Biological chip sample application method and sample application device

Abstract

The invention provides a sample application method and a sample application device for a biological chip. The sample application method for the biological chip comprises the following steps: placing a biological molecule solution with a preset volume at a preset position on a solid-phase support in a contact or non-contact manner to form a sample application point array; wherein the sample application point array comprises a plurality of sample application points which are uniformly or basically uniformly distributed, each sample application point comprises a tiny sample application point group, and each tiny sample application point group comprises a plurality of tiny sample application points which are uniformly or basically uniformly distributed so as to inhibit the coffee ring effect. According to the biological chip spotting method provided by the invention, a single spotting point is decomposed into a plurality of tiny spotting points, so that the problem that the signal uniformity of a detection site is influenced by a coffee ring effect is solved, the detection signal is more uniform, and in addition, the detection point signal can be accurately determined after the coffee ring effect is overcome, so that quantitative analysis can be realized; and the application scene of the biochip is expanded.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a biochip spotting method and a spotting device. Background Art

[0002] Biospot chip, also often called biochip, microarray chip or gene chip, is a high-tech platform that fixes a large number of biological macromolecules such as DNA, RNA, peptides, proteins, and even cells, tissue sections, etc. in an orderly form on a solid support (such as silicon wafer, glass, nylon membrane, polyacrylamide gel, etc.), forming a tight two-dimensional molecular grid, providing a fast, efficient and large-scale way to detect cells, genes, proteins and other biological components.

[0003] Biological spotting chips have remarkable features such as micro-quantification, high throughput, and automation, allowing a large amount of biological information to be analyzed on a very small area. Its application range is extremely wide, such as gene expression spectrum analysis (study the function and regulatory mechanism of genes by comparing the expression levels of genes under different conditions); SNP detection (detection of single base variations in the genome, used for disease diagnosis, pharmacogenomics, etc.); proteomics research (study the expression level, interaction and modification of proteins in cells or tissues); pathogen detection (rapid and accurate detection of various pathogens, such as viruses, bacteria, etc.); drug screening (high-throughput screening of drug molecules to accelerate the development of new drugs), etc.

[0004] Biochips are usually made by the method of pre-synthesis followed by spotting. In this method, a pre-synthesized biomolecule solution is precisely spotted on a solid support by a high-speed spotting robot. This process is relatively simple and cost-effective, and is currently the mainstream method for biochip production.

[0005] When spotting samples on a biochip, the droplets need to dry on the surface of a solid substrate, and then scan the substances remaining on the solid surface or visually inspect them after staining. However, during the evaporation of the solvent, the outward capillary flow inside the droplet will carry the solute or suspended particles to the edge of the droplet and deposit at the edge to form a "coffee-ring effect" (see Figure 6 ). This phenomenon often leads to uneven deposition of solutes or particles, which seriously affects the signal-to-noise ratio of biochip detection results. There are currently two common solutions to the "coffee ring effect". One is to use a solid surface substrate with relatively weak resistance to droplets to suppress the coffee ring effect; the other is to optimize the droplet composition, such as using droplets with low surface tension. However, in actual applications, due to different usage scenarios and / or detection requirements, it is actually impossible to choose the solid substrate and droplet composition at will.

[0006] In addition, existing biological spotting chips are rarely used for quantitative analysis. One of the reasons is that the "coffee ring effect" affects the uniformity of the signal at the detection site, making quantitative analysis impossible. Summary of the invention

[0007] In view of the defects in the prior art, the purpose of the present invention is to provide a biochip spotting method and a spotting device, which decomposes a single spotting point into a number of tiny spotting points, suppresses the coffee ring effect, and solves the problem of the coffee ring effect affecting the uniformity of the detection site signal, making the detection signal more uniform. In addition, after overcoming the coffee ring effect, the detection point signal can be accurately determined, thereby realizing quantitative analysis and expanding the application scenarios of biochips.

