt to the space histology analysis of tissue slices with larger area, realizes the breakthrough in the space histology technical field, greatly reduces the equipment cost for carrying out the space histology detection by adopting the MEMS device and the like in the prior art, and simultaneously improves the sequencing flux which can be realized by adopting the MEMS device as the space histology detection tool, can realize the processing and marking of DNA by directly electrifying and introducing biochemical reaction solvents, has simple operation and is beneficial to the space histology detection efficiency.

In particular, the spatial resolution accuracy of a spatial histology detection also refers to the spatial resolution, i.e. the smallest dimension that can be resolved in a spatial histology detection.

Specifically, each detection site electrode 121 comprises a circular wall and a solid circular protrusion, the solid circular protrusion is located in the inner diameter of the circular wall, a preset distance exists between the inner diameter of the circular wall and the circumference of the solid circle, a circular groove formed by the preset distance is used for electrochemically synthesizing the tag nucleic acid sequence 112, when the tag nucleic acid sequence 112 is electrochemically synthesized, the solid circular protrusion is used as an anode, the circular wall is used as a cathode, hydrogen ions are generated near the solid circular protrusion after the circular protrusion is electrified and used for synthesizing the tag nucleic acid sequence 112, the generated hydrogen ions are absorbed near the circular wall, and the movement of the hydrogen ions generated in one detection site electrode 121 to the adjacent detection site electrode 121 is avoided, so that the synthesis of the tag nucleic acid sequences 112 in other detection site electrodes 121 is influenced.

In one embodiment, the distance between the inner diameter and the outer diameter of the annular wall is 500 nanometers and the outer diameter of the solid circular protrusion is 1 micrometer.

In this embodiment, the space histology detection tool includes n site-powered structures, each site-powered structure includes a connection wire 131 and an external pad 132 that are electrically connected, the external pad 132 is electrically connected to the control system 140, each detection site electrode 121 is electrically connected to the connection wire 131 of one site-powered structure, and the control system 140 controls the power-on state of each detection site electrode 121 through the connection wire 131 and the external pad 132.

In the invention, the control of the electrified state of the detection site electrode 121 is directly realized through the connecting wire 131 and the external bonding pad 132, so that the cost of integrated circuit streaming is further reduced, and the space group science detection cost is reduced.

In one embodiment, the space histology detection tool further comprises a probe card, wherein the probe card is used for packaging the microelectrode array 120 and the site power-on structure, the probe card comprises n probes and a PCB circuit, one end of each probe is electrically connected with the external bonding pad 132 corresponding to one detection site electrode 121, the other end of each probe is electrically connected with the external bonding pad 132 through the PCB circuit to lead out the electrical connection of the external bonding pad 132 to a packaging pin on the outer surface, and the control system 140 is electrically connected with the packaging pin to control the power-on state of the detection site electrode 121.

The invention omits the wiring requirement on the integrated circuit by using the detachable probe card as the packaging structure of the space group science detection tool, improves the packaging efficiency, and simultaneously, as the probe card can be directly detached to package the new microelectrode array 120 and the site electrification structure, the replacement of the integrated circuit inside the space group science detection tool can be facilitated, the maintenance and replacement cost is further reduced, and the maintenance efficiency is improved.

In one embodiment, the control system 140 is a single chip microcomputer or FPGA (FieldProgrammable GATE ARRAY ), and the package pins are electrically connected to the control system 140 through dupont wires.

In one embodiment, the control system 140 may be configured to electrically connect to all the detection site electrodes 121 by using a single power source including only a controllable switch, which has a simple structure, but cannot perform a single targeted control on the detection site electrodes 121, and may be selected according to application requirements.

In one embodiment, n is 5000 or more.

According to the invention, the space histology detection tool is realized by using the MEMS structure, more than 5000 detection site electrodes 121 can be arranged on the same space histology detection tool, so that the space resolution precision of space histology detection is greatly improved, and the space histology analysis requirement of a tissue slice with a larger area can be met.

In one embodiment, the distance between two adjacent detection site electrodes 121 is 10 micrometers or less, or the distance between two adjacent detection site electrodes 121 is 5 micrometers or less.

The invention obtains the space histology detection tool by using the MEMS structure and the MEMS process, and can realize the integration density of the detection site electrode 121 with the distance less than or equal to 10 microns, even less than or equal to 5 microns, thereby greatly improving the space resolution precision of the space histology detection.

