A novel high-throughput closed microarray printing system

Abstract

The present invention relates to the field of biochip technology, and specifically to a novel high-throughput closed microarray printing system, comprising: a microarray printing module, including a microarray printing plate and a temperature control unit, for printing nucleic acids, proteins, etc. in a microarray production solution onto a chip surface while providing an optimal adsorption temperature; a microarray printing liquid path system, including a sample addition pump, a liquid supply pump and a valve, for continuously adding a fixed volume of microarray production solution to the microarray printing module; and a microarray printing motion system, for moving the microarray printing module. The present invention can improve the uniformity between microarray spots, avoid cross contamination between spots, and especially improve the protein fixation amount of spots in a protein microarray, so as to break through the bottleneck restricting the development of biochips.

Description

Technical Field

[0001] The invention relates to the technical field of biochips, and in particular to a novel high-throughput closed microarray printing system. Background Art

[0002] Biochip is a detection method that uses a microarray made on a substrate to achieve specific functions. It can be applied to a wide range of fields including genomics, proteomics, lipidomics, metabolomics and cytomics. Due to the advantages of high detection throughput, high sensitivity, low sample / reagent consumption and easy use, biochip-related technologies are widely concerned in the fields of life sciences, medicine, pharmacy and in vitro diagnosis. As an extremely important emerging scientific and technological platform and a potential strategic field for industrial transformation at the national level, biochip is at a very important stage of development.

[0003] The complex process of biochip production and the low yield of production products are the bottlenecks that restrict the development of biochips. At present, especially in the production of protein chips, products such as Aurora VERSA 10 Spotter, Genetix Qarray®, BioJet™ Non-Contact Nanoliter to Microliter Solenoid Dispenser mostly use pin printing, jet printing, and piezoelectric jet printing to produce microarrays. In addition to existing products, patents such as US6594432 and US6110426 use capillaries to make microarrays, but their operations are similar to those performed by pin printing. Patents such as US6623696, US6391625, and JP10084639 use a spin coating method. However, coffee rings and comet tailing often occur in microarrays produced by the above-mentioned technical means. Moreover, in the process of microarray production, factors such as ambient temperature, humidity, buffer viscosity, surface tension, ionic strength, etc. will cause problems such as poor uniformity between microarray spots and cross contamination between spots. Especially in the process of making protein microarrays, the concentration and purity of proteins will seriously limit the fixed amount of spots produced by current technology. Therefore, solving the above problems is an important direction to break through the bottleneck of biochip development. Summary of the invention

[0004] To solve the above problems, the present invention provides a novel high-throughput closed microarray printing system.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a novel high-throughput closed microarray printing system, comprising: a microarray printing module, including a microarray printing plate and a temperature control unit, for printing nucleic acids, proteins, etc. in a microarray production solution onto a chip surface while providing an optimal adsorption temperature; a microarray printing liquid path system, including a sample addition pump, a liquid supply pump and a valve, for continuously adding a fixed volume of microarray production solution to the microarray printing module; and a microarray printing motion system, for moving the microarray printing module.

[0006] Furthermore, the microarray printing plate carries a plurality of microarray printing plate internal flow paths and microarray printing plate internal liquid storage chambers, and each microarray printing plate internal flow path corresponds to a microarray solution of a different type or concentration.

[0007] Furthermore, a quality control sensor is installed in the microarray printing plate, which may be, but not limited to, a pressure sensor, a Hall sensor, a photoelectric sensor, a displacement sensor, a force sensor, etc. and a combination thereof.

[0008] Furthermore, the liquid supply pump and the sample adding pump adopt but are not limited to pressure pumps, injection pumps, peristaltic pumps, gear pumps, screw pumps, plunger pumps, diaphragm pumps, etc. and their combinations, and the valves adopt but are not limited to solenoid valves, hydraulic valves, pneumatic valves, stop valves, plug valves, ball valves, butterfly valves, rotary distribution valves, etc. and their combinations.

[0009] Further, the microarray solution includes, but is not limited to, proteins, nucleic acids including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), cells, peptides, lectins, modified polysaccharides, synthetic complex macromolecules, functionalized nanostructures, synthetic polymers, modified / blocked nucleotides / nucleosides, synthetic oligonucleotides, modified / blocked amino acids, fluorophores, chromophores, ligands, chelates, haptens, drug compounds, antibodies, sugars, lipids, liposomes, tissues, viruses, any nano- or micro-sized objects, and any combination thereof.