[0008] The first aspect of the present invention provides a biochip spotting method, comprising the following steps: a preset volume of biomolecule solution is placed at a predetermined position on a solid support via contact or non-contact means to form a spotting point array; wherein the spotting point array comprises a plurality of spotting points that are uniformly or substantially uniformly distributed, each of the spotting points comprises a micro spotting point group, and the micro spotting point group comprises a plurality of micro spotting points that are uniformly or substantially uniformly distributed, thereby improving the distribution consistency of the deposited molecules in the center and at the edge of the spotting point to suppress the coffee ring effect.

[0009] In the biochip spotting method provided by the present invention, a large spotting point is decomposed into multiple tiny spots while keeping the overall size unchanged, so that a large coffee ring can be evenly decomposed into multiple small coffee rings, making the deposition more uniform overall and improving the detection signal-to-noise ratio.

[0010] Furthermore, the sample points are decomposed into multiple smaller tiny sample points so that the diameter of the coffee ring effect formed by the tiny sample points is smaller than the recognition resolution of the detection tool (such as a visual system), which can also suppress the coffee ring effect in the detection results.

[0011] Furthermore, the size of the tiny sampling points is close to or smaller than the limit size of the coffee ring effect, and the evaporation speed of the droplets is greater than the movement speed of the solute or solid particles in the droplets, so as to eliminate the coffee ring effect.

[0012] The sample point is decomposed into multiple smaller micro-sample points, making them close to or smaller than the limit size of the coffee ring effect. The evaporation rate of the droplet is greater than the movement speed of the solute or solid particles in the droplet. Before the liquid evaporates, the particles do not have enough time to settle into a ring structure, and the micro-point coffee ring effect will no longer appear. The coffee ring effect is completely suppressed.

[0013] In one embodiment of the present invention, the maximum linear dimension of each of the sampling points is 0.2-0.5 mm, the diameter of each of the micro sampling points is 0-200 μm and not equal to 0, and the spacing between two adjacent micro sampling points is 0-200 μm and not equal to 0.

[0014] Furthermore, the diameter of each of the micro-dot sampling points is 10-200 μm, and the distance between two adjacent micro-dot sampling points is 5-100 μm.

[0015] In one embodiment of the present invention, each of the spot sampling points includes at least one layer of micro spot sampling point groups stacked in layers.

[0016] In the biochip spotting method provided by the present invention, after the spotting points are decomposed into micro-spots, the liquid volatilization speed is accelerated, and two or more layers of micro-spots can be used for spotting, so as to increase the deposition amount and enhance the detection signal.

[0017] Preferably, the present invention avoids self-aggregation between the micro-sampling points by specifically setting the diameter of the micro-sampling points and the spacing between adjacent micro-sampling points, thereby further ensuring the uniformity of the detection signal.

[0018] In one embodiment of the present invention, the maximum linear dimension of each of the sampling points is 0-10 mm and not equal to 0, the diameter of each of the micro sampling points is 0-500 μm and not equal to 0, and the spacing between two adjacent micro sampling points is 0-500 μm and not equal to 0.

[0019] In one embodiment of the present invention, the diameter of each of the micro dot sampling points is 10-30 μm, and the distance between two adjacent micro dot sampling points is 5-30 μm.

[0020] In one embodiment of the present invention, among the plurality of sampling points, at least one sampling point comprises one or more layers of stacked micro sampling point groups.

[0021] The second aspect of the present invention provides the use of the above-mentioned biochip spotting method in qualitative and / or quantitative analysis of target biomolecules.

[0022] The third aspect of the present invention provides a spotting device, comprising: A central controller is configured to set array parameters of sampling points, decompose each sampling point into a plurality of uniformly or non-uniformly distributed micro sampling points, wherein the plurality of micro sampling points constitute a micro sampling point group, and collect array parameters of the micro sampling point group; The execution module executes the above biochip spotting method based on the array parameters of the spotting points and the array parameters of the micro spotting points set in the central controller.

[0023] In one embodiment of the present invention, the execution module includes a liquid storage container for storing a biomolecule solution, and at least one spotting head, wherein the spotting head is one of a pl-level spotting head, an nl-level spotting head, and a μl-level spotting head.

