Concentration gradient micro-droplet array generation device for molecular interaction research

Abstract

The invention provides a concentration gradient micro-droplet array generation device for molecular interaction research, the device comprises a liquid suction mechanism and a sample application mechanism, the liquid suction mechanism respectively sucks a sample solution and a solvent, so that the sample solution and the solvent are continuously fused in a piston tube of the liquid suction mechanism, and the sample application mechanism is used for applying the sample solution and the solvent. The micro-droplet array is formed on the surface of the chip, and the concentration of each micro-droplet is accurate and controllable, so that the concentration gradient micro-droplet array can be directly generated by the simplest structure and the most convenient method, and the micro-droplet array can be applied to the surface of the chip. The method can be used for accurately detecting the affinity between the aptamer and the target protein, and can also be used for interaction research of other molecules. The device is simple in structure, the cost is reduced to a great extent, the use difficulty of the SPRi technology is reduced, the application prospect of the SPRi technology is widened, and the device is suitable for large-scale production and application.

Description

Technical Field

[0001] The present invention relates to multiple fields such as microfluidics, biomolecule affinity, SPRi, etc., and specifically to a device for generating a microdroplet array for multi-concentration gradient detection of affinity between proteins and nucleic acid aptamers and a method for using the same. Technical Background

[0002] SPRi (Surface Plasmon Resonance imaging) is a technology that can detect the interaction between ligands and analytes on biosensor chips. Based on the surface plasmon resonance phenomenon, this technology detects the interaction of biomolecules in real time by monitoring the changes in light signals caused by the binding of biomolecules to metal surfaces. It can then characterize the affinity between aptamers and other molecules to screen high-affinity aptamers, and is therefore suitable for drug screening, biomarker detection, interaction research, sensors and other fields. Compared with other affinity determination methods, SPRi technology has the advantages of high sensitivity, real-time monitoring and label-free. However, although SPRi can be used for high-throughput screening of aptamers, the spotting process requires a high-tech and expensive dispenser, which is an automated machine that drips and applies fluids on the surface or inside of a product. It is widely used in manufacturing industries such as electronics, electrical appliances, automobiles, and medical devices. The purchase cost of the dispensing machine is high, and it is not easy to promote and apply it at the grassroots level. The maintenance cost is high, and specific cleaning agents and tools are required for regular maintenance and maintenance, which in turn increases the production cost. In addition, the operation and maintenance of the dispensing machine also have certain requirements on the operator's skills. In short, the cost and technical requirements of using a dispensing machine have hindered the development and application of SPRi technology to a certain extent. In order to reduce costs, a pipette is often used instead of a dispensing machine, which results in a droplet volume of up to 0.25μL, greatly reducing the sample scale and chip utilization, thereby reducing detection efficiency and resulting in excessive costs.

[0003] Microdroplet array technology is an important technology that has attracted much attention in the biomedical field in recent years. In the process of detecting the affinity of proteins and nucleic acid aptamers, this technology can quickly and accurately screen out ligands with high affinity, and can achieve efficient and large-scale sample processing. Therefore, in biomedical research, high-throughput screening detection technology is widely used in drug discovery, gene function research, protein interaction analysis, etc. Compared with traditional spot detection technology, microfluidic technology has major advantages, including reducing sample / reagent consumption and reducing detection costs; more importantly, its ability to accurately operate small liquid volumes (4-10nL) enhances the applicability of single-cell analysis in various microfluidic technologies, because it uses nanoliter oil-in-water emulsion droplets as microreactors, preventing cross-contamination between different samples and reducing adverse interactions between the chip surface and reagents.

[0004] Molecular interaction studies, such as affinity detection between nucleic acid aptamers and target proteins, require detection through microdroplet arrays with concentration gradients. However, existing dispensing machines and other equipment are difficult to directly dispense microdroplets with concentration gradients. Usually, sample solutions of different concentrations need to be prepared separately to achieve this. The sampling channel needs to be repeatedly cleaned and the internal solution replaced. The operation process is time-consuming and laborious. Even with automated equipment, it takes a long time to prepare samples. For example, the Chinese patent "A Micro-Nanoliter Spotting Device" (Publication (Announcement) No. CN114522747B) provides a device for nanoliter spotting using capillaries, but the device can only spot samples quickly and cannot directly spot samples with concentration gradients.

[0005] Therefore, there is an urgent need to find a droplet array spotting technology as an effective alternative to liquid guns and dispensers, which can be used to generate a micro-droplet concentration gradient matrix with a concentration gradient. Summary of the invention

[0006] In view of the above problems, the present invention aims to provide a micro-droplet array generation device with functions such as gradient concentration generation, precise droplet manipulation, and micro-droplet array generation, as well as non-destructive micro-droplet array generation for nucleic acid aptamers, and the micro-droplet array has a concentration gradient and can be used for molecular interaction research. The device includes a liquid suction mechanism and a spotting mechanism, and the sample solution and the solvent are respectively sucked by the liquid suction mechanism, so that the sample solution and the solvent are continuously fused in the piston tube of the liquid suction mechanism, and while fusion, micro-droplets of different concentrations are dripped out through the glass capillary of the spotting mechanism to form a micro-droplet array on the chip surface, and the concentration of each micro-droplet is accurately controllable, thereby realizing the direct generation of a concentration gradient micro-droplet array with the simplest structure and the most convenient method, which can be used to accurately detect the affinity between nucleic acid aptamers and target proteins, and can also be used for other molecular interaction studies. The device has a simple structure, greatly reduces costs, reduces the difficulty of using SPRi technology, broadens the application prospects of SPRi technology, and is suitable for large-scale production applications.

[0007] On the one hand, the present invention provides a device for generating a micro-droplet concentration gradient array, the device comprising a liquid aspiration mechanism and a spotting mechanism, the liquid aspiration mechanism being used to aspirate a sample solution and a solvent respectively; the spotting mechanism comprising a capillary for exporting liquid from the liquid aspiration mechanism and directly spotting a micro-droplet array with a concentration gradient on a plane; the sample solution contains a macromolecular sample.

[0008] In some embodiments, the macromolecular sample refers to a substance with a molecular particle size greater than 1 nm.

[0009] Compared with those without concentration gradient, the application of microdroplet arrays with concentration gradient in the field of molecular interactions will increase the amount of experimental data and credibility. By deeply mining the data, the target interacting molecules can be screened in a short time, and even the screening purpose can be achieved through one experiment without repeatedly conducting the same experiment, shortening the experimental cycle. This feature can speed up the process of drug development, because in the process of drug development, it is often necessary to screen substances that bind to cell surface receptors, including but not limited to compounds, in order to achieve the purpose of isolating or activating signal transmission between cells. For example, when screening the most affinity nucleic acid aptamer for the target protein, it is also necessary to achieve this through the binding and dissociation of the target protein with the nucleic acid aptamer with a concentration gradient. Therefore, how to accurately prepare concentration gradient microdroplet arrays in a simpler way is of great significance.

