CN113561647A

CN113561647A - Novel high-throughput closed microarray printing system

Info

Publication number: CN113561647A
Application number: CN202111029597.9A
Authority: CN
Inventors: 王倩; 宋书香; 王静; 徐波; 许迎利; 闪烨; 朱先伟
Original assignee: Zhenghui Biotechnology Ningbo Co ltd; Peking University
Current assignee: Peking University
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2021-10-29

Abstract

The invention relates to the technical field of biological chips, in particular to a novel high-flux closed microarray printing system, which comprises: the microarray printing module comprises a microarray printing plate and a temperature control unit, and is used for printing nucleic acid, protein and the like in a microarray manufacturing solution on the surface of a chip and providing an optimal adsorption temperature; the microarray printing liquid path system comprises a sample adding pump, a liquid supply pump and a valve, and is used for continuously adding a microarray manufacturing solution with a fixed volume to the microarray printing module; and the microarray printing motion system is used for moving the microarray printing module. The invention can improve the uniformity among the spots of the microarray, avoid the cross contamination among the spots, and particularly improve the protein fixing amount of the spots in the protein microarray so as to break through the bottleneck of limiting the development of the biochip.

Description

Novel high-throughput closed microarray printing system

Technical Field

The invention relates to the technical field of biochips, in particular to a novel high-throughput closed microarray printing system.

Background

The biochip is a detection method for realizing specific functions by utilizing a microarray manufactured on a substrate, and can be applied to wide fields including genomics, proteomics, lipidomics, metabonomics and cytomics. Because the biochip has the advantages of high detection flux, high sensitivity, less sample/reagent consumption, convenient use and the like, the related technology of the biochip is widely concerned by the fields of life science, medicine, pharmacy, in-vitro diagnosis and the like. Biochips are in a very important development stage as emerging scientific and technological platforms and potential strategic fields of national-level industrial transformation which are extremely important in the present generation.

The biochip manufacturing process is complex, and the low yield of manufactured products is a bottleneck limiting the development of the biochip. At present, in particular, in the manufacture of protein chips, for example, Aurora VERSA 10 Spotter, Genetix Qarray, Biojet Non-Contact Nanoliter to Microliter Solenoid dispere, etc., are mostly manufactured into microarrays by using pin printing, jet printing, piezo jet printing techniques. In addition to existing products, capillary-based microarrays are used in patents such as US6594432, US6110426, etc., but the operation is similar to that performed by pin printing. US6623696, US6391625, JP10084639 et al utilize a spin coating method. However, the microarray manufactured by the above-mentioned technical means often has a coffee ring (coffee ring) and comet tailing (comet tailing). In addition, in the microarray manufacturing process, problems such as poor uniformity among microarray spots and cross contamination among spots will be caused by factors such as ambient temperature, humidity, buffer viscosity, surface tension, and ionic strength. In particular, during the process of making protein microarrays, the concentration and purity of the protein can severely limit the fixed amount of spots produced by current techniques. Therefore, solving the above problems is an important direction to break through the development bottleneck of biochips.

Disclosure of Invention

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

In order to achieve the purpose, the invention adopts the technical scheme that: a novel high-throughput closed microarray printing system, comprising: the microarray printing module comprises a microarray printing plate and a temperature control unit, and is used for printing nucleic acid, protein and the like in a microarray manufacturing solution on the surface of a chip and providing an optimal adsorption temperature; the microarray printing liquid path system comprises a sample adding pump, a liquid supply pump and a valve, and is used for continuously adding a microarray manufacturing solution with a fixed volume to the microarray printing module; and the microarray printing motion system is used for moving the microarray printing module.

Furthermore, a plurality of microarray printing plate internal flow paths and microarray printing plate internal liquid storage cavities are carried in the microarray printing plate, and each microarray printing plate internal flow path corresponds to a different type or concentration of microarray solution.

Further, a quality control sensor is additionally arranged in the microarray printing plate, and the microarray printing plate adopts, but is not limited to, a pressure sensor, a Hall sensor, a photoelectric sensor, a displacement sensor, a force sensor and the like and a combination thereof.

Further, the liquid feeding 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 and the like and combinations thereof, and the valves adopt, but are not limited to, electromagnetic valves, hydraulic valves, pneumatic valves, stop valves, plug valves, ball valves, butterfly valves, rotary distribution valves and the like and combinations thereof.

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, pharmaceutical compounds, antibodies, sugars, lipids, liposomes, tissues, viruses, any nano-or micro-sized object, and any combination thereof.