[0024] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: The biochip spotting method and spotting device provided by the present invention suppress the coffee ring effect by decomposing a single spotting point into a plurality of tiny spotting points, thereby solving the problem of the coffee ring effect affecting the uniformity of the detection site signal, and making the detection signal more uniform. As an optimization, the present invention specifically sets the diameter of the tiny spotting points and the spacing between adjacent tiny spotting points, thereby avoiding self-aggregation between the tiny spotting points, and further ensuring the uniformity of the detection signal.

[0025] The main reason why existing spotting chips are difficult to quantify is that the "coffee ring effect" causes uneven molecular distribution, background noise affects the low signal-to-noise ratio and the dynamic range is narrow. The biochip spotting method and spotting device provided by the present invention can realize multi-layer spotting, suppress the coffee ring effect, make the target molecules deposit evenly in the spot, eliminate the problems of signal imbalance and background noise interference, and can accurately determine the detection point signal, thereby realizing quantitative analysis and expanding the application scenarios of biochips. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of a spotting process in one embodiment of the present invention; Figure 2 is a schematic diagram of a spotting process in another embodiment of the present invention; Figure 3 Schematic diagram of a spotting process in another embodiment of the present invention; Figure 4 Schematic diagram of the sample array in Application Example 2; Figure 5 It is the signal detection curve diagram in application example 2; Figure 6 It is a schematic diagram of the coffee ring effect of an existing single sampling point; Figure 7 This is a schematic diagram of the coffee ring effect in the present invention; Figure 8 It is a schematic diagram of the signal intensity gradient of the multi-layer overlapping dots in the present invention; Fig. 9 This is a schematic structural diagram of the spotting device provided by the present invention. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0028] The maximum linear dimension is the longest line length of the object (here, the dot unit) in any direction. That is, it is the maximum linear extension length of the dot in any direction. For circular dots, the maximum linear dimension is the diameter. If the long side of a rectangular dot is 12 microns and the short side is 4 microns, then its maximum linear dimension is 12 microns.

[0029] The micro-spot sampling point group in the present invention includes a plurality of uniformly or substantially uniformly distributed micro-spot sampling points, wherein the "substantially uniform" mentioned in the present invention means that the spacing deviation between each micro-spot sampling point does not exceed ±5% of the theoretical value, so as to ensure that each reaction site receives a uniform input signal, thereby maintaining the detection accuracy of the biochip.

[0030] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] The present invention is further described below by means of specific examples.

[0032] Example 1 This embodiment provides a biochip spotting method, comprising the following steps: According to the usage scenario and usage requirements, preset sampling parameters are designed; wherein, the array parameters of the sampling points are first determined, and then the single sampling point is decomposed into a plurality of evenly distributed micro-sampling point arrays, and the array parameters of the micro-sampling points are obtained. The sampling parameters in this embodiment include the array parameters of the sampling points and the array parameters of the micro-sampling points; Based on the preset spotting parameters, a preset volume of biomolecule solution is placed at a predetermined position on the solid support via a contact or non-contact method to form a spotting point array.

[0033] In this embodiment, the array parameters of the sampling points include at least one of the following: Array type: The array type refers to the overall arrangement of the spots on the chip surface, such as rectangular, square or hexagonal arrangement; Point spacing: Point spacing refers to the distance between adjacent sampling points in the array. The point spacing may include the distance between the geometric centers of two adjacent sampling points and / or the distance between the closest edge portions of two adjacent sampling points. Spot size: Spot size refers to the size of each individual spot sampled droplet or deposit area; Array size and density: determine the overall detection coverage and flux; array size refers to the range of the entire dot pattern distribution area, including row length (total length from the first row to the last row) and column length (total width from the first column to the last column); array density refers to how many dot points can be accommodated per unit area.

