Liquid drop positioning printing method and single cell analysis platform

Abstract

The invention provides a liquid drop positioning printing method and a single cell analysis platform, and the method is used for calibrating the position of a nozzle before liquid drop printing, and comprises the following steps: pre-printing a plurality of liquid drops on a base material by using at least two specified nozzles, and obtaining the position of a pre-printed liquid drop point; and correcting the theoretical value coordinate of the nozzle position based on the drop point position to obtain a correction value coordinate of the nozzle position. According to different types of printing heads, high-efficiency and high-precision liquid drop arrangement operation can be completed under different liquid and different printing conditions, so that high-throughput and high-accuracy single cell operation can be completed by utilizing the single cell sorting device. The single cell analysis platform comprises a control assembly, a single cell sorting and printing device, a dielectric wetting chip and an optical module, and can realize pretreatment of single cell capture, fixation, dyeing and the like on a micro platform at a micron-to-millimeter level, so as to realize high-throughput analysis of cell morphology, genomics, transcriptomics and proteomics.

Description

Technical Field

[0001] The invention belongs to the field of microfluidics and biotechnology, and relates to a droplet positioning printing method and a single cell analysis platform. Background Art

[0002] With the development of microfluidics technology, biological droplets are gradually becoming carriers and tools for cell analysis, especially for single-cell application research. Digital chips and digital microfluidics chips change the wettability of the droplet interface through electrodes, can individually control tiny droplets, and can merge, mix, separate and distribute droplets.

[0003] In single-cell research, digital chips and digital microfluidics can realize the transport of single-cell droplets, assist in cell lysis and a series of biological reactions, and provide more possibilities for multi-dimensional analysis of single cells. However, the efficiency of single-cell droplet introduction of digital chips and digital microfluidics chips is often low.

[0004] Among the current mainstream single-cell sorting methods, flow cytometers rely on electric field deflection for single-cell sorting. Other printing methods, such as microfluidic chips, mostly print targets for 96-well plates (hole diameter is about 6.3mm) and 384-well plates (hole diameter is about 3mm). Printing and electric field deflection plus open-loop motion can meet the millimeter-level accuracy requirements of existing equipment. Therefore, these devices do not have software processes and hardware for droplet landing point calibration. Therefore, when the accuracy requirements are increased, there is a problem that single cells cannot be accurately positioned, for example, the accuracy of less than 0.1mm cannot be met. Digital chips and digital microfluidic chips are smaller in size, and the volume of the controlled droplets is at the picoliter (pL) level (the size is approximately at the micrometer (μm) level). General sorters cannot meet their precise and high-throughput cell introduction requirements.

[0005] Therefore, how to provide a droplet positioning printing method and a single-cell analysis platform to achieve precise positioning and control of single cells based on single-cell printing chips has become an important technical problem that needs to be urgently solved by technical personnel in this field.

[0006] It should be noted that the above introduction to the technical background is only for the convenience of providing a clear and complete description of the technical solutions of the present application and for the convenience of understanding by those skilled in the art. It cannot be considered that the above technical solutions are well known to those skilled in the art simply because they are described in the background technology section of the present application. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a droplet positioning printing method and a single cell analysis platform to solve the problems in the prior art such as the inability to accurately control droplet printing and the low droplet introduction efficiency.

[0008] To achieve the above-mentioned object and other related objects, the present invention provides a droplet positioning printing method, comprising the following steps:

[0009] Solution loading step: applying the solution into multiple nozzles of the print head;

[0010] Nozzle position calibration step: pre-printing a plurality of droplets on a substrate using at least two designated nozzles of the print head, obtaining the landing positions of the plurality of droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position;

[0011] Droplet printing step: obtaining a target printing position, and moving the print head to the target printing position to perform droplet printing.

[0012] Optionally, the solution includes a cell suspension, and the print head is used for single cell printing.

[0013] Optionally, the multiple nozzles of the print head are arranged into at least two columns, each column includes a plurality of nozzles arranged at intervals, and in the nozzle position calibration step, a first designated nozzle and a second designated nozzle are used to print multiple droplets on the substrate, and the first designated nozzle and the second designated nozzle are located in the same column.

[0014] Optionally, the first designated nozzle and the second designated nozzle are located at two ends of a column respectively.