Furthermore, when the microarray is manufactured, firstly, a microarray manufacturing solution with a certain volume is added into an inner flow path of the microarray printing plate through the microarray printing liquid path system, then the microarray manufacturing solution in the inner flow path of the microarray printing plate is pushed to an inner liquid storage cavity of the microarray printing plate through the microarray printing liquid path system, at the moment, the microarray manufacturing solution is suspended in the inner liquid storage cavity of the microarray printing plate due to the action of the surface tension of the liquid and the external atmospheric pressure, then the microarray printing plate is contacted with the chip to form a sealed cavity until the adsorption of nucleic acid, protein and the like in the surface of the chip and the microarray manufacturing solution is completed, and finally, the microarray printing plate is separated from the chip and simultaneously the solution is added onto the chip through the microarray printing liquid path system.

Further, microarray printing motion system adopts but not limited to unipolar arm, multiaxis arm, unipolar slip table, multiaxis slip table, objective table and combination thereof, and, in order to facilitate the laminating of microarray printing module and chip, is provided with and is used for realizing the fixed anchor clamps of chip centre gripping.

The invention has the following beneficial effects: can improve the uniformity among the spots of the microarray, avoid the cross contamination among the spots, and particularly improve the protein fixing amount of the spots in the protein microarray so as to break through the bottleneck of limiting the development of the biochip.

Drawings

FIG. 1 is a schematic diagram of a novel high throughput closed microarray printing system according to an embodiment of the present invention;

FIG. 2 is a schematic illustration of single pass printing in a novel high throughput closed microarray printing system according to an embodiment of the present invention;

FIG. 3 is a schematic view of a microarray printing plate according to an embodiment of the present invention.

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

As shown in fig. 1-2, a novel high throughput closed microarray printing system of the present invention comprises: a microarray printing module 1 including a microarray printing plate 11 and a temperature control unit 12 for providing an optimum adsorption temperature while printing nucleic acids, proteins, etc. in a microarray manufacturing solution on the surface of the chip 4; the microarray printing liquid path system 2 comprises a sample adding pump 21, a liquid supply pump 22 and a valve 23, and is used for continuously adding a microarray manufacturing solution with a fixed volume to the microarray printing module; and a microarray printing motion system 3 for moving the microarray printing module 1.

In this embodiment, as shown in fig. 3, a plurality of microarray printing plate internal channels 111 and microarray printing plate internal liquid storage chambers 112 are mounted in the microarray printing plate 11, and each microarray printing plate internal channel 111 corresponds to a different kind or concentration of microarray solution. 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, pharmaceutical compounds, antibodies, sugars, lipids, liposomes, tissues, viruses, any nano-or micro-sized object, and any combination thereof. The shape of the flow path 111 in the microarray printing plate may be, but not limited to, circular, rectangular, rhombic, triangular, and other various shapes according to different uses; the diameter/width dimension of the flow path 111 in the microarray printing plate may take an appropriate value as required. The inner liquid storage cavity 112 of the microarray printing plate is arranged at the tail end of the microarray printing plate 11, the arrangement of the liquid storage cavity is favorable for controlling the flexible control of the size and the shape of the microarray spots, and the shape of the inner liquid storage cavity 112 of the microarray printing plate can adopt various shapes such as but not limited to a circle, a rectangle, a diamond, a triangle, a hexagon, an octagon, a polygon and the like. And the design of the liquid storage cavity can also flexibly control the finally formed pattern of the microarray. In addition, the design of the liquid storage cavity is beneficial to saving the use amount of the solution for manufacturing the microarray. Most importantly, the design of the liquid storage cavity can effectively reduce the solvent evaporation when the microarray manufacturing solution is fixed on the chip, thereby improving the quality of the microarray. However, in different requirements, the design of the liquid storage cavity can be eliminated. The material of the microarray printing plate can be made of any suitable material compatible with the microarray fabrication solution, and can be made of, but not limited to, silicon dioxide, gallium arsenide, glass, ceramic, quartz, neoprene, polytetrafluoroethylene polymers, perfluoroalkoxy polymers, fluorinated ethylene propylene polymers, tetrafluoroethylene copolymers, polyethylene elastomers, polybutadiene/SBR, Polydimethylsiloxane (PDMS), and the like, and combinations thereof. In the examples, the microarray printing plate used Polydimethylsiloxane (PDMS).

In this embodiment, a quality control sensor 13 may be additionally installed in the microarray printing plate 11 as needed.