[0034] In this embodiment, the array parameters of the micro-dot sampling points include at least one of the following: Array type of micro-dots: describes the arrangement of micro-dots within each dot, such as rectangular, square or hexagonal arrangement; The maximum linear size of each spot; The diameter of each tiny spot; The distance between two adjacent micro-dots; Microarray size: refers to the total area or range occupied by each micro-dot array. It is usually reflected in the following aspects: row length (the total length from the first row to the last row of the microarray), column length (the total width from the first column to the last column of the microarray), total area (the total size of the area covered by the entire microarray (e.g. mm²)); Array density: refers to how many tiny sample points can be accommodated per unit area.

[0035] In this embodiment, the spot sampling point array includes a plurality of evenly distributed spot sampling points, each of which includes a micro spot sampling point group, and the micro spot sampling point group includes a plurality of evenly or substantially evenly distributed micro spot sampling points, thereby improving the distribution consistency of the deposited molecules in the center and the edge of the spot sampling points to suppress the coffee ring effect; wherein, the deposited molecules refer to biological molecules or chemical molecules fixed at specific positions on the chip substrate by spot sampling technology, and these molecules are usually used as probes for subsequent detection, identification or interaction analysis (for example, binding with target molecules in the sample to be tested).

[0036] There is a size limit for the coffee ring effect. This is because as the droplet size decreases, the evaporation rate of the droplet will increase greatly, while the movement speed of the solid particles in the droplet will not change much. If the droplet size is small enough, the evaporation rate of the droplet will be much greater than the movement speed of the solid particles. Before the droplet evaporates, the particles will not have enough time to settle into a ring structure. These small particles that do not have time to move will be deposited evenly on the entire area covered by the droplet rather than on the edge of the droplet. The DNA probes deposited on the biochip have a particle diameter of between tens of nanometers and hundreds of nanometers. For dissolving small particle droplets with a diameter of 10 to 100 nanometers, the coffee ring effect is avoided in this application by decomposing a single sampling point into tiny sampling points (tiny droplets) of about 10 microns.

[0037] Furthermore, the sample point can be decomposed into multiple smaller micro-sample points, so that the inner diameter of the coffee ring effect formed by the micro-sample points is smaller than the recognition resolution of the detection tool (such as a visual system), which can also suppress the coffee ring effect in the detection results. Figure 6 As shown, without using the spotting process provided in the present invention, after spotting directly according to the spotting point size, the spotting point has an obvious coffee ring effect. Figure 7 As shown, after decomposing the sample point into a plurality of smaller micro sample points in the present invention, the coffee ring effect is effectively suppressed.

[0038] Specifically, the size of the tiny sampling point in this embodiment is close to or smaller than the limit size of the coffee ring effect, and the evaporation rate of the droplet is greater than the movement rate of the solute or solid particles in the droplet. The "limit size" of the coffee ring effect mainly refers to some scales related to the ring formation characteristics in the ring deposition driven by droplet evaporation, including: the deposition ring width (minimum / maximum width), the minimum distance between particles in the ring, or the characteristic resolution.

[0039] Furthermore, the sample point is decomposed into multiple smaller micro-sample points, making them close to or smaller than the limit size of the coffee ring effect. The evaporation rate of the droplet is greater than the movement speed of the solute or solid particles in the droplet. Before the liquid evaporates, the particles do not have enough time to settle into a ring structure, and the micro-point coffee ring effect will no longer appear. The coffee ring effect is completely suppressed.

[0040] Furthermore, after the sample point is decomposed into micro sample points, the liquid evaporation speed is accelerated, and two or more layers of micro sample points can be used to stack the sample points to increase the deposition amount and enhance the detection signal. Figure 8 As shown, from right to left are single-layer spotting, 2-layer spotting, 3-layer spotting, 4-layer spotting, 5-layer spotting, 6-layer spotting, 7-layer spotting, and 8-layer spotting. Figure 8 As shown, from right to left, as the number of sample layers increases, the detection signal intensity gradually increases, and there is no coffee ring effect.