[0010] The present invention has proved through a large number of studies that when macromolecular substances are mixed with water to form a solution, there is a relatively stable gradual fusion process, which will gradually reduce the concentration of the macromolecular substances as the fusion time is prolonged until a complete equilibrium is finally reached. However, small molecules are extremely fast in fusion with water and quickly disperse when they encounter water, and this process cannot be achieved. Therefore, if this characteristic of macromolecular substances can be utilized, that is, in the process of fusion with water, directly spotting macromolecular microdroplets containing different concentrations can be obtained. For this reason, the present invention has developed a microdroplet concentration gradient array generation device, which can fully grasp, utilize and control the process of fusion of macromolecular substances with water, and directly form a microdroplet array with controllable concentration gradient through the simplest structure and the simplest method.

[0011] The way of absorbing the solution through the aspiration mechanism is the key to generating a concentration gradient micro-droplet array. Because the aspiration mechanism does not absorb the pre-mixed sample solution with a stable concentration, but absorbs the sample solution and the solvent separately, the latter causes the concentration of the solution inside the aspiration mechanism after absorbing the required liquid to change with the mixed concentration of the two solutions. When the sample solution is absorbed first and then the solvent, the concentration of the droplets dripping from the capillary increases from low to high over time, so the micro-droplet array formed is also from low concentration to high concentration; conversely, if the solvent is absorbed first and then the sample solution, a micro-droplet array from high concentration to low concentration is formed.

[0012] Furthermore, the macromolecular sample includes nucleic acid or protein; and the solvent includes water and / or buffer.

[0013] The controllable fusion process of macromolecules and water requires the following three conditions to be met: first, the macromolecules can be evenly dispersed or dissolved in water; second, the entire process of dispersion or dissolution can be carried out at a uniform speed; third, the time required for the macromolecules to disperse or dissolve in water cannot be too long or too short. If it is too long, the time required for spotting will be too long, resulting in too long waiting time; if it is too short, it will be completely dispersed before the array is formed, making it difficult to control.

[0014] Macromolecular substances include nucleic acids, polypeptides, proteins, polysaccharides, etc., of which nucleic acids or proteins are preferred, which can more effectively control the time of fusion with water to achieve spotting. Among them, nucleic acids are most preferred, because their molecular weight is more suitable, easy to control, and the dispersion or dissolution speed is more uniform.

[0015] It is understandable that the microdroplet array generated by the device can be used for the study of molecular interactions, that is, the sample solution (such as nucleic acid aptamers, antibodies, etc.) is dripped into the array according to the concentration gradient and fixed in the array, and then combined with SPRi technology to screen the molecules that interact with the sample solution. The fixation methods of different samples are inconsistent. The sample solution contains nucleic acids or proteins, etc., and the screening objects for molecular interactions with them also include nucleic acids or proteins. Therefore, the microdroplet array can be used to explore the interactions between nucleic acids-nucleic acids, nucleic acids-proteins and proteins-proteins, such as for screening high-affinity nucleic acid aptamers, selecting suitable antibodies or antigens, etc.

[0016] Furthermore, the macromolecular sample includes nucleic acid aptamers, and the base length of the nucleic acid aptamer is 40 to 180 bp.

[0017] Nucleic acid aptamers are small oligonucleotide sequences or short polypeptides obtained through in vitro screening, which can bind to the corresponding ligand with high affinity and strong specificity. Under the appropriate base length, the dispersion or dissolution speed of nucleic acid aptamers in water is relatively uniform, and the required overall dispersion time can be controlled within 90 to 120 seconds, so that microdroplets of different concentrations can be dripped out every 10 seconds or so, and the droplet concentration remains basically stable within 4 seconds.

[0018] Furthermore, the concentration of the nucleic acid aptamer in the sample solution is 1 to 20 μmol / L; and the volume ratio of the sample solution to the solvent is 1:3 to 1:10.

[0019] Furthermore, the capillary is a glass capillary with an inner diameter of 20 to 40 μm and a length of 40 to 50 mm.

[0020] Glass capillaries with appropriate pore size and uniform thickness can be used to accurately dispense microdroplets with a specific concentration gradient. If there is uneven thickness or the pore size fluctuates, it will directly affect the control of the concentration gradient. Therefore, in order to ensure that the pore size of the glass capillary is appropriate and the thickness is uniform, the preparation process of the glass capillary is very important.

[0021] It is understandable that the length of the glass capillary should not be too long, as a too long length will prolong the time the droplets stay there and will not be conducive to controlling the concentration of the droplets being dripped in, because there is a certain time delay from the start of concentration adjustment to the arrival of the droplets of the adjusted concentration; conversely, if the length of the glass capillary is too short, the volume of the droplets stored in the glass capillary will be too small. If the volume of the solution stored at one time is less than the total volume of droplets required for the same concentration (3 to 5 droplets are required for the same concentration), the concentration of droplets in the same batch in the array will vary greatly, thereby failing to achieve the purpose of generating a droplet array with a concentration gradient, and then affecting the experimental results and the credibility of the experimental results.

[0022] Furthermore, the glass capillary is prepared by drawing a glass tube, breaking the needle and subjecting it to hydrophobic treatment, wherein the hydrophobic treatment includes treatment with octyltrichlorosilane and 1H,1H,2H,2H-perfluorooctyltrichlorosilane.

[0023] The capillary is made by drawing, breaking and hydrophobicizing existing glass capillaries and is used for spotting samples.

[0024] The hydrophobic treatment refers to the hydrophobic treatment of both the inner and outer walls of the capillary tip. The hydrophobic treatment can prevent the droplets from adhering to the inner wall of the capillary when passing through the capillary, and then the droplets ejected from the capillary are three-dimensional, round, and have strong surface tension, and can ensure that the volume of the droplets ejected from the capillary each time is consistent, reducing the differences between different droplets, and then reducing the errors between different experimental groups.

[0025] In some embodiments, the mass ratio of xylene to octadecyltrichlorosilane is 99:1; and the density of the 1H,1H,2H,2H-perfluorooctyltrichlorosilane is 1.3 g / mL.

[0026] Furthermore, the liquid aspiration mechanism includes a ball screw pusher and a piston tube, and the piston tube sucks or pushes out the liquid solution under the action of the ball screw pusher.

[0027] Since multiple aspirations are required, in order to more accurately control the amount of liquid sucked in and pushed out by the piston tube, the present invention adopts a ball screw pusher to suck and push out the sample solution, thereby minimizing the loss caused by instrument friction and improving the control accuracy of trace liquids.

[0028] Furthermore, the spotting mechanism also includes a three-axis spotting module and a capillary fixing fixture; the plane includes the surface of the chip; the capillary is fixed by the capillary fixing fixture, and the three-axis movement module can drive the capillary to move in the X-axis and Z-axis and drive the chip to move in the Y-axis.

[0029] The fusion process of the sample solution and the solvent in the piston tube is short, and the time for the droplets to drip out through the capillary is also very short. The spotting module is required to spot samples during this fusion process, and the process must be accurate and efficient. The present invention uses a three-axis moving module to quickly and accurately drive the spotting module and the metal chip to move, ensuring that each droplet is accurately dropped on the correct position on the chip surface through the capillary, and the time needs to be accurately controlled to ensure that the micro droplets dropped on the specific position are the required specific concentration of micro droplets.