Example 2:

this embodiment provides a space histology detection tool that is substantially identical to the other features of embodiment 1, except that:

In this embodiment, as shown in fig. 2-5, fig. 2 is a schematic perspective structure diagram of the space group chemical detection tool, fig. 3 is a schematic structure diagram of the site power-on structure, fig. 4 is a schematic electrical connection structure diagram of a single fet 220 in the M part of fig. 3, and fig. 5 is a side cross-sectional view of a semiconductor structure of each site power-on structure, wherein the microelectrode array 120 and the site power-on structure are MEMS integrated circuits, and the site power-on structure includes n capacitors 210, n identical fets 220 arranged in an a-row b-column array, a-row control lines 231, b-column control lines 232, and n external pads 132, a, b are integers greater than or equal to 1;

Each gate 222 in each row of the field effect transistors 220 is commonly connected with a row control line 231, each source 223 in each column of the field effect transistors 220 is commonly connected with a column control line 232, and one end of each row control line 231 and one end of each column control line 232, which are not connected with the field effect transistor 220, are electrically connected with 1 external bonding pad 132;

Each capacitor 210 is connected with 1 corresponding field effect transistor 220 and 1 detection site electrode 121, each capacitor 210 comprises a first electrode 211, a second electrode 212 and an insulating medium 213, the first electrode 211 is electrically connected with the corresponding detection site electrode 121, the second electrode 212 is electrically connected with a drain 221 of the corresponding field effect transistor 220, the insulating medium 213 is located between the first electrode 211 and the second electrode 212, and the control system 140 controls the energizing state of the corresponding detection site electrode 121 by controlling input electric signals of a row control line 231 and a column control line 232 of the field effect transistor 220.

The micro-electrode array 120 and the site electrification structure are prepared through the MEMS integrated circuit in an integrated way through the structures of the field effect transistor 220 and the capacitor 210, the number of the required external bonding pads 132 is greatly reduced, the packaging difficulty is reduced, the density of the detection site electrode 121 is further improved, and the spatial resolution precision of space histology detection can be further improved, but meanwhile, compared with the mode of directly connecting the micro-electrode array 120 with the external bonding pads 132 through the connecting wire 131 in the embodiment 1 by adopting the MEMS integrated circuit integrated micro-electrode array 120 and the site electrification structure, the preparation cost is higher, and the technical personnel can select in two schemes according to the requirements of cost and spatial resolution precision, but the scheme cost in the embodiment is far lower than the cost required under the condition of realizing the same spatial resolution precision in the prior art as a whole.

Specifically, as shown in fig. 5, between the source 223 and the drain 221 is a channel region 224 of the fet 220.

In one embodiment, as shown in FIGS. 2-3, row control line 231 is coupled to row address integrated circuit 234 and column control line 232 is coupled to column address integrated circuit 235. The row addressing integrated circuit 234 and the column addressing integrated circuit 235 can be connected with the external bonding pad 132 through the row control line 231 and the column control line 232 and then controlled by the control system 140 through the external bonding pad 132, the row addressing integrated circuit 234 and the column addressing integrated circuit 235 can also be connected with the row control line 231 and the column control line 232 through being connected with the external bonding pad 132, and the row addressing integrated circuit 234 and the column addressing integrated circuit 235 can be placed in the control system 140 so as to further reduce the line density required to be set in the MEMS integrated circuit formed by the microelectrode array 120 and the site power-on structure, and can only replace the MEMS integrated circuit when the subsequent problems occur without replacing the control system 140, thereby reducing the maintenance cost.

Specifically, when the row control line 231 and the column control line 232 to which one detection site electrode 121 is connected are both selected, the detection site electrode 121 is energized, otherwise, in a power-off state.

In one embodiment, as shown in fig. 5, the first electrode 211 is a polysilicon region 215 at a predetermined position around the drain 221 correspondingly connected to the capacitor 210, the second electrode 212 is the drain 221 correspondingly connected to the capacitor 210, and the insulating medium 213 is a silicon dioxide layer 214 between the first electrode 211 and the second electrode 212.

The invention uses the drain electrode 221 of the field effect transistor 220 as the second electrode 212 of the capacitor 210, and realizes the structure of the capacitor 210 through the polysilicon and the silicon dioxide layer 214 outside the polysilicon in the conventional semiconductor structure, thereby directly preparing the capacitor by using the mature process and equipment in the existing MEMS integrated circuit process without special custom materials or tools, further improving the possibility of the production popularization and use of the space histology detection tool and reducing the preparation cost.

In one embodiment, as shown in fig. 4, the fet 220 is an NMOS transistor.