[0015] Optionally, the nozzle position calibration step includes:

[0016] Printing a first droplet using the first designated nozzle and printing a second droplet using the second designated nozzle;

[0017] Move the print head along the X-axis by a preset distance, and use the first designated nozzle to print a third droplet;

[0018] Collecting an image of the droplet landing point, measuring an angle A between a line connecting the first droplet and the second droplet and a selected substrate reference line based on the image, and measuring an angle B between a line connecting the first droplet and the third droplet and the substrate reference line;

[0019] Calculate the angle C between the Y axis of the mechanical motion of the print head and the base line based on the angle B;

[0020] Based on the angle A and the angle C, the theoretical value coordinates of each nozzle position are corrected.

[0021] Optionally, the print head mechanical movement Y axis and the print head mechanical movement X axis are perpendicular to each other, C=90-B.

[0022] Optionally, the correction of the theoretical coordinates of each nozzle position based on the angle A includes: calculating the offset between the actual nozzle position and the theoretical position, wherein the absolute value S of the X-axis offset is X =L·sinA, the absolute value of the Y-axis offset S Y =L·(1-cosA), L is the straight-line distance between the corresponding nozzle and the preset nozzle, and L is an integer multiple of the nozzle spacing in the column direction.

[0023] Optionally, correcting the theoretical coordinates of each nozzle position based on the angle C includes: rotating the substrate platform by the angle C in a clockwise or counterclockwise direction to make the substrate platform coordinate system consistent with the print head mechanical motion coordinate system.

[0024] Optionally, in the nozzle position calibration step, a theoretical value coordinate matrix of the nozzle position of a print head of a corresponding model stored in a control component is acquired based on the model of the print head, and a correction value coordinate matrix of the nozzle position is obtained after correction.

[0025] The present invention also provides a single-cell analysis platform, which includes a control component and a single-cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The single-cell analysis platform is used to execute the droplet positioning printing method described in any one of the above items to introduce single cells into the designated position of the dielectric wetting chip through the single-cell sorting and printing device for analysis and / or cultivation.

[0026] Optionally, the single cell sorting and printing device comprises a thermal bubble printing chip.

[0027] Optionally, the analysis comprises single-cell transcriptome analysis or single-cell sequencing analysis.

[0028] As described above, the droplet positioning printing method of the present invention first calibrates the nozzle position before performing droplet printing. The calibration step includes using at least two designated nozzles of the print head to pre-print multiple droplets on the substrate, obtaining the landing positions of the multiple droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position. Using the droplet positioning printing method of the present invention, high-efficiency and high-precision droplet arrangement operations can be completed for different types of print heads, under different liquids and different printing conditions. In the single-cell positioning printing test, the deviation between the landing point of the single-cell droplet printed after the nozzle position is calibrated and the target is less than ±50μm, so that high-throughput and high-accuracy single-cell operations can be completed using a single-cell sorting device. The single-cell analysis platform of the present invention includes a control component and a single-cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The platform adopts the above-mentioned droplet positioning printing method, and can complete high-throughput and high-accuracy single-cell operations through the single-cell sorting and printing device, and realize single-cell capture, fixation, staining and other pretreatments on a tiny platform at the micron to millimeter level, thereby realizing the construction of a micro high-throughput analysis platform for cell morphology, genomics, transcriptomics and proteomics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is a flow chart of the droplet positioning printing method of the present invention.

[0030] Figure 2 Shown is a flow chart for single-cell printing using the droplet positioning printing method of the present invention.

[0031] Figure 3 Shown is a schematic diagram of the nozzle arrangement of a high-throughput single-cell printhead.

[0032] Figure 4 Shown is a schematic diagram of an image captured after pre-printing three droplets.

[0033] Figure 5 Shown is a schematic diagram of measurements based on the image.

[0034] Figure 6 Displayed as the coordinate change of point P after the coordinate system is rotated two-dimensionally by an angle θ.

[0035] Figure 7 The diagram shows a method of using an optical module to take a picture of a side of a thermal bubble printing chip having a nozzle.

[0036] Figure 8 Shown is a schematic diagram of using the optical module to take a photo of the front side of the printing substrate.

[0037] Fig. 9Shown is a schematic diagram of using an optical module to take a picture of the back side of a printed substrate.

[0038] Fig.10 Shown is a schematic diagram of the structure of the optical module.