[0030] Furthermore, the surface of the chip is coated with a coating and subjected to a hydrophobic treatment to be hydrophobic, wherein the hydrophobic treatment comprises treatment with an ethanol solution.

[0031] In some embodiments, the ethanol solution is a 99% ethanol solution.

[0032] The chip must first be prepared with a microporous metal surface using a one-step electrodeposition method, and then treated with a 99% ethanol solution for hydrophobicity. Treatment with ethanol can also better utilize the one-step electrodeposition method to prepare a chip with a microporous metal surface, so that the dodecyl chains are "pulled" out, and the surface of the metal chip is transformed into a superhydrophobic surface, which can increase the contact angle of the microdroplets, making the droplets more concentrated and full, making the droplets easier to form after spotting, and allowing more liquid to be carried above the unit contact area, that is, containing more solutes, which is conducive to the fixation of nucleic acid aptamers.

[0033] In some embodiments, the coating is a gold coating.

[0034] It should be understood that when the concentration gradient microdroplet array is applied to the study of the interaction between nucleic acids and proteins, the use of a chip made of gold can greatly reduce the difficulty of fixing nucleic acid aptamers and shorten the fixation time, because nucleic acids can be fixed by self-assembly, that is, when synthesizing DNA / RNA chains, one end of the nucleic acid chain is directly modified with a thiol group (-SH), and the Au-S bond formed by the covalent binding of the thiol group and the gold (Au) surface is used to directly fix the DNA / RNA on the gold surface, so the fixation process can be simplified, the fixation time can be shortened, and the detection efficiency can be improved.

[0035] In another aspect, the present invention provides a method for preparing a concentration gradient microdroplet array, wherein the method is prepared using the above-mentioned device and comprises the following steps:

[0036] (1) aspirating the solution through the liquid aspiration mechanism, first aspirating the sample solution and then aspirating the solvent, or first aspirating the solvent and then aspirating the sample solution;

[0037] (2) A concentration gradient microdroplet array is spotted on the chip surface through a spotting mechanism.

[0038] In some embodiments, when the solution is aspirated by the aspiration mechanism in step (1), the sample solution is aspirated first and then the solvent.

[0039] It should be understood that if the sample solution is aspirated first and then the solvent, according to the placement of the piston tube when it is working, the sample solution is located in the upper layer and the solvent is located in the lower layer. The solution with high density will diffuse downward under the action of gravity, accelerating the mixing process of the two solutions, making the mixing process of the sample and the solvent more uniform, and more conducive to generating an accurate concentration gradient microdroplet array; on the contrary, if the sample solution is located in the lower layer and the solvent is located in the upper layer, the speed at which the sample solution diffuses upward to the solvent will slow down under the action of gravity, and the internal space of the piston tube is limited, resulting in the fusion process of the two solutions being hindered, which will prolong the time to generate the concentration gradient microarray, and may even lead to inconsistent local concentration gradients, affecting the concentration accuracy of the concentration gradient microdroplets. Therefore, it is more preferred to adopt the method of aspirating the sample solution first and then the solvent.

[0040] Furthermore, step (2) includes the spotting mechanism performing discontinuous spotting on the chip surface, that is, after completing the first round of spotting, discarding the second round of droplets flowing out of the capillary, and using the third round of droplets for spotting to ensure that the droplet array has a concentration gradient.

[0041] It takes a certain amount of time for the sample solution to mix with the solvent. In order to ensure that the concentration gradient between the droplets formed on the chip surface is accurately controllable, the chip must discard the next round of 3 to 6 droplets (about 3 to 5 seconds) after collecting a round of 3 to 5 droplets (about 3 to 4 seconds), and then collect the third round of droplets, thereby ensuring that there is a controllable concentration difference between the droplets formed in the first and third rounds, and the cycle is repeated in sequence to form a stable concentration gradient microarray.

[0042] The discontinuous spotting method requires the cooperation of a three-axis moving module, that is, when it is necessary to discard droplets, the moving device in the Y-axis direction will drive the metal chip out of the spotting range of the spotting module, and the spotting module will not move. At the same time, the liquid pusher of the ball screw structure continues to discharge liquid at the original rate, and 3 to 6 droplets will be discarded.

[0043] In another aspect, the present invention provides a use of a nucleic acid aptamer solution for preparing a reagent for directly spotting a concentration gradient micro-droplet array through a capillary, wherein the base length of the nucleic acid aptamer is 40 to 180 bp.

[0044] On the other hand, the present invention provides a use of a capillary for preparing a device for directly dotting a concentration gradient micro-droplet array on a plane, wherein the capillary is a hydrophobic glass capillary with an inner diameter of 20 to 40 μm and a length of 40 to 50 mm; the glass capillary is prepared by drawing a glass tube, breaking the needle and subjecting it to a hydrophobic treatment, wherein the hydrophobic treatment includes treatment with octyltrichlorosilane and 1H,1H,2H,2H-perfluorooctyltrichlorosilane.

[0045] The present invention combines SPRi technology with micro-nanofluidics technology and uses a micro-droplet array for spotting, that is, spotting a micro-droplet array with a concentration gradient on the surface of a cleaned gold chip, and detecting the interaction of biomolecules in real time by monitoring the changes in optical signals caused by the binding of biomolecules to the metal surface. The beneficial effects include:

[0046] 1. The liquid pusher with ball screw structure can be used with capillary to dispense tiny droplets of 4-10nL, which is much smaller than the 0.25μL dispensed by the liquid gun;

[0047] 2. The three-axis mobile spotting device can spot a large area of ​​small volume droplet arrays, greatly increasing the number of samples that can be tested at one time, providing a large amount of effective test data for subsequent SPRi testing; at the same time, the automated operation is both accurate and convenient, and does not rely on expensive dispensing equipment;

[0048] 3. The use of glass capillaries greatly simplifies the cumbersome spotting operation of traditional dispensing machines. It does not require multiple suction and cleaning of the liquid outlet like a dispensing machine. Only one suction is required to achieve the setting of the liquid concentration gradient in the droplet, thereby reducing the difficulty of spotting, greatly saving the time of spotting, improving the layout of the concentration gradient dot array required in affinity research and improving the overall efficiency of detection;

[0049] 4. The cost of the device provided by the present invention is only 10% of that of the piezoelectric ceramic actuator of the traditional dispensing machine, but it can also dispense nL level liquids, and the whole process is low-cost, automated control, and the data is highly reliable;

[0050] 5. The capillary tube treated with hydrophobicity first absorbs the sample solution, and then absorbs pure water or buffer to dilute the sample solution; combined with the discontinuous spotting method, the sample solution and the solvent are fused while dripping out microdroplets through the capillary tube, and the concentration of each microdroplet is accurately controllable, thereby achieving the direct generation of concentration gradient microdroplet arrays with the simplest structure and the most convenient method, which can be used to accurately detect the affinity between nucleic acid aptamers and target proteins, and can also be used for other molecular interaction studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1 : Overall schematic diagram of the device described in Example 2