[0010] Furthermore, when making a microarray, a fixed volume of microarray making solution is first added to the flow path in the microarray printing plate by the microarray printing liquid path system, and then the microarray printing liquid path system pushes the microarray making solution in the flow path in the microarray printing plate to the liquid storage cavity in the microarray printing plate. At this time, due to the surface tension of the liquid and the external atmospheric pressure, the microarray making solution is suspended in the liquid storage cavity in the microarray printing plate. Subsequently, the microarray printing plate contacts the chip to form a sealed cavity until the adsorption of nucleic acids, proteins, etc. on the chip surface and the microarray making solution is completed. Finally, while the microarray printing plate is separated from the chip, the microarray printing liquid path system adds the solution to the chip.

[0011] Furthermore, the microarray printing motion system adopts but is not limited to a single-axis robotic arm, a multi-axis robotic arm, a single-axis slide, a multi-axis slide, a stage and a combination thereof, and, in order to facilitate the lamination of the microarray printing module and the chip, a fixture for clamping and fixing the chip is provided.

[0012] The present invention has the following beneficial effects: it can improve the uniformity between microarray spots, avoid cross contamination between spots, and especially increase the protein fixation amount of spots in protein microarray, so as to break through the bottleneck restricting the development of biochips. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the structure of a novel high-throughput closed microarray printing system according to an embodiment of the present invention;

[0014] Figure 2 This is a schematic diagram of single-channel printing in a novel high-throughput closed microarray printing system according to an embodiment of the present invention;

[0015] Figure 3 Schematic diagram of the structure of the microarray printing plate in an embodiment of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] like Figure 1-Figure 2 As shown, a novel high-throughput closed microarray printing system of the present invention comprises: a microarray printing module 1, comprising a microarray printing plate 11 and a temperature control unit 12, for printing nucleic acids, proteins, etc. in a microarray production solution onto the surface of a chip 4 while providing an optimal adsorption temperature; a microarray printing liquid path system 2, comprising a sample addition pump 21, a liquid supply pump 22 and a valve 23, for continuously adding a fixed volume of microarray production solution to the microarray printing module; and a microarray printing motion system 3, for moving the microarray printing module 1.

[0018] In this embodiment, Figure 3As shown, the microarray printing plate 11 carries a plurality of microarray printing plate internal flow paths 111 and a microarray printing plate internal liquid storage chamber 112, and each microarray printing plate internal flow path 111 corresponds to a microarray solution of different types or concentrations. The microarray solution includes but is not limited to proteins, nucleic acids including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), cells, peptides, lectins, modified polysaccharides, synthetic composite macromolecules, functionalized nanostructures, synthetic polymers, modified / blocked nucleotides / nucleosides, synthetic oligonucleotides, modified / blocked amino acids, fluorophores, chromophores, ligands, chelates, haptens, drug compounds, antibodies, sugars, lipids, liposomes, tissues, viruses, any nano- or micron-sized objects and any combination thereof. Corresponding to different uses, the shape of the microarray printing plate internal flow path 111 can be, but not limited to, circular, rectangular, diamond, triangular and other forms; the diameter / width size of the microarray printing plate internal flow path 111 can be an appropriate value according to needs. The liquid storage chamber 112 in the microarray printing plate is arranged at the end of the microarray printing plate 11. The arrangement of the liquid storage chamber is conducive to the flexible control of the size and shape of the microarray spots. The shape of the liquid storage chamber 112 in the microarray printing plate can be adopted but not limited to a variety of shapes such as a circle, a rectangle, a rhombus, a triangle, a hexagon, an octagon, a polygon, etc. And the design of the liquid storage chamber can also flexibly control the graphic pattern finally formed by the microarray. In addition, the design of the liquid storage chamber is conducive to saving the amount of microarray production solution used. Most importantly, the design of the liquid storage chamber can effectively reduce the solvent evaporation when the microarray production solution is fixed to the chip, thereby improving the quality of the microarray. However, in different requirements, the design of the liquid storage chamber can also be cancelled. The material of the microarray printing plate can be made of any suitable material compatible with the microarray production solution, and can be adopted but not limited to silicon, silicon dioxide, gallium arsenide, glass, ceramics, quartz, chloroprene rubber, polytetrafluoroethylene polymer, perfluoroalkoxy polymer, fluorinated ethylene propylene polymer, tetrafluoroethylene copolymer, polyethylene elastomer, polybutadiene / SBR, polydimethylsiloxane (PDMS), etc. and combinations thereof. In an embodiment, polydimethylsiloxane (PDMS) is used as the microarray printing plate.