[0039] Component number description

[0040] 101 First designated nozzle

[0041] 102 Second designated nozzle

[0042] 201 The First Drop

[0043] 202 The Second Drop

[0044] 203 The Third Drop

[0045] 301 Optical Module

[0046] 302 Thermal Bubble Printing Chip

[0047] 303 Nozzle

[0048] 304 Printing substrate

[0049] 305 Positive light source

[0050] 306 Backlight DETAILED DESCRIPTION

[0051] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0052] See also Figures 1 to 10 It should be noted that the illustrations provided in this embodiment are only used to illustrate the basic concept of the present invention in a schematic manner, and the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0053] Embodiment 1

[0054] This embodiment provides a droplet positioning printing method, see Figure 1 , shown as a flow chart of the method, comprising the following steps:

[0055] Solution loading step: applying the solution into multiple nozzles of the print head;

[0056] Nozzle position calibration step: pre-printing a plurality of droplets on a substrate using at least two designated nozzles of the print head, obtaining the landing positions of the plurality of droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position;

[0057] Droplet printing step: obtaining a target printing position, and moving the print head to the target printing position to perform droplet printing.

[0058] Specifically, since the landing point of picoliter droplets is directly related to the printing conditions and environment, such as solution ratio, solution viscosity, printing speed, ambient humidity, etc., which will directly affect the landing point of the printed droplets, the droplet positioning printing method of this embodiment first performs a nozzle position calibration step after the print head is clamped, the solution is loaded, and before formal printing, so that high-efficiency and high-precision droplet arrangement operations can be completed for different types of print heads, under different liquids and different printing conditions.

[0059] As an example, before correcting the nozzle position, the mechanical accuracy and optical accuracy of the equipment can be calibrated and compensated first. This is mainly done by using a precision optical calibration ruler to correct the mechanical installation error of the optical lens of the equipment during the equipment initialization phase. This is not part of each calibration process.

[0060] As an example, the solution includes a cell suspension, and the print head is used to perform single cell printing.

[0061] As an example, in the nozzle position calibration step, the theoretical value coordinate matrix of the nozzle position of the corresponding model print head stored in the control component is obtained based on the model of the print head, and the correction value coordinate matrix of the nozzle position is obtained after correction.

[0062] See also Figure 2 , showing a flow chart for single-cell printing using the droplet positioning printing method of this embodiment, which is generally divided into two steps: droplet position calibration and single-cell precision printing. Among them, the droplet position calibration sequentially executes print head model selection, droplet printing on the substrate, and obtains the droplet position and calibrates the accuracy. The single-cell precision printing sequentially executes obtaining the target printing position and executing single-cell droplet printing.

[0063] As an example, the multiple nozzles of the print head are arranged into at least two columns, each column includes a plurality of nozzles arranged at intervals, and in the nozzle position calibration step, a first designated nozzle and a second designated nozzle are used to print multiple droplets on the substrate, and the first designated nozzle and the second designated nozzle are located in the same column.

[0064] See also Figure 3, which is a schematic diagram of nozzle arrangement of a high-throughput single-cell print head. In this embodiment, the first designated nozzle 101 and the second designated nozzle 102 are located at two ends of the column respectively.

[0065] As an example, the nozzle position calibration step includes:

[0066] Print a first droplet using the first designated nozzle 101 and print a second droplet using the second designated nozzle 102;

[0067] Move the print head along the X-axis by a preset distance, and use the first designated nozzle 101 to print a third droplet;

[0068] Collecting an image of the droplet landing point, measuring an angle A between a line connecting the first droplet and the second droplet and a selected substrate reference line based on the image, and measuring an angle B between a line connecting the first droplet and the third droplet and the substrate reference line based on the image;

[0069] Calculate the angle C between the Y axis of the mechanical motion of the print head and the base line based on the angle B;

[0070] Based on the angle A and the angle C, the theoretical value coordinates of each nozzle position are corrected.

[0071] See also Figure 4 , which is a schematic diagram of an image collected after pre-printing three droplets, shows the landing points of the first droplet 201 , the second droplet 202 , and the third droplet 203 .

[0072] See also Figure 5 , which is a schematic diagram of measurement based on the image, wherein a substrate reference line L, a line connecting the first droplet 201 and the second droplet 202, a line connecting the second droplet and the third droplet, an angle A, and an angle B are illustrated.