When the microarray is manufactured, firstly, a microarray manufacturing solution with a fixed volume is added into the inner flow path 111 of the microarray printing plate by the microarray printing liquid path system 2, then the microarray manufacturing solution in the inner flow path of the microarray printing plate is pushed to the inner liquid storage cavity 112 of the microarray printing plate by the microarray printing liquid path system, at the moment, the microarray manufacturing solution is suspended in the inner liquid storage cavity of the microarray printing plate due to the surface tension of the liquid and the external atmospheric pressure, then the microarray printing plate is contacted with the chip to form a sealed cavity until the adsorption of nucleic acid, protein and the like in the surface of the chip and the microarray manufacturing solution is completed, and finally, the microarray printing plate is separated from the chip and simultaneously the solution is added to the chip by the microarray printing liquid path system.

In order to realize the process, firstly, a microarray manufacturing solution is added by a liquid supply pump and a sample adding pump and enters a microarray printing module through a pipeline and the like. The amount of the solution used for preparing the microarray may be controlled by a pump alone, or may be controlled by a combination of a pump and a valve. The liquid supply pump and the sample adding pump can adopt but are not limited to a pressure pump, an injection pump, a peristaltic pump, a gear pump, a screw pump, a plunger pump, a diaphragm pump and the like and combinations thereof according to requirements, and the valves can adopt but are not limited to an electromagnetic valve, a hydraulic valve, a pneumatic valve, a stop valve, a plug valve, a ball valve, a butterfly valve, a rotary distribution valve and the like and combinations thereof according to requirements. In the embodiment, the liquid supply pump and the sample adding pump use a pressure pump and a syringe pump, and the valve uses an electromagnetic valve.

The temperature control unit 12 can provide an advantageous constant temperature condition during the fabrication of the microarray. The design of the temperature control unit can avoid the inactivation of biomacromolecules caused by temperature in the process of manufacturing the microarray. And, the temperature control unit can provide a good low temperature environment, thereby effectively reducing solvent evaporation while the microarray manufacturing solution is fixed to the chip, and thus improving the quality of the microarray. The temperature control unit can be realized in various forms, and can adopt, but is not limited to, solid phase (metal plate, heat conducting plate, etc.) heat conduction temperature control, liquid phase (water, silicon oil, etc.) heat conduction temperature control, gas phase (inert gas) heat conduction temperature control, full wrapping type heat conduction temperature control, half wrapping type heat conduction temperature control, local heat conduction temperature control, and the like, and combinations thereof. The temperature control element may be any reliable temperature control method, including but not limited to peltier, convective temperature control, cyclic temperature control, cooling coils, air compression, and the like, and combinations thereof. In general, in order to accurately control the temperature, a temperature sensor may be used to correct a deviation of the temperature.

In the present invention, the microarray printing plate needs to be closely attached to the chip during the microarray manufacturing process. Therefore, the quality control sensor can detect whether the microarray printing plate is tightly attached to the chip or not in the manufacturing process of the microarray, and can correct the microarray printing plate according to the result of the quality control sensor, so that the quality of the microarray is finally improved. The quality control sensor can use various types of sensors according to specific use environments, and can adopt, but is not limited to, a pressure sensor, a hall sensor, a photoelectric sensor, a displacement sensor, a force sensor and the like and a combination thereof. In an embodiment, a pressure sensor is used as the quality control sensor.

The microarray printing motion system 3 is used to move the microarray printing module. Thus, the microarray printing motion system may use any mechanical structure that enables movement of the microarray printing module, even manually. The microarray printing motion system may employ, but is not limited to, a single axis robotic arm, a multi-axis robotic arm, a single axis slide, a multi-axis slide, an object stage, and the like, and combinations thereof. Also, in order to facilitate the attachment of the microarray printing module to the chip, any type of jig may be used for the fixation of the chip. In addition, in order to facilitate the attachment of the microarray printing module to the chip, the novel high-throughput closed microarray printing system can be mounted or arranged on a base or a platform which can be adjusted in balance in any mode.