[0041] In this embodiment, when spotting multiple layers, a continuous spotting method can be used, that is, after spotting one layer, the second layer is immediately spotted; in addition, after spotting one layer, wait for it to dry before spotting another layer.

[0042] In this embodiment, the spacing between adjacent micro-dots can be configured to avoid self-aggregation between adjacent micro-dots.

[0043] Specifically, in this embodiment, the maximum linear dimension of each of the sampling points is 0.2-0.5 mm, the diameter of each of the micro-sampling points is 0-200 μm and is not equal to 0, and the spacing between two adjacent micro-sampling points is 0-200 μm and is not equal to 0. Further preferably, the diameter of each of the micro-sampling points is 10-200 μm, and the spacing between two adjacent micro-sampling points is 5-100 μm. By setting the maximum linear dimension of each sampling point, the diameter of each micro-sampling point, and the spacing between two adjacent micro-sampling points, not only the coffee ring effect is suppressed, but also the self-aggregation between adjacent micro-sampling points is avoided, which affects the detection effect.

[0044] In the prior art, the diameter of the sample point is 0.2-0.5mm, in order to ensure that the sample point is as small as possible and visible to the naked eye. In the present invention, the single sample point is composed of multiple tiny sample points, which ensures that the single sample point is as small as possible and visible to the naked eye while overcoming the problem of the coffee ring effect.

[0045] In one embodiment, the maximum linear dimension of each of the sampling points is 0.5 mm, the diameter of each of the micro-sampling points is 12 μm, and the distance between two adjacent micro-sampling points is 21 μm.

[0046] In one embodiment, the biomolecule solution includes at least one of DNA, RNA, polypeptide, protein, and antibody.

[0047] In one embodiment, the biomolecule solution comprises particles with a diameter of 10-100 nm. The particles described in this embodiment include biomolecule particles, such as DNA, RNA, polypeptide, protein, antibody, etc.

[0048] The biochip spotting method in this embodiment is implemented by the following spotting device, referring to Fig. 9 As shown, the spotting device includes a solid phase substrate component, a spotter, and a central controller.

[0049] The solid substrate assembly comprises a solid substrate surface and a motion drive system controlled by a central controller. The solid substrate surface is a surface for carrying sample droplets. The solid substrate surface can be stationary or movable under the control of a central controller.

[0050] The sample spotter comprises a liquid storage container and a sample spotting head. The liquid storage container is used to store the biomolecule solution. The sample spotting head is a mechanism for transferring the biomolecule solution in the liquid storage container to the surface of the solid phase substrate according to a specified volume. The sample spotting head is a p1-level sample spotting head.

[0051] The execution module is used to carry the solid substrate surface and is controlled by the central controller to drive the solid substrate surface to move.

[0052] The central controller is configured to set array parameters of the spotting points, decompose each spotting point into a number of tiny spotting points, and form a tiny spotting point group, collect array parameters of the tiny spotting point group, and control the spotter to transfer a set volume of biomolecule solution to a specified position on the surface of the solid substrate according to the parameters.

[0053] In one embodiment, the spotter can be contact inkjet spotting, thermal inkjet spotting, piezoelectric inkjet spotting, electrofluidic inkjet spotting, electrostatic inkjet spotting, ultrasonic inkjet spotting, or pneumatic inkjet spotting.

[0054] In one embodiment, the spotter can be a single-hole, double-hole or multi-hole array.

[0055] In one embodiment, if Figure 3 If you need to sample an array of 8 points in both the horizontal and vertical directions, the sample point size is required to be a 0.5mm diameter circle with a sample point spacing of 0.8mm.

[0056] First, set the sampling parameters of 8*8 sample matrix and single sample diameter of 0.5mm through the central controller. After enabling the "coffee ring effect" suppression function, the central controller automatically decomposes the 0.5mm diameter sample data into the following according to the preset program: Figure 3 The central controller then controls the solid substrate to move from the left side to the right side of the spotting head, and at the same time controls the corresponding nozzles of the spotter to spray ink and form a solid substrate surface. Figure 3 The sample point is completed.