[0073] Since the Y axis of the print head mechanical movement is perpendicular to the X axis of the print head mechanical movement, the angle C=90-B.

[0074] As an example, the correction of the theoretical coordinates of each nozzle position based on the angle A includes: calculating the offset between the actual nozzle position and the theoretical position, wherein the absolute value S of the X-axis offset X =L·sinA, the absolute value of the Y-axis offset S Y =L·(1-cosA), L is the straight-line distance between the corresponding nozzle and the preset nozzle, and L is an integer multiple of the nozzle spacing in the column direction.

[0075] As an example, assuming that the theoretical coordinates of the nozzle position are (Xn, Yn), and the corrected coordinates of the nozzle position after correction are (Xn', Yn'), then Xn'=Xn±Ln·sinA, Yn'=Yn-Ln·(1-cosA), where n is the nozzle number, and Ln is the straight-line distance between nozzle number n and the preset nozzle.

[0076] Specifically, in the formula Xn'=Xn±Ln·sinA, the addition and subtraction of the offset can be automatically adjusted according to the direction of the offset. For example, Figure 5 In the case where the second droplet deviates from the first droplet counterclockwise, Xn'=Xn-Ln·sinA, and if the second droplet deviates from the first droplet clockwise, Xn'=Xn+Ln·sinA.

[0077] The following is an example of a print head with 320 nozzles in each column. If the nozzle spacing is 42 microns, the straight-line distance from nozzle No. 1 to nozzle No. 320 is 319×42=13398 microns.

[0078] If the theoretical coordinates of the position of nozzle No. 1 are (X1, Y1), and the theoretical coordinates of the position of nozzle No. 320 are (X320, Y320), then X320=X1, Y320=Y1+13398.

[0079] If the correction value coordinates of the position of nozzle No. 320 are (X320', Y320'), then according to the above formula, if the second droplet is Figure 5 As shown, deviating counterclockwise from the first droplet, X320'=X1-13398·sinA, Y320'=Yn-13398·(1-cosA).

[0080] As an example, correcting the theoretical coordinates of each nozzle position based on the angle C includes: rotating the substrate platform by the angle C in a clockwise or counterclockwise direction to make the substrate platform coordinate system consistent with the print head mechanical motion coordinate system.

[0081] As an example, if you need to calculate the two-dimensional rotation of a coordinate system, you can do this through the following process:

[0082] See also Figure 6 , which shows the coordinate change of point P after the coordinate system rotates counterclockwise by angle θ, where O is the origin of the coordinate system, and A, B, C, D, E, F, and G are auxiliary points.

[0083]

[0084]

[0085] Depend on Figure 6 It can be seen that x b =xa ·cosθ+y a sinθ,y b =-y a sinθ+y a ·cosθ, that is

[0086]

[0087] Since there is a physical distance between the print heads, only integer multiples of the physical distance can be compensated, so the angle C is mostly compensated by rotating the substrate platform.

[0088] Specifically, after completing the nozzle position calibration step, the target printing position is obtained, and the print head is moved to the target position for printing through the motion axis (moving along the mechanical movement X-axis direction and the mechanical movement Y-axis direction). In this way, the single cell sorting device can be used to complete high-throughput and high-accuracy single-cell operations.

[0089] The droplet positioning printing method of this embodiment first calibrates the nozzle position before performing droplet printing. The calibration step includes using at least two designated nozzles of the print head to pre-print multiple droplets on the substrate, obtaining the landing positions of the multiple droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position. Using the droplet positioning printing method of the present invention, high-efficiency and high-precision droplet arrangement operations can be completed for different types of print heads, under different liquids and different printing conditions. In the single-cell positioning printing test, the deviation between the landing point of the single-cell droplet printed after the nozzle position is calibrated and the target is less than ±50μm, so that high-throughput and high-accuracy single-cell operations can be completed using a single-cell sorting device.

[0090] Embodiment 2

[0091] A single-cell analysis platform is provided in this embodiment, and the single-cell analysis platform includes a control component and a single-cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The single-cell analysis platform is used to execute the droplet positioning printing method described in Example 1 to introduce single cells into the designated position of the dielectric wetting chip through the single-cell sorting and printing device for analysis and / or cultivation.