Specifically, the field effect tube 220 may be a PMOS tube or other types of field effect tubes 220, the specific connection structure of which may be adjusted according to the requirements, but the NMOS tube as the field effect tube 220 realizes the control of the power on state of the detection site electrode 121, and the characteristics of low power consumption and high reaction speed of the space histology detection tool may be realized by using a smaller on-resistance and a faster switching speed, so as to improve the use efficiency of the space histology detection tool.

Specifically, the field effect transistor 220 may be replaced by other suitable semiconductor switch structures, so as to realize the power-on control of the detection site electrode 121, which is within the protection scope of the present invention.

Preferably, when the field effect transistor 220 is an NMOS transistor, the source 223 and the drain 221 are heavily doped in N type, the substrate 310 is a P type silicon substrate, and the polysilicon region 215 is electrically connected to the ground line 233, so as to achieve that the electric potentials of the drains 221 of all the field effect transistors 220 are the same, and ensure the reliability of controlling the energizing state of the detection site electrode 121.

Example 3:

the present embodiment provides a method for detecting a space histology, which is performed using the space histology detection tool of any one of embodiments 1-2.

The method for detecting the space histology of the present invention will be described in detail with reference to the accompanying drawings, wherein it should be noted that the above sequence does not strictly represent the sequence of the method for detecting the space histology of the present invention, and those skilled in the art can vary depending on the actual preparation steps.

Firstly, step A1 is performed, the liquid exchange micro-channel 111 in the micro-fluidic liquid exchange system 110 is controlled to feed label raw materials to the detection site electrode 121 in the micro-electrode array 120, and the detection site electrode 121 fed with the label raw materials is controlled to be electrified by the control system 140 so as to electrochemically synthesize the label raw materials of the detection site electrode 121 into the label nucleic acid sequence 112 shown in FIG. 6.

Specifically, as shown in fig. 6, each tag nucleic acid sequence 112 includes a plurality of different tag structures 113, only one tag nucleic acid sequence 112 is shown to include a plurality of tag structures 113, and in fact each tag nucleic acid sequence 112 includes a plurality of different tag structures 113.

Then, step A2 is performed, the detection site electrode 121 is controlled to be powered off by the control system 140, and the tissue slice to be detected is transferred to the detection site electrode 121 after the cell membrane of the tissue slice to be detected is perforated.

Specifically, by performing cell membrane perforation on the tissue slice to be detected, the tissue slice to be detected can be transferred to the detection site electrode 121, and then the internal DNA can be smoothly discharged for processing, so that the detection efficiency is improved, and the reliability of the detection result can be ensured.

Next, step A3 is performed to unbind and cut DNA from the tissue slice to be detected transferred to the detection site electrode 121 to obtain a plurality of DNA fragments, so that each DNA fragment obtained after cutting is captured by the tag nucleic acid sequence 112 in the corresponding detection site electrode 121.

Specifically, the nucleic acid transferred to the detection site electrode 121 of the tissue slice to be detected is captured by the tag nucleic acid sequence 112 on the detection site electrode 121 at the corresponding position in a hybridization manner.

In one embodiment, the DNA is solubilized and unbound by a chromosomal histone. In particular, other suitable methods may also be used to unbind the DNA.

In one embodiment, the DNA is cleaved into a plurality of DNA fragments by Tn5 transposase cleavage of the DNA. Specifically, other suitable methods for cleaving DNA may also be used.

Then, step A4 is performed, wherein the tag nucleic acid sequence 112 of the DNA fragment captured by the detection site electrode 121 is subjected to library preparation so that the DNA fragment captured by the tag nucleic acid sequence 112 replicates the tag structure 113 of the corresponding tag nucleic acid sequence 112, and the DNA fragment with the tag structure 113 is collected and processed into a structure that can be sequenced, thereby obtaining a transcript.

Specifically, the number of the tag nucleic acid sequences 112 included in each detection site electrode 121 is set to be greater than the number of the DNA fragments after unbinding and cutting, so as to ensure that enough tag nucleic acid sequences 112 can capture the DNA fragments obtained after unbinding and cutting in one-to-one manner in each detection site electrode 121.

In one embodiment, 10 ⁷-10¹¹ tag nucleic acid sequences 112 are provided within each of the detection site electrodes 121.

Finally, step A5 is carried out, and high-throughput sequencing is carried out on the DNA fragment with the tag structure 113 in the transcript obtained by library preparation and collection treatment, so that the spatial position information of the DNA fragment in the tissue section to be detected is obtained.