[0053] Figure 2 : Front view of the device described in Example 2

[0054] Figure 3 : A side view of the device described in Example 2

[0055] Figure 4 : The connection between the capillary tube 23 and the piston tube 35 of the device described in Example 2

[0056] Figure 5 : Schematic diagram of the three-axis mobile module 1 described in Example 2

[0057] Figure 6 : Schematic diagram of the spotting module 2 described in Example 2

[0058] Figure 7 : Schematic diagram of the capillary 23 described in Example 2

[0059] Figure 8 : Schematic diagram of the liquid push module 3 described in Example 2

[0060] Fig. 9 : Schematic diagram of the metal chip 4 described in Example 2

[0061] Explanation of the numbers in the accompanying drawings: three-axis moving module 1, three-axis device table 11, X-axis direction screw 12, cylindrical rod 13, moving bracket 14, Z-axis direction screw 15, stepper motor 16, Y-axis direction moving platform 17, cylindrical rod 18; spotting module 2, dropper clamp 21, fixing rod 22, glass capillary 23, capillary body 231, capillary tip 232, clamp 24, screw 25; liquid pushing module 3, stepper motor 31, fixing plate 32, screw 33, ball nut 34, piston tube 35; metal chip 4, metal chip coating 41, glass prism 42, micro droplet array 43. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0063] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0064] Example 1: Preparation and hydrophobic treatment of glass capillaries

[0065] This embodiment provides a preparation method and a hydrophobic treatment method for a glass capillary, and the specific steps are as follows:

[0066] (1) Using a tube puller (Sutter P1000), a round glass capillary tube (World Precision Instruments, Inc.) with a length of 10 to 12 cm (preferably 12 cm in this embodiment) and an outer diameter and an inner diameter in the range of 0.91 to 1.06 mm (preferably 1 mm in this embodiment) and 0.55 to 0.60 mm (preferably 0.6 mm in this embodiment) was pulled. 1B100-6) is stretched and broken into 2-3 sections, and the stretched tip is used for subsequent broken needle treatment; the broken glass capillary is placed on a broken needle instrument (M4-01, laboratory-made-China University of Metrology), with the tip facing the silicon ball, the silicon ball is embedded on the platinum wire, the power supply is turned on, the power supply voltage is set to 1.5V-2.0V (preferably 2.0V in this embodiment), and the three-axis platform (China Runjia LD60) is moved to break the tip, and the broken needle diameter can be measured under an optical microscope (Shanghai Liwan Optoelectronic Equipment Co., Ltd. LW300LFT) until the required width of the experiment is reached, that is, 20-40μm (preferably 25μm in this embodiment);

[0067] (2) After the needle is broken, the end of the needle that is not broken is connected to a capillary Teflon tube, and pure water is passed through it for cleaning to prevent residual impurities from clogging the capillary tube. Then nitrogen is passed through it for drying;

[0068] (3) Octyltrichlorosilane (OTS, Shanghai MacLean Biochemical Technology Co., Ltd.) and 1H,1H,2H,2H-perfluorooctyltrichlorosilane (Shanghai Enfujia Technology Co., Ltd.) (the preferred density of 1H,1H,2H,2H-perfluorooctyltrichlorosilane in this embodiment is 1.3 g / mL) with a mass ratio of xylene to octadecyltrichlorosilane of 99:1 (preferably 14.85 g xylene and 0.15 g octadecyltrichlorosilane in this embodiment) were used to hydrophobically treat the glass capillary after the needle was broken, so as to better spot the sample onto the droplet array. The tip of the glass capillary needs to be treated with double-wall hydrophobic treatment, that is, both the inner and outer walls of the capillary need to be treated. The end to be treated is inserted into the OTS solution, soaked for 60 to 90 seconds, then taken out (preferably 60 seconds in this embodiment), and placed on a hot plate at 150 to 180°C for 40 to 60 minutes (preferably 60 minutes in this embodiment); after heating, cool and stand for 5 to 10 minutes (preferably 8 minutes in this embodiment), and then the treated end is inserted into a 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane solution and soaked for 50 to 80 seconds (preferably 60 seconds in this embodiment), then taken out, and placed on a hot plate at 100 to 160°C for 30 to 40 minutes (preferably 30 minutes in this embodiment);

[0069] (4) After heating is completed, the glass capillary is tested for water flow. The tail end of the capillary that has completed the hydrophobic treatment is connected to the capillary Teflon tube, and a syringe filled with pure water is slowly pushed. If water can flow out from the tip, it means that the capillary is not blocked and subsequent experiments can be carried out. Otherwise, the processing fails and the above steps are repeated to reprocess.

[0070] Example 2: A simple micro-droplet concentration gradient array generation device for molecular interaction research

[0071] The present invention provides a simple micro-droplet concentration gradient array generation device for molecular interaction research ( Figures 1 to 3 ), the device is divided into four parts: a three-axis moving module 1, a spotting module 2 with an embedded stepping motor, a liquid pushing module 3 with a ball screw structure, and a metal chip 4. The three-axis moving module 1 and the spotting module 2 are connected together by a screw 15 and a fixed rod 22; the three-axis moving module 1 is spliced ​​with the liquid pushing module 3 by a moving bracket 14 and a fixed plate 32; the capillary 23 in the spotting module 2 and the piston tube 35 in the liquid pushing module 3 use the difference in inner and outer diameters (the outer diameter of the capillary 23 is slightly smaller than the inner diameter of the piston tube) and the elasticity of the piston tube to directly insert the capillary 23 into the end of the piston tube 35 (such as Figure 4 and the metal chip is located below the capillary in the spotting module 2, which is placed above the Y-axis moving platform 17 and is located within the moving range of the spotting module 2 (the central area of ​​the moving range is preferred in this embodiment).

[0072] like Figure 5 As shown, the three-axis mobile module 1 mainly includes eight parts 11 to 18. There is an X-axis screw rod 12 with a length of 20 cm and a diameter of 1 cm above the three-axis device platform 11; there are a cylindrical rod 13 and a cylindrical rod 18 with a length of 12 cm and a diameter of 0.8 cm above and below the X-axis screw rod 12; the X-axis screw rod 12 and the cylindrical rod 13 pass through the mobile bracket 14, which mainly plays a fixing role. A Z-axis screw rod 15 with a length of 6 cm and a diameter of 0.8 cm is fixed on the front of the mobile bracket 14. On the right side is a square stepping motor 16 with a length of nearly 8 cm. The stepping motor 16 is connected to the X-axis screw rod 12. When the motor 16 is working, it drives the mobile bracket 14 and the X-axis screw rod 12 to move in the X-axis direction; below the mobile bracket 14 is a Y-axis mobile platform 17 with a length of 22 cm, a width of 16 cm, and a thickness of 2 cm. It can be understood that the function of the three-axis moving module 1 is to enable the spotting module 2 to move in the X-axis and Z-axis directions; because the metal chip 4 is placed on the Y-axis moving platform 17, the three-axis moving module 1 drives the metal chip 4 to move in the Y-axis direction.