[0092] Specifically, the control component controls the single cell sorting and printing device, the dielectric wetting chip and the optical module, and generally includes a customized control board and a host computer (PC).

[0093] Specifically, the control component includes a computer-readable storage medium on which a computer program is stored. When the computer program is executed by a processor, the droplet positioning printing method is implemented.

[0094] As an example, the single cell sorting printing device includes a thermal bubble printing chip, wherein the thermal bubble printing chip uses the instantaneous high temperature of the heating film to vaporize the liquid above the heating film, generate bubbles to drive the liquid to flow and eject from the nozzle. The thermal bubble printing nozzle is controlled by the bottom circuit.

[0095] As an example, the thermal bubble printing chip is provided with a flow channel connected to the nozzle, and the solution containing cells can be supplied to the nozzle via the flow channel. The layout of the flow channel and the size of the nozzle can be adjusted according to the size of the cells to be sorted. The present invention adds an appropriate volume of cells of an appropriate concentration to the thermal bubble printing chip, and utilizes the high-throughput, single-cell-level microfluidic design of the thermal bubble printing chip to naturally form a large number of single cells in multiple nozzles at one time, and can quickly and gently export the cells.

[0096] As an example, the thermal bubble printing chip can be assembled on an automated instrument including the control component, the automated instrument may include a base, the control component and a reservoir for storing a cell solution, the thermal bubble printing chip is assembled on the base and electrically connected to the control component. An appropriate volume of cells of an appropriate concentration is added to the thermal bubble printing chip through the reservoir, and a large number of single cells can be naturally formed in multiple nozzles at one time by utilizing the high-throughput, single-cell-level microfluidic design of the thermal bubble printing chip.

[0097] As an example, see Figure 7 , which is a schematic diagram showing the use of the optical module 301 to photograph the side of the thermal bubble printing chip 302 having the nozzle 303, so as to obtain the distribution of cells in multiple nozzles, thereby selecting the required single cell for printing.

[0098] As an example, see Figure 8 , which is a schematic diagram showing the use of the optical module 301 to take a picture of the front side of the printing substrate 304, so as to obtain the landing positions of multiple droplets pre-printed on the substrate.

[0099] As an example, see Fig. 9 , which is a schematic diagram showing the use of the optical module 301 to take a picture of the back side of the printing substrate 304. The optical module 301 is focused to the expected landing point of the single cell so as to obtain the morphology of the single cell just after being printed.

[0100] As an example, see Fig.10 , which is a schematic diagram of the structure of the optical module 301.

[0101] As an example, the optical module 301 includes a high-resolution, low-magnification, long-working-distance objective lens, a barrel mirror, a coaxial collimated light source, a multi-channel laser light source, a multi-channel filter, a large target camera, etc., and its connection relationship and parameters can be adjusted as needed to achieve a wide field of view and high-resolution imaging performance.

[0102] As an example, the optical module 301 adopts a plurality of switchable modes such as a positive light source 305, a backlight source 306, and phase contrast imaging, so that the optical module 301 can be applied to a variety of target observations, including transparent substrates, non-transparent substrates, reflective substrates, etc. The optical module 301 can achieve micron-level observations, and combined with the host computer software, it can accurately record the target position where the droplet needs to be printed. Among them, the principle of phase contrast imaging is that after the illumination light passes through a transparent sample (such as a cell), the imaging cannot be directly observed due to the small change in light intensity, but due to the different composition and density of the sample, different phase shifts will be produced when the light passes through. Phase contrast imaging uses the Abbe imaging principle to convert phase changes into amplitude changes, thereby realizing the observation of transparent objects.

[0103] As an example, the light type of the positive light source 305 can be annular light or coaxial light, depending on the requirements of the image.

[0104] The single-cell analysis platform of this embodiment includes a control component and a single-cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The platform adopts the droplet positioning printing method described in Example 1, and can complete high-throughput and high-accuracy single-cell operations through the single-cell sorting and printing device, and realize single-cell capture, fixation, staining and other pre-processing on a tiny platform at the micron to millimeter level, thereby realizing the construction of a micro high-throughput analysis platform for cell morphology, genomics, transcriptomics and proteomics.