The novel high-throughput closed microarray printing system can simultaneously add dozens to hundreds of different microarray preparation solutions of nucleic acid, protein and the like to the surface of the chip to complete the preparation of the microarray, so the system has extremely high microarray preparation throughput. Different microarray making solutions reach the surface of the chip through independent paths, and physical isolation is formed among the paths, so that cross contamination among the microarray making solutions is effectively avoided, and meanwhile, good addressability is achieved. During microarray fabrication, volatilization of solvents in the microarray fabrication solution is a major cause of coffee ring and comet tailing. When a novel high-flux closed microarray printing system is used for manufacturing a microarray, a microarray printing plate is in contact with a chip to form a sealed cavity, so that volatilization of solvents in the adsorption time process of nucleic acid, protein and the like on the surface of the chip and in a microarray manufacturing solution is avoided, and the phenomenon of trailing of coffee circles and comets can be effectively avoided. In addition, the adsorption time and temperature of nucleic acid, protein and the like in the chip surface and the microarray preparation solution can be accurately controlled, so that the fixing amount of biological macromolecules in spots can be increased, and the inactivation of nucleic acid, protein and the like in the microarray preparation solution caused by temperature change can be reduced. In addition, different from common microarray processing products, the liquid storage cavity in the microarray printing plate of the novel high-throughput closed microarray printing system can be designed into different patterns according to requirements, so that microarray spots of various geometric patterns such as circles, squares, triangles, diamonds and the like can be manufactured.

Example (b):

1. preparation of microarray printing plate: a single-side polished silicon wafer was used as the substrate for the SU-8 mold. The wafer was preheated at 95 ℃ for 10 min to drive off surface water and improve adhesion. After the wafer had cooled, SU-8 was added to the wafer and spun at 1300 rpm for 60 s to produce a 100 μm thick layer. The wafer was baked at 65 ℃ for 3 min, then at 95 ℃ for 2 h, and taken out to cool. A photomask (mask) was applied to the wafer with the emulsion side facing SU-8 and covered with a glass plate. 430 mJ/cm mask alignment system using 365 nm²Exposure to the dose of (c). And baking at 65 ℃ for 3 min and at 95 ℃ for 15 min after exposure to complete the crosslinking of the exposed resist. After the wafer was cooled, the wafer was immersed in pgmea (propylene glycol monomethylether acetate) and developed for 20 min, and then, after the wafer was washed with isopropyl alcohol, it was dried in a nitrogen drying oven. After the wafer is dried, the wafer is put into a vacuum chamber of a fluorosilane (tridefluoro-1, 1,2, 2-tetrahydroctyl-1-triethoxysilane) to be evaporated for 2 hours, so that the surface reaction forms a monomolecular surface layer at a controlled rate.

PDMS (polydimethylsiloxane, Sylgard 184) was selected and 40 ml of a base resin was thoroughly mixed with a curing agent at 10: 1 (v/v) according to the product specifications. The prepolymer was left in vacuo for 1 h to remove any bubbles. The prepolymer was added to the wafer and allowed to settle uniformly. The wafer was then placed under vacuum for 1 hour to remove air bubbles remaining between the mold and the prepolymer. After all air bubbles were drawn out of the mold, the wafer was baked with the prepolymer at 65 ℃ for 2 h to cure the PDMS microarray printing plate. Immediately after curing was completed, the PDMS microarray printing plate was peeled off the wafer, washed with isopropanol, and dried in a nitrogen dry box.

The PDMS microarray printing plate was placed in a clean container and a hole was opened on the PDMS microarray printing plate using a drill with a diameter of 0.6 mm. The treated surfaces of the PDMS microarray printing plates were brought together to form an immediate bond between the PDMS microarray printing plates after activating the PDMS microarray printing plates at 273 mTorr for 20 s using 73W of oxygen plasma. The PDMS microarray printing plate was then baked at 65 ℃ for 2 h to form a hermetic seal. The PDMS microarray printing plate was placed in a clean container and excess PDMS was cut off.

2. Constructing a novel high-flux closed microarray printing system: a novel high-throughput closed microarray printing system was set up as shown in schematic FIG. 1. In this embodiment, the microarray printing solution path system is used as a microarray manufacturing solution flow power source for the entire novel high-throughput closed microarray printing system to add microarray manufacturing solution to the microarray printing module. After the required amount (volume) of the microarray preparation solution enters the microarray printing module, the electromagnetic valve switches the flow path, and the sample adding pump pushes the microarray preparation solution in the flow path in the microarray printing plate to the liquid storage cavity in the microarray printing plate. And the microarray printing motion system enables the microarray printing plate to be in contact with the chip, and determines that the microarray printing plate is in contact with the chip to form a sealed cavity according to the feedback of the quality control sensor until the adsorption of the surface of the chip and nucleic acid, protein and the like in the microarray manufacturing solution is completed. In the whole process, namely the whole process that the microarray manufacturing solution enters the microarray printing plate to complete the adsorption of nucleic acid, protein and the like on the surface of the chip and in the microarray manufacturing 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, a positive pressure is provided by the sample adding pump to add the microarray manufacturing solution to the chip.