[0073] like Figure 6 As shown, the spotting module 2 includes five parts 21 to 25. The main body is a dripper fixture 21 with an edge length of 6 cm and an embedded square stepper motor with an edge length of nearly 5 cm. The top of the dripper fixture is a rectangular fixed rod 22 with a length of 5 cm and a diameter of 1 cm, and the end is a fixed rod 22 with a screw hole. The Z-axis screw 15 can be inserted into the screw hole of the fixed rod 22, and then the three-axis moving module 1 is connected to the dripper fixture 21. Then, driven by the stepper motor 16, the dripper fixture 21 can be moved in the Z-axis direction; the front of the dripper fixture 21 has a groove, which is mainly used to place the glass capillary 23 prepared and treated according to the method described in Example 1, with an outer diameter ranging from 0.8 to 1.2 mm (preferably 1 mm in this embodiment), and the lengths of the capillary body 231 and the capillary tip 232 are 40 to 50 mm (preferably 45 mm in this embodiment) and 20 to 50 μm (preferably 30 μm in this embodiment), respectively. The structure of the capillary 23 is as follows Figure 7As shown. The silicon content and boron content of the capillary 23 reach 90% and 10% respectively, so that its linear expansion coefficient reaches the effect of easy needle breaking and easy needle pulling, which is beneficial to better point out the droplet array; it is understandable that the outer diameter of the glass capillary 23 is too large to pull the needle and break the needle to the required caliber, so the above outer diameter range is most suitable. The diameter range of the tip 232 of the glass capillary after the needle pulling and breaking operation is 20 to 30μm (preferably 30μm in this embodiment) to point out nL-level droplets that meet the requirements. The volume of the droplet is 30 to 50nL, which is much smaller than the 0.25μL pointed out by the liquid gun; in addition, the clamp 24 clamps the glass capillary 23 and is fixed to the clamp 24 with screws 25.

[0074] Depend on Figure 8 As shown, the liquid pushing module 3 with ball screw structure mainly includes five parts 31 to 35. The stepper motor 31 is in the shape of a cube with an edge length of nearly 4 cm, and below it is a fixed plate 32, which is used to be fixed on the mobile bracket 14 of the three-axis mobile module 1 and glued with quick-drying glue, and moves with the mobile bracket 14; the screw 33 is a cylindrical structure with a length of 6 cm and a diameter of 0.6 cm. The ball nut 34 moves under the drive of the stepper motor 31. The ball nut 34 is used to help push the piston tube 35 fixed at the tail end of the screw 33 and detachable. Its maximum volume is 1 mL, which is used to load the sample liquid.

[0075] like Fig. 9 As shown, the metal chip 4 mainly includes three parts 41 to 43, and the material of the metal chip is preferably gold. Among them, the metal chip 4 is a cuboid with a length of 2.5 cm, a width of 1 cm, a thickness of 0.1 cm, and a gold coating 41 on the surface; the metal chip 4 has been hydrophobically treated, that is, treated with 99% ethanol, so that the dodecyl chain is "pulled" out, and then transformed into a super-hydrophobic surface, which can increase the contact angle of the droplet, making the droplet more concentrated and full. At the same time, the ethanol treatment can also better utilize the one-step electrodeposition method to prepare a chip 4 with a microporous metal surface; under the chip is a glass prism 42, which is used for the light refraction required for SPRi detection, and the chip 4 and the prism 42 are adhered with refractive index oil (Thermo Fisher); the micro-droplet array 43 is an array with a concentration gradient that is sampled using the device described in this embodiment, and the interval between the droplets ranges from 1 to 2 mm (preferably 2 mm in this embodiment).

[0076] The steps for establishing a microdroplet array with a concentration gradient using the above device are as follows:

[0077] (1) According to the experimental requirements, 0.2-0.3 mL (preferably 2 mL in this embodiment) of the configured aptamer solution is first sucked into a piston tube 35 with a specification of 1 mL. The aptamer refers to a short segment of oligonucleotide or short polypeptide obtained by in vitro screening, which has the characteristics of high affinity and strong specificity; then, according to the experimental requirements, 0.1-0.2 mL of pure water is first sucked and then a buffer solution is sucked (preferably 2 mL of PBS buffer solution is sucked in this embodiment), so as to achieve a concentration gradient from high to low inside the piston tube 35;

[0078] (2) The moving speed of the stepper motor of the dripper fixture 21 is 0.5-1 mm / s (preferably 1 mm / s in this embodiment), and the single moving distance of the dripper fixture 21 is set to 1-2 mm (preferably 2 mm in this embodiment), and the round trip distance is 3-4 mm (preferably 4 mm in this embodiment);

[0079] (3) The glass capillary 23 touches the surface of the metal chip 4 downward once every 2 seconds to complete the semi-contact spotting and form a droplet with a volume of 30 to 50 nL; it returns to the initial position and continues the next spotting. After 3 to 5 droplets are spotted each time (preferably 5 droplets in this embodiment), the moving platform 14 in the Y-axis direction will move 2 to 4 cm (preferably 4 cm in this embodiment), that is, the metal chip 4 leaves the spotting range of the spotting module 2, and the stepper motors 16 in the X-axis direction and the Z-axis direction stop working, but the liquid pusher module 3 with a ball screw structure continues to discharge liquid at the original rate, that is, 3 to 6 droplets (preferably 5 droplets in this embodiment) are wasted; the moving platform 14 in the Y-axis direction moves back to the previous position in the opposite direction; at the same time, the stepper motors 16 in the X-axis direction and the Z-axis direction start working to continue spotting; this process is a discontinuous acceptance of droplets;

[0080] (4) Repeating the spotting operation of step (3) will form different concentration gradient microdroplet arrays; the concentration gradient achieved is in the range of 1-20 μmol / L, and the aptamer and water are completely fused in the range of 90s to 120s (preferably 120s in this embodiment), so the spotting needs to be completed within this time range.

[0081] It should be understood that the reason why the micro-droplet array with a concentration gradient can be pointed out is that the concentration of the droplets dripped from the capillary 23 is inconsistent at different times. This is because the piston tube 35 first absorbs the nucleic acid aptamer solution and then absorbs pure water or buffer, rather than pre-preparing the nucleic acid aptamer solution to a fixed concentration. Therefore, with the change of time, the degree of mixing of the nucleic acid aptamer solution and pure water or buffer is also changing, that is, the concentration of the solvent (pure water or buffer) near one end of the capillary 23 will become higher and higher, and then the concentration of the droplets dripping from the capillary 23 will become higher and higher. Therefore, the droplets collected on the metal chip 4 are arranged from low to high concentration; and because it takes a certain amount of time for the nucleic acid aptamer solution to mix with pure water or buffer, in order to ensure that there is a certain difference in concentration between the droplets collected by the metal chip 4 one after another, after the metal chip collects a round (3 to 5) of droplets each time, it discards the next round (3 to 6) of droplets, and then collects the third round of droplets, and the cycle is repeated.