[0105] The single-cell analysis platform of this embodiment can reduce the precision of single-cell manipulation from the millimeter level to the 50-micron level. The designated area of ​​the dielectric wetting chip is about 100 microns in size. If the single cell cannot be precisely manipulated, the size of the downstream dielectric wetting chip will increase, the cost will increase, or the cell droplets will most likely not land at the designated location, resulting in cell movement failure. In addition, compared with the traditional microfluidic clamp flow sorting of single cells, the precise manipulation of single cells can effectively reduce the loss of single cells and improve the sorting efficiency of single cells. It is not affected by the type of sorting magnetic beads and can be combined with various sequencing platforms for a variety of tests.

[0106] The single-cell analysis platform of this embodiment is based on the microfluidic characteristics of high-throughput printing chips, the algorithm of AI image recognition and the high-precision automation strategy, which can sort out single cells with high efficiency and high precision, and realize the multi-omics analysis of single-cell automation and high throughput in combination with the digital microfluidic chip system (dielectric wetting). Compared with the traditional well plate single-cell analysis, the full-process automation of the platform greatly reduces the time-consuming process of the original cell sorting and other links, and avoids the impurity pollution introduced by the multi-step experiment. The precise calibration of the position of micro-volume droplets and the precise printing of single cells can realize the construction of a high-throughput reaction system with a uniform and controllable number of tens of thousands on the microfluidic platform. Through the micron-level droplet and single-cell drop point control, the synchronous detection of multiple samples or the full process or multi-index detection of a single sample can be carried out within the selected area, which greatly saves the analysis time cost, improves the accuracy of the detection, the traceability of the reaction sample and the repeatability of the experiment.

[0107] Embodiment 3

[0108] This embodiment uses the single-cell analysis platform described in Example 2 to perform single-cell transcriptome analysis. First, the cell line or dissociated tissue is fully cleaned, filtered, resuspended, and adjusted to the corresponding cell suspension concentration. A single cell sorting printing device is used to separate individual cells from the cell suspension and accurately print them to the designated area of ​​the dielectric wetting chip so that they are evenly arranged, and then the cell lysate and magnetic beads are driven to the single-cell droplet area, and mRNA is captured while the cells are lysed by dielectric wetting. Finally, the eluted captured mRNA droplets are driven to the qRT-PCR area, and the dielectric wetting chip is thermally cycled to perform reverse transcription steps and qPCR. After the reaction is completed, qPCR atlas data is obtained, and relevant data processing and analysis are performed to achieve a system from single cell separation to mRNA purification and gene expression analysis.

[0109] Embodiment 4

[0110] This embodiment uses the single cell analysis platform described in Example 2 to perform single cell sequencing analysis. First, a single cell droplet is sprayed onto the sample loading area of ​​the dielectric wetting chip using a single cell sorting printing device, and the single cell suspension is evenly mixed with the cell preservation solution under the drive of the dielectric wetting chip. Secondly, the dielectric wetting drives the mixed solution to the reaction area under the electric driving force, and mixes and reacts evenly with the cell lysis buffer solution. Subsequently, the above reaction complex is uniformly driven to the chip waiting area (containing the termination reaction solution) to terminate the lysis reaction. In the reaction pool on the other side of the chip, a primer mixture, an enzyme mixture and purified water are sequentially introduced to prepare a mixed solution. After the dielectric wetting is evenly mixed, it is fully mixed with the single cell solution in the above waiting area by the dielectric wetting effect, and then moved to the constant temperature area for reaction for 2 hours. After the reaction is completed, the temperature is raised to 65 degrees Celsius to inactivate the enzyme. At this point, the amplified product is a high-concentration genomic DNA, which is diluted to a suitable concentration using nuclease-free double distilled water (Nuclease-freeddH2O) or TE buffer solution (Tris-EDTA buffer solution) for downstream experiments. Its amplified products can be widely used in whole genome and exome sequencing, microsatellite analysis, qPCR analysis, gene chip analysis, microarray comparative genomic hybridization technology (Array-CGH), etc.