[0057] Compared with directly using a single droplet of continuous phase with a diameter of 0.5 mm as a detection sample point, since a single detection sample point of the present invention is composed of 104 tiny sample points of the dispersed phase, the solute or particle deposition after the solvent evaporates is more uniform, which effectively suppresses the "coffee ring effect" and greatly improves the signal-to-noise ratio of subsequent diagnostic detection.

[0058] Application Example 1 The spotting method and device described in Example 1 were used for spotting.

[0059] like Figure 3 As described above, in this application example, an array of 8 sampling points in both the horizontal and vertical directions needs to be sampled, the size of a single sampling point is required to be a circle with a diameter of 0.5 mm, and the spacing between adjacent sampling points is 0.8 mm.

[0060] The steps for spotting are as follows: First, set the sampling parameters through the central controller: 8×8 sampling matrix ( Figure 1), the sampling parameters of a single sampling point with a diameter of 0.5 mm, and then the central controller decomposes the sampling point data of each 0.5 mm diameter into Figure 3 The data of 104 tiny droplets (micro-dot sampling points) shown in the single sample point morphology image are used to obtain the array parameters of the micro-dot sampling points. Among them, the diameter of a single tiny droplet (micro-dot sampling point) is 12μm, the distance between two adjacent tiny droplets is 21 microns, and 104 tiny droplets form a single sample point with a diameter of 0.5mm; Based on the array parameters of the above-mentioned spotting points and the array parameters of the micro-spotting points, the spotting head places the biomolecule solution stored in the liquid storage container at a predetermined position on the solid support to form a spotting point array.

[0061] Compared with directly using a single droplet of continuous phase with a diameter of 0.5 mm as the detection sample point, the single detection sample point in this application example is composed of 104 tiny droplets of dispersed phase. The solute or particle deposition after solvent evaporation is more uniform, which effectively suppresses the "coffee ring effect" and greatly improves the signal-to-noise ratio of subsequent diagnostic detection.

[0062] Among them, in specific applications, the number of tiny sampling points that a single sampling point is decomposed into can be further determined by technical personnel based on the type of molecules, detection requirements, etc. As long as the coffee ring effect is suppressed to a certain extent and the detection signal reaches a certain signal-to-noise ratio, the application requirements are met.

[0063] Application Example 2: Quantitative Detection by Spot Chip Gradient Method The spotting method and device described in Example 1 can be used for spotting, which can effectively suppress the coffee ring effect, thereby making the spotting chip signal more uniform, improving the signal-to-noise ratio, and achieving quantitative detection.

[0064] Furthermore, in this application example, a multi-layer spotting method is simultaneously used to realize a concentration gradient change of the spotting point detection probe (protein, etc.), thereby achieving the purpose of quantitative detection.

[0065] like Figure 4 As shown, 1, 2, 3, 4, 5, 6, 7, 8 layers of multi-layer spotting are performed from right to left for different columns, and then signal detection is performed. The signal detection is shown below Figure 5 The detection signal is analyzed and a standard curve is fitted, and the concentration of the detection sample is calculated according to the standard curve to achieve quantitative detection.

[0066] Spot chip is a biomolecule detection tool, usually used for high-throughput screening and identification. However, most traditional spot chips lack quantitative functions, and the main reasons can be summarized as follows: 1. Uneven distribution of droplets during spotting In the traditional spotting process, tiny droplets can cause uneven distribution of target molecules on the chip surface due to internal fluid dynamics (such as the "coffee ring effect").

[0067] The coffee-ring effect refers to the passive confinement of solutes to the edge area during the evaporation of a droplet, rather than uniform distribution in the entire spot area. This makes the effective signal of each point likely to be concentrated in certain micro-positions, while the signal in other areas is significantly weakened, resulting in deviations in the detection results.

[0068] 2. Uneven signal distribution The "coffee ring" or disordered distribution after spotting will cause the detection signal to vary in strength. Even for the target molecules with the same concentration, the signal intensities cannot maintain a linear relationship.