[0019] In this embodiment, a quality control sensor 13 can be installed in the microarray printing plate 11 as needed.

[0020] In the present specific implementation, when making a microarray, the microarray printing liquid path system 2 first adds a fixed volume of microarray making solution into the flow path 111 in the microarray printing plate, and then the microarray printing liquid path system pushes the microarray making solution in the flow path in the microarray printing plate to the liquid storage cavity 112 in the microarray printing plate. At this time, due to the surface tension of the liquid and the external atmospheric pressure, the microarray making solution is suspended in the liquid storage cavity in the microarray printing plate. Subsequently, the microarray printing plate contacts the chip to form a sealed cavity until the adsorption of nucleic acids, proteins, etc. on the chip surface and the microarray making solution is completed. Finally, while the microarray printing plate is separated from the chip, the microarray printing liquid path system adds the solution to the chip.

[0021] To achieve the above process, firstly, the solution for making the microarray is added by the liquid supply pump and the sample adding pump, and the solution enters the microarray printing module through the pipeline or the like. The amount of solution added for making the microarray can be controlled by the pump alone, or the amount of solution added for making the microarray can be controlled by the method of joint control of the pump and the valve. Here, the liquid supply pump and the sample adding pump can be used according to the needs but not limited to pressure pump, injection pump, peristaltic pump, gear pump, screw pump, plunger pump, diaphragm pump, etc. and their combinations, and the valve can be used according to the needs but not limited to solenoid valve, hydraulic valve, pneumatic valve, stop valve, plug valve, ball valve, butterfly valve, rotary distribution valve, etc. and their combinations. In the embodiment, the liquid supply pump and the sample adding pump use a pressure pump and a injection pump, and the valve uses a solenoid valve.

[0022] The temperature control unit 12 can provide a favorable constant temperature condition during the production process of the microarray. The design of the temperature control unit can avoid the inactivation of biomacromolecules caused by temperature during the production process of the microarray. In addition, the temperature control unit can provide a good low temperature environment, thereby effectively reducing the solvent evaporation when the microarray production solution is fixed to the chip, thereby improving the quality of the microarray. The temperature control unit can be implemented in a variety of forms, and can be used but not limited to solid phase (metal plate, heat conduction plate, etc.) thermal conduction temperature control, liquid phase (water, silicone oil, etc.) thermal conduction temperature control, gas phase (inert gas) thermal conduction temperature control, full-wrapped thermal conduction temperature control, semi-wrapped thermal conduction temperature control, local thermal conduction temperature control, etc. and their combinations. The temperature control element can use any reliable temperature control method, and can be used but not limited to Peltier, convection temperature control, circulation temperature control, cooling coil, air compression, etc. and their combinations. Usually, in order to accurately control the temperature, a temperature sensor can be used to correct the temperature deviation.

[0023] In the present invention, the microarray printing plate needs to be closely fitted to the chip during the production process of the microarray. Therefore, the quality control sensor can detect whether the microarray printing plate is closely fitted to the chip during the production process of the microarray, and can be corrected according to the results of the quality control sensor, thereby ultimately improving the quality of the microarray. The quality control sensor can use various types of sensors according to the specific use environment, and can use but is not limited to pressure sensors, Hall sensors, photoelectric sensors, displacement sensors, force sensors, etc. and combinations thereof. In the embodiment, a pressure sensor is used as a quality control sensor.

[0024] The microarray printing motion system 3 is used for moving the microarray printing module. Therefore, the microarray printing motion system can use any mechanical structure that can realize the movement of the microarray printing module or even manually realize the movement of the microarray printing module. The microarray printing motion system can adopt but not limited to a single-axis mechanical arm, a multi-axis mechanical arm, a single-axis slide, a multi-axis slide, a stage, etc. and a combination thereof. And, for the convenience of the lamination of the microarray printing module with the chip, any form of fixture can be used for the fixing of the chip. In addition, for the convenience of the lamination of the microarray printing module with the chip, the novel high-throughput closed microarray printing system can also be installed or placed on a base or platform that can adjust the balance in any form.