[0111] In summary, the droplet positioning printing method of the present invention first calibrates the nozzle position before performing droplet printing. The calibration step includes using at least two designated nozzles of the print head to pre-print multiple droplets on the substrate, obtaining the landing positions of the multiple droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position. Using the droplet positioning printing method of the present invention, high-efficiency and high-precision droplet arrangement operations can be completed for different types of print heads, under different liquids and different printing conditions. In the single-cell positioning printing test, the deviation between the landing point of the single-cell droplet printed after the nozzle position is calibrated and the target is less than ±50μm, so that a single-cell sorting device can be used to complete high-throughput and high-accuracy single-cell operations. The single cell analysis platform of the present invention includes a control component and a single cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The platform adopts the above-mentioned droplet positioning printing method, and can complete high-throughput and high-accuracy single cell operations through the single cell sorting and printing device, and realize single cell capture, fixation, staining and other pretreatments on a micro-platform at the micron to millimeter level, and realize the construction of a micro high-throughput analysis platform for cell morphology, genomics, transcriptomics, and proteomics. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0112] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A droplet positioning printing method, characterized in that: The following steps are involved: Solution loading step: applying the solution into multiple nozzles of the print head; Nozzle position calibration step: pre-printing a plurality of droplets on a substrate using at least two designated nozzles of the print head, obtaining the landing positions of the plurality of droplets pre-printed on the substrate, and correcting the theoretical value coordinates of the nozzle position of the print head based on the landing position to obtain the corrected value coordinates of the nozzle position; Droplet printing step: obtaining a target printing position, and moving the print head to the target printing position to perform droplet printing.

2. The droplet positioning printing method according to claim 1, characterized in that: The solution includes a cell suspension, and the print head is used for single cell printing.

3. The droplet positioning printing method according to claim 1, characterized in that: The multiple nozzles of the print head are arranged in at least two rows, each row includes multiple nozzles arranged at intervals, and in the nozzle position calibration step, a first designated nozzle and a second designated nozzle are used to print multiple droplets on the substrate, and the first designated nozzle and the second designated nozzle are located in the same row.

4. The droplet positioning printing method according to claim 3, characterized in that: The first designated nozzle and the second designated nozzle are respectively located at two ends of a column.

5. The droplet positioning printing method according to claim 3, characterized in that: The nozzle position calibration step comprises: Printing a first droplet using the first designated nozzle and printing a second droplet using the second designated nozzle; Move the print head along the X-axis by a preset distance, and use the first designated nozzle to print a third droplet; Collecting an image of the droplet landing point, measuring an angle A between a line connecting the first droplet and the second droplet and a selected substrate reference line based on the image, and measuring an angle B between a line connecting the first droplet and the third droplet and the substrate reference line; Calculate the angle C between the Y axis of the mechanical motion of the print head and the base line based on the angle B; Based on the angle A and the angle C, the theoretical value coordinates of each nozzle position are corrected.

6. The droplet positioning printing method according to claim 5, characterized in that: The print head mechanical movement Y axis and the print head mechanical movement X axis are perpendicular to each other, C=90-B.

7. The droplet positioning printing method according to claim 5, characterized in that: Correcting the theoretical coordinates of each nozzle position based on the angle A includes: calculating the offset between the actual nozzle position and the theoretical position, wherein the absolute value S of the X-axis offset X =L·sinA, the absolute value of the Y-axis offset S Y =L·(1-cosA), L is the straight-line distance between the corresponding nozzle and the preset nozzle, and L is an integer multiple of the nozzle spacing in the column direction.

8. The droplet positioning printing method according to claim 5, characterized in that: Correcting the theoretical coordinates of each nozzle position based on the angle C includes: rotating the substrate platform by the angle C in a clockwise or counterclockwise direction to make the substrate platform coordinate system consistent with the print head mechanical motion coordinate system.

9. The droplet positioning printing method according to claim 1, characterized in that: In the nozzle position calibration step, the theoretical value coordinate matrix of the nozzle position of the corresponding model print head stored in the control component is obtained based on the model of the print head, and the correction value coordinate matrix of the nozzle position is obtained after correction.

10. A single cell analysis platform, characterized in that: The single-cell analysis platform includes a control component and a single-cell sorting and printing device connected to the control component, a dielectric wetting chip and an optical module. The single-cell analysis platform is used to execute the droplet positioning printing method as described in any one of claims 1 to 9 to introduce single cells into the designated position of the dielectric wetting chip through the single-cell sorting and printing device for analysis and / or cultivation.

11. The single cell analysis platform according to claim 10, characterized in that: The single cell sorting and printing device comprises a thermal bubble printing chip.

12. The single cell analysis platform according to claim 10, characterized in that: The analysis includes single-cell transcriptome analysis or single-cell sequencing analysis.

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