[0082] Since the larger the motion range of the three-axis platform, the lower its accuracy, the overall length, width and height of the spotting device designed in the present invention are 25 cm, 10 cm and 20 cm respectively; considering the spotting operation and accuracy, the moving range of the spotting module 2 is more appropriately 3 to 5 times the corresponding size of the chip (preferably 3 times in this embodiment).

[0083] In summary, the printing of a micro-droplet array with a concentration gradient requires the cooperation of a three-axis (X, Y and Z axis) moving device, a discontinuous droplet receiving device, a hydrophobic treated glass capillary and a piston tube 35 for absorbing liquid; the hydrophobic treatment method of the glass capillary is the same as that described in Example 1, and the piston tube 35 absorbs liquid by first absorbing the aptamer solution and then absorbing pure water or buffer solution to dilute the aptamer solution.

[0084] After establishing a microarray with an aptamer concentration of 0.8-1 μM on the cleaned chip surface according to the above-mentioned spotting method, the microarray is placed in a closed and humid environment (humidity is 80%-90%) for 20-24 hours (preferably 20 hours in this embodiment), and then placed in a dry environment at 25° C. overnight to complete the fixation of the aptamer, the aptamer is a protein or a nucleic acid, and the specific steps of protein and nucleic acid fixation are as follows:

[0085] Protein immobilization can be achieved by using the carboxyl magnetic bead method, which is a method of covalently coupling proteins to the surface of magnetic beads. First, carboxyl magnetic beads (Beijing Biolab Technology Co., Ltd.) were incubated with an activator in 2-(morpholino)ethanesulfonic acid buffer (MES buffer, pH 6.0, 0.02M) at room temperature for 0.5h to obtain activated magnetic beads, wherein the activator was a mixture of 0.03M EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.03M NHS (N-hydroxysuccinimide) in a volume ratio of 1:1; the activated magnetic beads were incubated with the protein to be fixed in 0.02M MES buffer (pH6.0) for 1h at 25°C to obtain magnetic beads coupled with the protein; the magnetic beads coupled with the protein were treated with a blocking solution for 2h to block the active sites on the protein surface, wherein the blocking solution was a 0.05%-5% bovine serum albumin buffer (Thermo Fisher); finally, the obtained magnetic beads were shaken and stored at 4°C for later use.

[0086] If the recognition element is an antibody, one of the most commonly used methods for fixing the antibody is to fix the antibody on a solid surface by bioconjugation. For example, biotin and streptavidin can be combined to form a stable biotin-streptavidin complex, and then the antibody can be coupled to the solid material. The specific process includes antibody labeling, preparation of the solid support, and antibody fixation. First, according to the instructions of the biotinylation reagent (Thermo Fisher), add the biotinylation reagent to the antibody solution (buffer solution is 0.1M PBS at pH 7.2, and the molar ratio of biotin to antibody molecules is 1:20), and react at room temperature for 1-2 hours; use centrifugal filtration to remove unbound biotin; prepare a solid support (such as a streptavidin-coated microplate); according to the product instructions, prepare a streptavidin solution, usually at a concentration of 1-10μg / mL; add the streptavidin solution to the solid support and incubate for 1 hour; add the biotinylated antibody to the streptavidin-coated solid support and incubate for 1-2 hours to ensure that the antibody is bound to the solid phase.

[0087] The methods for fixing nucleic acids mainly include covalent fixation, adsorption fixation, self-assembly, etc., which can be selected according to specific application requirements. Self-assembly is generally used to fix nucleic acids on gold chips, that is, when synthesizing DNA / RNA chains, one end of the nucleic acid chain is directly modified with a thiol group (-SH), and the Au-S bond formed by the covalent binding of the thiol group and the gold (Au) surface is used to directly fix the DNA / RNA on the gold surface. The specific operation steps are as follows: the thiol-modified nucleic acid is dissolved in 0.01M PBS buffer, and the nucleic acid solution is deposited on the surface of the gold chip with a volume of 0.25μL per droplet, and then the biochip is placed in a dark, room temperature, 80% humidity environment and incubated for 2h, and then dried overnight at room temperature to complete the fixation of the oligonucleotide sequence; finally, the gold chip with the fixed nucleic acid is immersed in a PEG 2000 solution (Sigma-aldrich) with a concentration of 10-150μM for blocking to block non-specific binding sites.

[0088] After the biochip is simply cleaned and dried, it is placed in a surface plasmon resonance imaging spectrometer (Open PleX, Horiba, France) for detection. Different concentrations (25-200nM) of target protein solutions are injected and flowed through the chip surface at a flow rate of 50μL / min, respectively, to specifically bind to the aptamer, and the signal response value generated by each spot at different concentrations of target protein is recorded. Sensor chips are generally regenerative, and the target-aptamer dissociation can be carried out by injecting 50mM NaOH. After dissociation, the chip surface is rinsed with running buffer (HEPES10mM, MgCl2 5mM, NaCl 150mM, pH=7.4), and the next concentration of analyte is injected for detection.

[0089] Because proteins are easily degraded at room temperature, thus affecting the experimental results, when the above-mentioned system for detecting molecular interactions contains proteins, it is more appropriate to conduct experimental operations under low temperature conditions. Therefore, it can be understood that the spotting device provided in this embodiment can be used under low temperature conditions and can also be used in conjunction with a refrigeration device.

[0090] In summary, the spotting device provided in this embodiment has the characteristics of low cost, easy operation, automation, etc., and can be used to generate microdroplet arrays with concentration gradients, which can be used for molecular interaction research, including nucleic acid-protein interaction and protein-protein interaction.

[0091] Example 3: Specific implementation of the device described in Example 2 combined with SPRi technology

[0092] This example provides a specific implementation scheme of combining the device described in Example 2 with the SPRi technology. The device mainly uses a semi-contact spotting method of a glass capillary for spotting. The specific steps are as follows:

[0093] (a) Pre-treating the DNA sample. To prevent the DNA powder from adhering to the tube wall, centrifuge it first, then add a certain amount of PBS buffer (the existing 10×PBS (Shanghai Aladdin), add a certain amount of water to make it 1×PBS), prepare a 10 μM solution, and oscillate on a vortex for 5 minutes;

[0094] (b) Use a piston tube 35 (Shenzhen Baitong Tools Co., Ltd.) with a specification of 1 mL to first absorb 0.2 mL of the aptamer solution prepared in step (a), and then absorb 0.2 mL of pure water, so as to achieve a concentration gradient from high to low inside the piston tube 35. The piston tube 35 is fixed to the tail of the ball screw 33. After debugging and calculation, the movement speed of the ball nut 34 is preferably 0.5 mm / s, and the liquid is pushed in a top-to-bottom order;

[0095] (c) Place the gold chip 4 on the Y-axis moving platform 17, and fix the hydrophobic treated glass capillary 23 on the dripper fixture 21. The tail end of the capillary that has not been pulled or broken faces upward and is connected to the liquid outlet of the piston tube 35. The tip of the glass capillary that has been pulled or broken faces downward and is used for liquid discharge. Fix the device and proceed to the next step after the inspection is completed.