[0025] The novel high-throughput closed microarray printing system of the present invention can simultaneously add dozens to hundreds of different nucleic acid, protein and other microarray production solutions to the chip surface to complete the production of the microarray, so it has an extremely high microarray production throughput. Different microarray production solutions reach the chip surface through independent paths, and each path forms a physical isolation, so it has good addressability while effectively avoiding cross contamination between microarray production solutions. In the microarray production process, the volatilization of the solvent in the microarray production solution is the main cause of the coffee ring and comet tailing phenomenon. When the novel high-throughput closed microarray printing system produces a microarray, the microarray printing plate contacts the chip to form a sealed cavity, avoiding the volatilization of the solvent during the adsorption time of nucleic acid, protein and the like on the chip surface and in the microarray production solution, so the occurrence of coffee ring and comet tailing phenomenon can be effectively avoided. In addition, the time and temperature of the adsorption of nucleic acid, protein and the like on the chip surface and in the microarray production solution can be accurately controlled, so that the fixed amount of biological macromolecules in the spots can be increased, and the inactivation of nucleic acid, protein and the like in the microarray production solution caused by temperature changes can be reduced. In addition, unlike general microarray processing products, the liquid storage cavity in the microarray printing plate of the new high-throughput closed microarray printing system can be designed into different patterns according to needs, so that microarray spots with various geometric shapes such as circle, square, triangle, diamond, etc. can be produced.

[0026] Example:

[0027] 1. Fabrication of microarray printing plates: Use single-sided polished silicon wafers as the substrate for the SU-8 mold. Preheat the wafer at 95°C for 10 min to drive off surface water and improve adhesion. After the wafer cools, add SU-8 to the wafer and spin at 1300 rpm for 60 s to produce a 100 μm thick layer. Bake the wafer at 65°C for 3 min, then at 95°C for 2 h, take it out and cool it. Cover the wafer with a photomask, with the emulsion side facing the SU-8, and cover it with a glass plate. Use a 365nm mask alignment system 430 mJ / cm 2 The wafer was exposed to a dose of . After exposure, it was baked at 65°C for 3 min and at 95°C for 15 min to complete the cross-linking of the exposed resist. After the wafer was cooled, it was immersed in PGMEA (propylene glycolmonomethyl ether acetate) for 20 min, then washed with isopropanol and dried in a nitrogen drying oven. After the wafer was dried, it was placed in a vacuum chamber of fluorosilane (Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-triethoxysilane) for 2 h to allow the surface reaction to form a monomolecular surface layer at a controlled rate.

[0028] PDMS (polydimethyl siloxane, Sylgard 184) was selected. According to the product instructions, 40 ml of the base resin and the curing agent were thoroughly mixed at a ratio of 10:1 (v / v). The prepolymer was placed in a vacuum for 1 h to remove all bubbles. The prepolymer was added to the wafer and allowed to settle evenly. The wafer was then placed in a vacuum for 1 hour to remove bubbles remaining between the mold and the prepolymer. After all bubbles were extracted from the mold, the wafer and the prepolymer were baked at 65°C for 2 h to cure the PDMS microarray printing plate. After curing, the PDMS microarray printing plate was immediately peeled off the wafer, cleaned with isopropanol, and placed in a nitrogen drying oven to dry.

[0029] Place the PDMS microarray printing plate in a clean container and use a 0.6 mm diameter drill to make holes in the PDMS microarray printing plate. After the sealed surface of the PDMS microarray printing plate is activated by 73 W oxygen plasma at 273 mTorr for 20 s, the treated surfaces of the PDMS microarray printing plate are placed together to form an instant bond between the PDMS microarray printing plates. The PDMS microarray printing plate is then baked at 65°C for 2 h to form an airtight seal. Place the PDMS microarray printing plate in a clean container and cut off the excess PDMS.

[0030] 2. Construction of a new high-throughput closed microarray printing system: Follow the instructions Figure 1 As shown, a novel high-throughput closed microarray printing system is constructed. In this embodiment, the microarray printing liquid path system serves as a microarray production solution flow power source of the entire novel high-throughput closed microarray printing system to add the microarray production solution to the microarray printing module. After the required amount (volume) of microarray production solution enters the microarray printing module, the solenoid valve switches the flow path, and the sample pump pushes the microarray production solution in the flow path in the microarray printing plate to the liquid storage cavity in the microarray printing plate. The microarray printing motion system makes the microarray printing plate contact the chip, and determines that the microarray printing plate contacts the chip to form a sealed cavity according to the feedback of the quality control sensor until the adsorption of nucleic acids, proteins, etc. on the chip surface and the microarray production solution is completed. In the whole process, that is, the whole process from the microarray production solution entering the microarray printing plate to the completion of the adsorption of nucleic acids, proteins, etc. on the chip surface and the microarray production solution, the temperature control unit provides a favorable constant temperature condition to improve the quality of the manufactured microarray. Finally, when the microarray printing plate is separated from the chip, the sample pump provides a positive pressure to add the microarray production solution to the chip.