3. Evaluation of novel high-throughput closed microarray printing systems: protein a microarrays were fabricated on Surface Plasmon Resonance (SPR) bare gold chips using a novel high-throughput closed microarray printing system. There are two main variables in microarray fabrication: protein a solution concentration and deposition time. For this purpose, protein A microarrays were prepared under different concentrations (0.01, 0.05, 0.1 mg/ml) of 0.1XPBS solution and different deposition times (5, 15, 30, 45, 60 min). Afterwards, the SPR bare gold chip was washed with 0.1x PBS solution to remove all loosely unbound protein a. After the protein a microarray was fabricated, the microarray was analyzed using surface plasmon resonance microscopy. Subsequently, various concentrations of Cy 3-labeled human IgG solutions were prepared using 0.2 mg/ml BSA in PBS. After blocking the protein A microarray for 10 min by using a 5 mg/ml BSA solution, adding Cy 3-labeled human IgG with different concentrations, incubating at 37 ℃ for 15 min, and then washing the SPR bare gold chip for 10 min by using a 0.1XPBS solution. The microarray was analyzed using surface plasmon resonance microscopy and fluorescence microscopy, respectively. Experimental results show that the novel high-flux closed microarray printing system effectively improves the quality of manufactured microarrays, and spots in the microarrays do not have coffee ring and comet trailing phenomena. Furthermore, the spots in the microarray fabricated under the same conditions had good uniformity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. A novel high-throughput closed microarray printing system is characterized in that: the method comprises the following steps: the microarray printing module (1) comprises a microarray printing plate (11) and a temperature control unit (12) and is used for printing nucleic acid and protein in a microarray manufacturing solution on the surface of a chip and providing an optimal adsorption temperature; the microarray printing liquid path system (2) comprises a sample adding pump (21), a liquid supply pump (22) and a valve (23), and is used for continuously adding a microarray manufacturing solution with a fixed volume to the microarray printing module; a microarray printing motion system (3) for moving the microarray printing module (1).

2. The novel high throughput closed microarray printing system of claim 1, wherein: the microarray printing plate (11) is internally provided with a plurality of microarray printing plate internal flow paths (111) and microarray printing plate internal liquid storage cavities (112), and each microarray printing plate internal flow path (111) corresponds to a different type or different concentration of microarray solution.

3. The novel high throughput closed microarray printing system of claim 1, wherein: a quality control sensor (13) is additionally arranged in the microarray printing plate (11), and a pressure sensor, a Hall sensor, a photoelectric sensor, a displacement sensor, a force sensor and a combination thereof are adopted.

4. The novel high throughput closed microarray printing system of claim 1, wherein: the liquid supply pump (22) and the sample adding pump (21) adopt but not limited to pressure pumps, injection pumps, peristaltic pumps, gear pumps, screw pumps, plunger pumps, diaphragm pumps and combinations thereof, and the valve (23) adopts but not limited to electromagnetic valves, hydraulic valves, pneumatic valves, stop valves, plug valves, ball valves, butterfly valves, rotary distribution valves and combinations thereof.

5. The novel high throughput closed microarray printing system of claim 2, wherein: 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, pharmaceutical compounds, antibodies, sugars, lipids, liposomes, tissues, viruses, any nano-or micro-sized object, and any combination thereof.

6. The novel high throughput closed microarray printing system of claim 1, wherein: when a microarray is manufactured, firstly, a microarray manufacturing solution with a fixed volume is added into a flow path (111) in a microarray printing plate by a microarray printing liquid path system (2), then the microarray manufacturing solution in the flow path in the microarray printing plate is pushed to a liquid storage cavity (112) in the microarray printing plate by the microarray printing liquid path system, at the moment, the microarray manufacturing solution is suspended in the liquid storage cavity in the microarray printing plate due to the action of the surface tension of the liquid and the external atmospheric pressure, then the microarray printing plate is contacted with a chip to form a sealed cavity until the adsorption of nucleic acid and protein in the microarray manufacturing solution on the surface of the chip is completed, and finally, the microarray printing plate is separated from the chip and simultaneously the solution is added to the chip by the microarray printing liquid path system.

7. The novel high throughput closed microarray printing system of claim 1, wherein: microarray printing moving system (3) adopt but not limited to unipolar arm, multiaxis arm, unipolar slip table, multiaxis slip table, objective table and combination, and, for the convenience of the laminating of microarray printing module and chip, are provided with and are used for realizing the fixed anchor clamps of chip centre gripping.