[0096] (d) The liquid push module 3 starts working and pushes the solution. When a stable and uniform droplet flows out of the needle mouth, the spotting operation can be performed. At the same time, the start button of the dripper fixture 21 and the stepper motor 16 of the three-axis device platform 11 is pressed to move in the three directions of X, Y and Z axes.

[0097] (e) The moving speed of the dripper fixture 21 is 1 mm / s, and the single moving distance of the dripper is set to 2 mm, and the round trip distance is 4 mm. The glass capillary 23 touches the gold chip surface 41 downward once every 2 seconds to complete the semi-contact spotting, forming a 30-50 nL droplet, returning to the initial position, and then continuing the next spotting;

[0098] (f) The moving speed of the stepper motor 16 of the three-axis device stage 11 is 0.1 mm / s. According to the sampling interval requirements, it can be selected to move once every 1s to 2s to arrange the sampling in the X-axis direction; the moving speed of the Y-axis moving platform 17 is 0.5 mm / s, and the sampling arrangement is carried out back and forth in the Y-axis direction;

[0099] (g) After 3 to 5 droplets are spotted each time, the chip 4 is removed, that is, after several droplets are wasted, the chip 4 receives the droplets again, thereby realizing the concentration gradient. The time required for the aptamer to completely merge with water is 90s to 120s, so it is necessary to complete the spotting of a 5×6 or other specification droplet matrix within this time range (a 5×6 matrix is ​​preferred in this embodiment); after the spotting is completed, the gold chip 40 with the droplet array is placed in a closed device with a humidity environment of 80%. After standing for 22 hours, it is taken out for detection.

[0100] After spotting, the generated microdroplet array is placed in the SPRi device to complete the detection. The specific steps are as follows:

[0101] (a) placing the spotted droplet array chip 4 in a sealed, room temperature wet box (humidity of about 80%) for incubation for 3 h to allow the aptamer to be completely fixed on the gold chip 4;

[0102] (b) After the incubation is completed, the gold chip surface 41 is cleaned with deionized water and blown dry;

[0103] (c) placing the functionalized gold surface 41 in a PEG 2000 solution and soaking it at room temperature for 1.5 h to block non-specific sites on the chip surface;

[0104] (d) After the soaking is completed, the mixture is rinsed again with deionized water and dried;

[0105] (e) cleaning the glass prism 42 substrate to prevent impurities from affecting the refraction of light, and drying it with nitrogen;

[0106] (f) The prism 42 base and the chip 41 are adhered and fixed with refractive index oil;

[0107] (g) On-machine testing: ① Turn on the surface plasmon resonance imaging spectrometer (Open PleX, Horiba, France), add pure water for cleaning, and run at high speed; ② Then add running buffer and run at low speed; ③ Create a work document in the user interface, define and name each point according to the order of spotting; ④ Calibrate the resonance angle; ⑤ Test the sample, inject a protein solution with a certain concentration, and the flow rate is 50 μL / min ⑥ Use sodium hydroxide solution to dissociate the protein-aptamer complex and regenerate the chip surface 41; ⑦ Add running buffer to rinse the residual sodium hydroxide solution; ⑧ Measure the next sample;

[0108] (h) Use software to process data and obtain the kinetic constants between the target protein and the aptamer.

[0109] Example 4: Effect of nucleic acid aptamers with different base numbers on the preparation of concentration gradient microarray

[0110] Different base lengths mean different molecular weights of nucleic acid aptamers, and molecular weights will affect the dissolution rate of the nucleic acid aptamers in the solvent, thereby affecting the preparation process of the concentration gradient microarray. Therefore, this embodiment uses the preparation and hydrophobic treatment method of the capillary in Example 1, the device described in Example 2, and the method for using the device described in Example 3 to generate a concentration gradient droplet microarray of nucleic acid aptamers of different lengths, and detects the concentration of droplets collected in the first and second rounds of the metal chip, wherein the initial concentration of the aptamer is 10 μM, the number of droplets measured in each round is 3, and the concentration of the droplets is measured using a micro-spectrophotometer (NanoDrop) (A 260 ) was measured. The specific results are shown in Table 1, where the first drop of liquid dripped out when the capillary first half-contacted the metal chip was counted as 0s, because at this time the sample solution and the solvent had just contacted, and the concentration of the generated droplets was too low, so the droplet concentration was measured starting from the second drop of liquid dripped out (2s), and the interval time between the droplets dripping was 2s, which was determined by the distance between the metal chip apertures and the moving speed of the dripper fixture; because the metal chip received the droplets in a discontinuous manner, the droplets collected by the metal chip in the second round were actually the third round of droplets dripped out by the capillary, so the time interval between the first round of droplets and the second round of droplets collected by the metal chip was 8s.

[0111] Table 1 Effect of aptamer base length on droplet concentration in concentration gradient microarray

[0112]

[0113] As shown in Table 1, as the number of bases of the aptamer increases, the concentration difference between the droplets dripped at different times becomes smaller, indicating that the increase in the number of bases will slow down the fusion rate of the sample solution and the solvent. On the one hand, this can make the concentration difference of the droplets in the same batch smaller (reflected as the standard deviation); on the other hand, if the fusion rate is too slow, it will also lead to a smaller difference in the concentration of droplets between batches, and then it is impossible to ensure that the micro-droplet array generated within a certain time has a certain concentration gradient, or in order to ensure the generation of a micro-array with a concentration gradient, the time for collecting droplets is extended and the number of discarded droplets is increased, which increases the time and money costs. In summary, combined with the experimental results in Table 1, the length of the aptamer base is more suitable between 120 and 150 bp, that is, the micro-droplet matrix generated by the nucleic acid solution with a base length in this range is better, among which 120 bp is the best.

[0114] Example 5: Effect of hydrophobic treatment of glass capillaries on preparation of concentration gradient microarray

[0115] The hydrophobic treatment of the glass capillary will cause the shape and size of the droplets dripping out, which will in turn affect the obtained concentration gradient microarray. Therefore, this embodiment uses the device described in Example 2, the method of using the device described in Example 3, different chemical reagents to perform hydrophobic treatment on the capillary (the specific operation is the same as that described in Example 1, but the reagents are different) and 120 bp nucleic acid aptamers to generate a concentration gradient droplet microarray, and uses the detection method described in Example 4 to detect the concentration of the droplets collected by the metal chip. The contact angle and spreading diameter of the droplets are photographed by a horizontal camera, and according to the spherical crown droplet volume formula The volume of the droplet is calculated. The specific experimental results are shown in Table 2.