[0069] Due to poor uniformity and high variation in experimental repetitions, the volatility and irreproducibility of the data make it difficult for the fluorescence signal between points to reflect the exact concentration of the actual molecule.

[0070] 3. Low signal-to-noise ratio and large background interference The lack of contrast between the detection signal of the spot chip and the background noise also limits the sensitive detection of concentration. When the concentration of the target molecule is low, the generated fluorescence signal is close to the background noise level (low signal-to-noise ratio), making it difficult to distinguish the real signal, which seriously restricts the accurate detection in the low concentration range.

[0071] This application fundamentally solves the problems existing in existing chips by improving the spotting method, achieving more uniform signal distribution and higher signal-to-noise ratio, thereby supporting the chip for quantitative analysis. The specific improvements are as follows: 1. Change the method of sampling to suppress the "coffee ring effect" In the spotting method of this application, the spotting process enables the target molecules to be evenly distributed in the droplets and form a highly consistent deposition pattern on the sample surface. This ensures that the signal intensity inside each spot is consistent relative to the area, and can reflect the actual concentration regardless of the center or edge of the sample.

[0072] 2. Improve signal uniformity The biomolecules after spotting and immobilization are evenly and stably distributed without overlapping or overly sparse areas, thus ensuring that the fluorescence signals measured at each point are relatively stable and consistent.

[0073] 3. Improve signal-to-noise ratio Based on the spotting method of this application, a more sensitive fluorescence detection technology is used, and the experimental steps are optimized (such as the washing process to reduce the adsorption of nonspecific impurities), which significantly reduces the background noise.

[0074] The improvement in signal-to-noise ratio not only enhances the ability to detect low concentrations, but also improves the reliability of the signal throughout the dynamic range, so that even small concentration changes can be significantly reflected in the signal response.

[0075] 4. Adapt to various detection applications The spotting method in this application is used for detection, maintaining the original universal design of the chip, and at the same time being compatible with different types of target molecule tests (such as protein and nucleic acid microarrays) under the new process, which not only improves the detection efficiency but also has universality.

[0076] Application example 3: Quantitative detection of spot chip The spotting method and device described in Example 1 can be used for spotting, which can effectively suppress the coffee ring effect, thereby making the spotting chip signal more uniform, improving the signal-to-noise ratio, and achieving quantitative detection.

[0077] like Figure 1 As described above, in this application example, an array of 15 sampling points in both the horizontal and vertical directions needs to be sampled, the size of a single sampling point is required to be a circle with a diameter of 0.5 mm, and the spacing between adjacent sampling points is 0.8 mm.

[0078] The spotting method in this embodiment is as follows: First, the central controller sets the sampling parameters of 8×8 sampling matrix and a single sampling point diameter of 0.5 mm. Then the central controller decomposes the sampling point data of each 0.5 mm diameter into the following Figure 1 The data of 144 tiny droplets (tiny dot sampling points) shown in the single sample point morphology image. The diameter of a single tiny droplet (tiny dot sampling point) is 25μm, the distance between two adjacent tiny droplets is 15 microns, and 144 tiny droplets form a single dot sampling point with a diameter of 0.5mm.

[0079] Reference Figure 1 As shown in the sample array in the figure, the leftmost column point is set as the internal reference point. Different internal reference points have different DNA sequences, but the spot concentration is the same. The DNA fragments detected by the internal reference point are mixed into the sample to be tested, and the concentration gradient is set, and then the subsequent detection process is carried out together. In the signal reading stage, the signal of the internal reference point is first collected, and the internal reference curve is drawn according to the signal intensity and the internal reference concentration. Then the signal of the detection point is read, and the concentration result in the sample is obtained according to the signal intensity and the internal reference curve.

[0080] Application example 4: Spotting of different DNA mixtures The spotting method and device described in Example 1 can effectively suppress the coffee ring effect, thereby making the spotting chip signal more uniform, improving the signal-to-noise ratio, and realizing multiple signal detection. At the same time, the multi-layer spotting method can realize in-situ spotting of multiple types of biological detection molecules, thereby improving the detection capability of the detection point.