[0031] 3. Evaluation of a novel high-throughput closed microarray printing system: A novel high-throughput closed microarray printing system was used to prepare protein A microarrays on surface plasmon resonance (SPR) bare gold chips. There are two main variables in microarray preparation: protein A solution concentration and deposition time. For this purpose, protein A microarrays were prepared under conditions of different concentrations (0.01, 0.05, 0.1 mg / ml) of protein A in 0.1XPBS solution and different deposition times (5, 15, 30, 45, 60 min). Afterwards, the SPR bare gold chip was washed with 0.1×PBS solution to remove all loose unbound protein A. After the protein A microarray was prepared, the microarray was analyzed using surface plasmon resonance microscopy. Subsequently, different concentrations of Cy3-labeled human IgG solutions were prepared using 0.2 mg / ml BSA in PBS. After blocking the protein A microarray with 5 mg / ml BSA solution for 10 min, different concentrations of Cy3-labeled human IgG were added and incubated at 37 °C for 15 min. The SPR bare gold chip was washed with 0.1XPBS solution for 10 min. The microarray was analyzed by surface plasmon resonance microscopy and fluorescence microscopy, respectively. The experimental results showed that the new high-throughput closed microarray printing system effectively improved the quality of the microarray, and the spots in the microarray had no coffee ring and comet tailing phenomenon. In addition, the spots in the microarray prepared under the same conditions had good uniformity.

[0032] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel high-throughput closed microarray printing system, characterized in that: include: A microarray printing module (1) comprises a microarray printing plate (11) and a temperature control unit (12), and is used to print nucleic acids or proteins in a microarray production solution onto a chip surface while providing an optimal adsorption temperature; A microarray printing liquid path system (2), comprising a sample addition pump (21), a liquid supply pump (22) and a valve (23), for continuously adding a fixed volume of microarray production solution to a microarray printing module; A microarray printing motion system (3) for moving the microarray printing module (1); When making a microarray, a microarray printing liquid path system (2) first adds a fixed volume of microarray making solution into the flow path (111) in the microarray printing plate, and then the microarray printing liquid path system pushes the microarray making solution in the flow path in the microarray printing plate into the liquid storage cavity (112) in the microarray printing plate. At this time, due to the surface tension of the liquid and the external atmospheric pressure, the microarray making solution is suspended in the liquid storage cavity in the microarray printing plate. Then, the microarray printing plate contacts the chip to form a sealed cavity until the adsorption of nucleic acids or proteins in the microarray making solution on the chip surface is completed. Finally, while the microarray printing plate is separated from the chip, the microarray printing liquid path system adds the solution to the chip.

2. A novel high-throughput closed microarray printing system as claimed in claim 1, characterized in that: The microarray printing plate (11) contains a plurality of microarray printing plate internal flow paths (111) and a microarray printing plate internal liquid storage cavity (112), and each microarray printing plate internal flow path (111) corresponds to a microarray solution of a different type or concentration.

3. A novel high-throughput closed microarray printing system as claimed in claim 1, characterized in that: The microarray printing plate (11) is equipped with a quality control sensor (13), which is selected from a pressure sensor, a Hall sensor, a photoelectric sensor, a displacement sensor, a force sensor and a combination thereof.

4. A novel high-throughput closed microarray printing system as claimed in claim 1, characterized in that: The liquid supply pump (22) and the sample adding pump (21) are selected from pressure pumps, injection pumps, peristaltic pumps, gear pumps, screw pumps, plunger pumps, diaphragm pumps and combinations thereof, and the valve (23) is selected from solenoid valves, hydraulic valves, pneumatic valves, stop valves, plug valves, ball valves, butterfly valves, rotary distribution valves and combinations thereof.

5. A novel high-throughput closed microarray printing system as claimed in claim 2, characterized in that: The microarray solution is selected from the group consisting of proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptides, modified / blocked nucleotides, synthetic oligonucleotides, modified / blocked amino acids, haptens, antibodies, and any combination thereof.

6. A novel high-throughput closed microarray printing system as claimed in claim 1, characterized in that: The microarray printing motion system (3) is selected from a single-axis robotic arm, a multi-axis robotic arm, a single-axis slide, a multi-axis slide, a stage and a combination thereof, and, in order to facilitate the bonding of the microarray printing module with the chip, a fixture for clamping and fixing the chip is provided.

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