[0116] Table 2 Effect of hydrophobic treatment reagents of glass capillaries on the preparation of concentration gradient microarrays

[0117]

[0118]

[0119] As shown in Table 2, different hydrophobic treatment methods of the capillary will affect the concentration and volume of the generated droplets. Among them, the hydrophobic treatment of the capillary using OTS and 1H,1H,2H,2H-perfluorooctyltrichlorosilane reagents has the best effect, that is, the difference in droplet concentration and volume within and between batches is not large, and the output droplets are relatively stable, indicating that the hydrophobic treatment is effective and is beneficial to the generation of concentration gradient microarrays; on the contrary, if the hydrophobic treatment of the capillary is not thorough, it will lead to the output droplet size and concentration. Unstable, that is, the volume and concentration are too low. This may be because the droplets flowing through the capillary hang on the capillary wall or are not easy to separate from the capillary tip. The concentration of the previous batch of droplets is lower than that of the next batch. When the high-concentration droplets pass through the capillary, they will merge with the previous batch of low-concentration droplets remaining in the capillary, and then the concentration will be diluted. Moreover, this dilution is random and uncontrollable, which will cause the concentration of droplets generated in the same batch to vary greatly, which is not conducive to the generation of concentration gradient microdroplet arrays. In summary, the use of capillaries treated with OTS and 1H,1H,2H,2H-perfluorooctyltrichlorosilane reagents to generate concentration gradient microdroplet arrays has the best effect.

[0120] Example 6: Effect of glass capillary length on preparation of concentration gradient microarray

[0121] The length of the glass capillary affects the volume of liquid that can be contained in the capillary. The solution in the piston tube is changing all the time. The capillary has a small capacity, weak liquid fluidity, and a small change in concentration in the tube. Therefore, in order to ensure that the concentration difference between the droplets of the same batch is small, the volume of liquid that can be contained in the capillary must be greater than the total volume of the droplets collected in one round. On the other hand, the length of the capillary should not be too long, otherwise the low-concentration droplets retained in the capillary will dilute the high-concentration droplets produced later, resulting in a large difference in the concentration of droplets in the same batch; it will also cause a delay in regulating the concentration of the droplets, that is, after issuing a control droplet instruction, the droplets generated under the instruction need to pass through a longer capillary (distance), and the time required will also increase; it will also increase the volume of the generating device and increase the production cost. Therefore, this embodiment uses the preparation and hydrophobic treatment method of the capillary in Example 1, the device described in Example 2, and the method for using the device described in Example 3 to explore the length of the glass capillary, and uses the detection method described in Example 4 to detect the concentration of the droplets. The specific results are shown in Table 3.

[0122] Table 3 Effect of glass capillary length on preparation of concentration gradient microarray

[0123]

[0124]

[0125] As shown in Table 3, when the capillary length is less than 40 mm, although the fusion state of the liquid in the piston tube can be reflected immediately, the concentration within the batch is high and the difference is large; when the capillary length is greater than 40 mm, although the difference in droplet concentration within the batch is not large, the difference in concentration between batches is small, and a microdroplet array with a significant concentration gradient cannot be generated. In summary, the most suitable length of the capillary is 40 mm.

[0126] Example 7: Effect of the order of piston tube aspirating solutions on the preparation of concentration gradient microarray

[0127] The order in which the piston tube draws the solution determines whether the droplets in the microarray are arranged from high concentration to low concentration or from low concentration to high concentration. The former can be achieved by first drawing the solvent and then drawing the sample solution, while the latter can be achieved by first drawing the sample solution and then drawing the solvent. This Example This Example uses the capillary preparation and hydrophobic treatment method in Example 1, the device described in Example 2, and the device use method described in Example 3 to explore the effect of the order in which the piston tube draws the solution on the preparation of the concentration gradient microarray, and uses the detection method described in Example 4 to detect the concentration of the droplets. The specific results are shown in Table 4.

[0128] Table 4 Effect of the length of the piston tube to draw solution on the preparation of concentration gradient microarray

[0129]

[0130] As shown in Table 4, the order in which the piston tube absorbs liquid does not affect the difference in droplet concentration within the same batch, but it will affect the concentration difference between batches, that is, the order of absorbing the solvent first and then the sample solution will reduce the concentration difference between batches. This may be because when the piston tube is placed in the working state, this way of absorbing liquid will cause the sample solution to sink to the bottom and is not easy to merge with the solvent, so the fusion speed of the two liquids slows down, and then the concentration difference formed in the same time interval is relatively small; on the contrary, if the sample solution is in the upper layer and the solvent is in the lower layer, due to the effect of gravity, the liquid with high density will move downward spontaneously, thus accelerating the fusion rate of the two solutions. In summary, whether the piston tube absorbs the sample solution first and then the solvent or absorbs the solvent first and then the sample solution, it can generate a droplet microarray with a concentration gradient, and the former generates a larger concentration gradient, while the latter generates a smaller one.

[0131] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A micro-droplet concentration gradient array generating device, characterized in that: It comprises a liquid aspiration mechanism and a spotting mechanism, wherein the liquid aspiration mechanism is used to absorb the sample solution and the solvent respectively; the spotting mechanism comprises a capillary tube, which is used to guide the liquid from the liquid aspiration mechanism and directly spot a micro-droplet array with a concentration gradient on a plane; The sample solution contains a macromolecular sample.

2. The device according to claim 1, characterized in that The macromolecular sample includes nucleic acid or protein; the solvent includes water and / or buffer.

3. The device according to claim 2, characterized in that The macromolecular sample includes nucleic acid aptamers, and the base length of the nucleic acid aptamers is 40 to 180 bp.

4. The device according to claim 3, characterized in that The concentration of the nucleic acid aptamer in the sample solution is 1 to 20 μmol / L; the volume ratio of the sample solution to the solvent is 1:3 to 1:

10.

5. The device according to claim 5, characterized in that The glass capillary is prepared by drawing a glass tube, breaking the needle and performing a hydrophobic treatment, wherein the hydrophobic treatment includes using octyltrichlorosilane and 1H,1H,2H,2H-perfluorooctyltrichlorosilane for treatment.

6. The device according to claim 1, characterized in that The liquid suction mechanism comprises a ball screw liquid pusher and a piston tube, and the piston tube sucks or pushes out the liquid solution under the action of the ball screw liquid pusher.

7. The device according to claim 1, characterized in that The spotting mechanism also includes a three-axis spotting module and a capillary fixing fixture; the plane includes the surface of the chip; the capillary is fixed by the capillary fixing fixture, and the three-axis moving module can drive the capillary to move in the X-axis and Z-axis and drive the chip to move in the Y-axis.

8. The device according to claim 8, characterized in that The surface of the chip is coated with a coating and treated with hydrophobicity to be hydrophobic, wherein the hydrophobic treatment includes treatment with an ethanol solution.

9. A method for preparing a concentration gradient microdroplet array, characterized in that: The method is prepared by using the device according to any one of claims 1 to 9, comprising the following steps: (1) aspirating the solution through the liquid aspiration mechanism, first aspirating the sample solution and then aspirating the solvent, or first aspirating the solvent and then aspirating the sample solution; (2) A concentration gradient microdroplet array is spotted on the chip surface through a spotting mechanism.

10. A use of a nucleic acid aptamer solution for preparing a reagent for directly spotting a concentration gradient microdroplet array through a capillary, characterized in that: The base length of the nucleic acid aptamer is 40 to 180 bp.

Images