In one embodiment, the high throughput sequencing is a second generation sequencing technique or a third generation sequencing technique. In particular, the scheme of the invention can also be applied to the first generation sequencing technology, but the first generation sequencing technology is not described because the effect of improving the spatial resolution accuracy of the invention cannot be fully reflected in the first generation sequencing technology.

The invention is applied to space histology technology by using MEMS structure and technology to obtain space histology detection tool in cross field, so that space bar code technology in space histology analysis is not dependent on imaging any more, but a nucleotide coding array (a labeled nucleic acid sequence 112 on a microelectrode array 120) captures genes (DNA on a tissue slice to be detected) to obtain transcripts, and then an NGS sequencing technology is combined to obtain sequence and position information of the DNA on the tissue slice to be detected.

Example 4:

This example provides a method for preparing a space histology detection tool for preparing the space histology detection tool of example 1, comprising:

step B1, providing a substrate 310 with a first oxide layer 311 on the upper surface;

step B2, disposing a first metal layer 321 on the first oxide layer 311;

step B3, patterning the first metal layer 321;

Step B4, covering the second oxide layer 312 on the gaps and the upper surface of the patterned first metal layers 321;

Step B5, patterning the second oxide layer 312 above the patterned first metal layer 321 to expose a part of the first metal layer 321 below, and disposing a second metal layer 322 in a gap between the patterned second oxide layers 312, wherein the second metal layer 322 and the first metal layer 321 below form corresponding electrical connection;

Step B6, disposing a third metal layer 323 on the surface of the second metal layer 322, where the third metal layer 323 covers the second metal layer 322 and the second oxide layer 312 with exposed surfaces;

And B7, patterning the third metal layer 323 to obtain a microelectrode array 120 and an external bonding pad 132 which are electrically connected with the second metal layer 322, wherein the microelectrode array 120 comprises n detection site electrodes 121 which are arranged in an array, each detection site electrode 121 is electrically connected with the first metal layer 321 and a corresponding external bonding pad 132 through the second metal layer 322 below, the detection site electrodes 121 are used for carrying out electrochemical synthesis on a label raw material which is introduced into the detection site electrodes 121 after being electrified through the external bonding pad 132, and the label nucleic acid sequence 112 is used for capturing DNA and preparing a library in a space histology technology.

The method for preparing the space histology detection tool of the present invention will be described in detail with reference to the accompanying drawings, wherein, the above-mentioned sequence is not strictly representative of the sequence of the preparation method of the space histology detection tool protected by the present invention, and the person skilled in the art can vary depending on the actual preparation steps.

First, as shown in fig. 7, step B1 is performed to provide a substrate 310 having a first oxide layer 311 provided on the upper surface.

In one embodiment, the substrate 310 is a silicon wafer.

In one embodiment, the first oxide layer 311 is a thermal oxide layer.

In one embodiment, the substrate 310 having the first oxide layer 311 is a6 inch silicon oxide wafer, wherein the first oxide layer 311 is silicon dioxide, and the thickness of the silicon dioxide is 1 micron or more.

Before step B2, the substrate 310 provided with the first oxide layer 311 is subjected to organic ultrasonic cleaning, and is subjected to hot baking at 120 ℃ for 5 minutes, so that the first oxide layer 311 and the substrate 310 are pretreated, and the surface is clean and pollution-free.

Then, as shown in fig. 8, step B2 is performed, and a first metal layer 321 is provided on the first oxide layer 311.

In one embodiment, the material of the first metal layer 321 may be gold, platinum, aluminum, or other suitable material.

In one embodiment, the first metal layer 321 is a titanium/gold composite layer, wherein the thickness of the titanium layer is 20 nm and the thickness of the gold layer is 200 nm.

In one embodiment, the first metal layer 321 is provided by a magnetron sputtering method.

Next, as shown in fig. 9, step B3 is performed to pattern the first metal layer 321.

In one embodiment, photoresist is disposed on the first metal layer 321, developing exposure is performed on the photoresist on the first metal layer 321 through a 6-inch step (stepper) photomask and the photoresist to expose a part of the first metal layer 321 under the photoresist, IBE (Ion beam etching) is performed on the exposed first metal layer 321 to realize patterning of the first metal layer 321, line width and line spacing of the photomask are all 500 nanometers, and after patterning of the first metal layer 321, NMP (N-methylpyrrolidone) is used to remove the residual photoresist.

Then, as shown in fig. 10, step B4 is performed to cover the second oxide layer 312 on the gaps and the upper surface of the patterned first metal layers 321.