[0081] DNA fragments with significant sequence differences are mixed in a spotting point for spotting chip preparation. The number of DNA fragments can be 2-4. Signal labeling is performed on the DNA sample to be detected, such as one can be labeled with Cy3 and the other can be labeled with Cy5. The sample to be detected and the spotting chip are hybridized and scanned and detected under the corresponding fluorescence field. If the signal intensity is low, antibody fluorescence cascade amplification technology can be used to enhance the fluorescence signal and improve the detection sensitivity.

[0082] Application Example 5: DNA and protein mixed spotting The spotting method and device described in Example 1 can effectively suppress the coffee ring effect, thereby making the spotting chip signal more uniform, improving the signal-to-noise ratio, and realizing multiple signal detection. At the same time, the multi-layer spotting method can realize in-situ spotting of multiple types of biological detection molecules, thereby improving the detection capability of the detection point.

[0083] DNA fragments and proteins are mixed in a spotting point for spotting chip preparation. The number of proteins and DNA fragments can be 2-4. For signal labeling in the sample to be detected, such as a mutually hybridized DNA can be labeled with Cy3, and an interactive protein can be labeled with Cy5. The sample to be detected and the spotting chip are hybridized and scanned and detected under the corresponding fluorescence field. If the signal intensity is low, antibody fluorescence cascade amplification technology can be used to enhance the fluorescence signal and improve the detection sensitivity. The mixed spotting chip can be used together or alone to detect DNA fragments or protein information.

[0084] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A biochip spotting method, characterized in that: The method comprises the following steps: a preset volume of biomolecule solution is placed at a predetermined position on a solid support via contact or non-contact means to form a spot array; wherein the spot array comprises a plurality of spot points that are evenly or substantially evenly distributed, each of the spot points comprises a micro spot group, and the micro spot group comprises a plurality of micro spot points that are evenly or substantially evenly distributed, so as to improve the distribution consistency of the deposited molecules in the center and at the edge of the spot, so as to suppress the coffee ring effect.

2. The biochip spotting method according to claim 1, characterized in that: And the diameter of the coffee ring formed by the tiny sample points is smaller than the recognition resolution of the detection tool.

3. The biochip spotting method according to claim 1 or 2, characterized in that: The size of the tiny sample points is close to or smaller than the limit size of the coffee ring effect, so as to eliminate the coffee ring effect.

4. The biochip spotting method according to claim 1, characterized in that: The maximum linear dimension of each of the sampling points is 0-10 mm and is not equal to 0, the diameter of each of the micro sampling points is 0-500 μm and is not equal to 0, and the spacing between two adjacent micro sampling points is 0-500 μm and is not equal to 0.

5. The biochip spotting method according to claim 4, characterized in that: The diameter of each of the micro-dot sampling points is 10-200 μm, and the distance between two adjacent micro-dot sampling points is 5-100 μm.

6. The biochip spotting method according to claim 1, characterized in that: Among the plurality of sampling points, at least one sampling point comprises one or more layers of stacked micro sampling point groups.

7. Use of the biochip spotting method according to any one of claims 1 to 6 in qualitative and / or quantitative analysis of target biomolecules.

8. A spotting device, characterized in that: include: A central controller is configured to set array parameters of sampling points, decompose each sampling point into a plurality of uniformly or non-uniformly distributed micro sampling points, wherein the plurality of uniformly or non-uniformly distributed micro sampling points constitute a micro sampling point group, and collect array parameters of the micro sampling point group; The execution module executes the biochip spotting method according to any one of claims 1 to 6 based on the array parameters of the spotting points and the array parameters of the micro spotting points set in the central controller.

9. The spotting device according to claim 8, characterized in that: The execution module includes a liquid storage container for storing a biomolecule solution and at least one spotting head, wherein the spotting head is one of a pl-level spotting head, an nl-level spotting head, and a μl-level spotting head.

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