In one embodiment, the first oxide layer 311 and the second oxide layer 312 are made of the same material, so as to ensure adhesion between the two oxide layers, so as to ensure reliability of the semiconductor structure.

In one embodiment, the second oxide layer 312 is provided by PECVD (Plasma-ENHANCED CHEMICAL Vapour Deposition, plasma enhanced chemical vapor deposition).

In one embodiment, the second oxide layer 312 has a thickness of 500 nanometers.

Next, as shown in fig. 11, step B5 is performed to pattern the second oxide layer 312 above the patterned first metal layer 321 to expose a portion of the first metal layer 321 below, and a second metal layer 322 is provided to a gap between the patterned second oxide layers 312, wherein the second metal layer 322 and the first metal layer 321 below form a corresponding electrical connection.

In one embodiment, photoresist is disposed on the second oxide layer 312, the photoresist on the second oxide layer 312 is exposed and developed by a 6-inch step (stepper) photomask, a portion of the second oxide layer 312 under the photoresist is exposed, RIE (Reactive Ion Etching ) is performed on the exposed second oxide layer 312, patterning of the second oxide layer 312 is achieved, the linewidth and line spacing of the photomask are all 500 nm, the alignment error of the photomask is less than 150 nm, and NMP (N-methylpyrrolidone) is used to remove the remaining photoresist.

In one embodiment, after patterning the second oxide layer 312, a second metal layer 322 is disposed on the second oxide layer 312, the second metal layer 322 covers the second oxide layer 312 and the exposed surface of the first metal layer 321, photoresist is disposed on the second metal layer 322, the photoresist on the second metal layer 322 is exposed and developed with a photolithography plate to expose a portion of the second metal layer 322 under the photoresist, IBE (Ion beam etching) is performed on the exposed second metal layer 322 to obtain the second metal layer 322 between the gaps of the second oxide layer 312, and NMP (N-methylpyrrolidone) is used to remove the remaining photoresist.

Specifically, the same photolithography mask may be used in patterning the second oxide layer 312 and the second metal layer 322.

In one embodiment, the material of the second metal layer 322 may be gold, platinum, aluminum, or other suitable material.

In one embodiment, the second metal layer 322 further includes a titanium layer, where the titanium layer contacts the first metal layer 321 to achieve better adhesion and improve the reliability of the structure.

In one embodiment, the titanium layer has a thickness of 20 nanometers and the metal layer on top of the titanium layer has a thickness of 200 nanometers.

Then, as shown in fig. 12, step B6 is performed, a third metal layer 323 is disposed on the surface of the second metal layer 322, and the third metal layer 323 covers the second metal layer 322 and the second oxide layer 312 exposed on the surface.

In one embodiment, the material of the third metal layer 323 is platinum, and the platinum is located on the upper surface of the third metal layer 323, so as to ensure that when the detection site electrode 121 performs electrochemical synthesis of the tag nucleic acid sequence 112, the detection site electrode 121 itself is not corroded by the synthesis solution, so as to ensure the reliability and the service life of the space histology detection tool, and ensure the reliability of the detection result.

In one embodiment, as shown in fig. 12, the material of the third metal layer 323 is a composite layer formed by a titanium layer 324/a platinum layer 325, where the titanium layer 324 contacts with the underlying second metal layer 322 to achieve a better adhesion effect and improve the reliability of the structure.

In one embodiment, the thickness of the titanium layer 324 in the third metal layer 323 is 35 nm, and the thickness of the platinum layer 325 is 200 nm.

Finally, as shown in fig. 13, step B7 is performed to pattern the third metal layer 323, so as to obtain a microelectrode array 120 and an external bonding pad 132 electrically connected with the second metal layer 322, where the microelectrode array 120 includes n detection site electrodes 121 arranged in an array, each detection site electrode 121 is electrically connected with the first metal layer 321 and a corresponding external bonding pad 132 through the second metal layer 322 below, the detection site electrodes 121 are used to perform electrochemical synthesis on the label raw material introduced into the detection site electrodes 121 after the external bonding pad 132 is electrified, and the label nucleic acid sequence 112 is used for capturing DNA and preparing a library in space histology technology.

The invention adopts the first metal layer 321 and the second metal layer 322 below the detection site electrode 121 and the external bonding pad 132 to carry out electric connection, thereby avoiding overlarge occupied area caused by too many single-layer metal layers, further improving the density of the detection site electrode 121 which can be realized by a single space histology detection tool and further improving the spatial resolution precision which can be realized.

In one embodiment, a photoresist is disposed on the second metal layer 322 before disposing the third metal layer 323, exposing and developing the photoresist on the second metal layer 322 by a 6 inch step (stepper) photomask to expose a portion of the second metal layer 322 under the photoresist, and performing metal sputtering on the patterned photoresist to obtain a third metal layer 323, wherein the third metal layer 323 fills the gaps of the photoresist and covers the exposed surface of the second metal layer 322, and removing the remaining photoresist while performing metal Lift-Off (Lift-Off) on the third metal layer 323 covered on the photoresist to leave the patterned third metal layer 323.

In one embodiment, after the step B7 is performed, the microfluidic liquid-exchanging system 110 is disposed on the microelectrode array 120, where the microfluidic liquid-exchanging system 110 includes a liquid-exchanging micro-channel 111 with controllable on-off state, and the liquid-exchanging micro-channel 111 is communicated with each detection site electrode 121 in the microelectrode array 120, so that a label raw material is introduced from the liquid-exchanging micro-channel 111 to the detection site electrodes 121. Specifically, the microfluidic liquid exchange system 110 may be configured between other suitable steps of the preparation, which are all within the scope of the present invention.

In summary, the space histology detection tool, the preparation method and the space histology detection method thereof can obtain the detection site electrode capable of capturing DNA by electrochemically synthesizing the tag nucleic acid sequence through adopting the MEMS device, so that the density of the detection site electrode is greatly improved, the space resolution precision of the space histology detection is reduced to a sub-single cell level, the space histology analysis of a tissue slice with larger area can be adapted, the equipment cost for the space histology detection is greatly reduced, the efficiency of the space histology detection is improved, meanwhile, the microelectrode array and the site electrification structure are integrated and prepared through the structures of the field effect transistor and the capacitor through the MEMS integrated circuit, the number of required conductive pads is reduced, the packaging difficulty is reduced, the further improvement of the density of the detection site electrode is facilitated, in addition, the density of the detection site electrode is further improved through electrically connecting the detection site electrode and the external pad from the bottom metal, the resolution of the space histology detection is improved, and finally the replacement efficiency of the space histology detection tool is improved through arranging the detachable probe card as a packaging structure.

Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. The space histology detection tool is characterized by comprising a microfluidic liquid exchange system, a microelectrode array, a site electrification structure and a control system;

2. The tool of claim 1, wherein the tool comprises n site-powered structures, each site-powered structure comprises a connecting wire electrically connected with an external bonding pad, the external bonding pad is electrically connected with the control system, each site electrode is electrically connected with the connecting wire of one site-powered structure, and the control system controls the energizing state of each site electrode through the connecting wire and the external bonding pad.

3. The space histology detection tool of claim 1, wherein the microelectrode array and the site-powered structure are MEMS integrated circuits, the site-powered structure comprises n capacitors, n identical field effect transistors arranged in an a-row b-column array, a-row control lines, b-column control lines and n external bonding pads, and a and b are integers greater than or equal to 1;

4. A tool for spatial histology according to claim 3, wherein the first electrode is a polysilicon region at a predetermined position around the drain to which the capacitor is correspondingly connected, the second electrode is the drain to which the capacitor is correspondingly connected, and the insulating medium is a silicon dioxide layer between the first electrode and the second electrode.

5. A tool for spatial histology according to claim 3, wherein the field effect transistor is an NMOS transistor.

6. The tool of any one of claims 1-5, further comprising a probe card for encapsulating the array of microelectrodes and the site-powered structure, the probe card comprising n probes and PCB circuitry, one end of each probe being electrically connected to the external bond pad corresponding to one of the site-tested electrodes, the other end of each probe being electrically connected to an encapsulation pin header on an external surface through the PCB circuitry, the control system being electrically connected to the encapsulation pin header for controlling the powered state of the site-tested electrodes.

7. The tool of any one of claims 1-5, wherein n is 5000 or more.

8. The tool of any one of claims 1-5, wherein a pitch between two adjacent ones of the detection site electrodes is 10 microns or less, or a pitch between two adjacent ones of the detection site electrodes is 5 microns or less.

9. A method of spatial histology, the method being performed using the spatial histology detection tool of any one of claims 1-8, the method comprising:

10. A method of preparing a space histology detection tool, the method for preparing the space histology detection tool of claim 2, the method comprising:

providing a substrate with a first oxide layer on the upper surface;

a first metal layer is arranged on the first oxide layer;

patterning the first metal layer;