A bionic super-hydrophobic-hydrophilic integrated well plate and a high-throughput automated nucleic acid library construction instrument based on the well plate

Abstract

The invention relates to a bionic super-hydrophobic-hydrophilic integrated well plate and a high-throughput automated nucleic acid library construction instrument based on the well plate, belonging to the technical field of nucleic acid detection. The sample loading well of the well plate of the invention is a through hole, the side wall of the through hole is super-hydrophobic, and the bottom of the through hole is a hydrophilic silicon wafer. Based on the principle of bionics, a bionic super-hydrophobic low-adhesion well plate with a surface micro-nano composite structure and ultra-low surface energy is designed. The inner wall of the well plate is super-hydrophobic and has low adhesion, which greatly reduces the adsorption and loss of samples and reagents; the bottom of the well plate is integrated with a silicon wafer with high thermal conductivity, good thermal stability and good hydrophilicity, which fully guarantees the temperature control in a PCR amplification process, and the hydrophilicity also strengthens the fusion reaction between the sample and the reagent, reduces bubbles, greatly reduces the adsorption and loss of reagents and products on the inner wall in the process of nucleic acid amplification and purification, and significantly improves the product amount of actual library construction.

Description

Technical Field

[0001] The invention provides a bionic super-hydrophobic-hydrophilic integrated well plate and a high-throughput automated nucleic acid library building instrument based on the well plate, belonging to the technical field of nucleic acid detection. Background Art

[0002] Polymerase chain reaction (PCR) is a technique that uses known oligonucleotide primers to guide the amplification of trace gene fragments in unknown fragments in vitro. Since PCR technology can obtain a large number of DNA fragments in a short period of time, with high sensitivity and strong specificity, it is widely used in the detection of pathogenic microorganisms, genetic disease-related genes, tumor-related genes, eugenics and other fields, but traditional PCR technology cannot obtain information on unknown sequences.

[0003] Next-generation sequencing (NGS) is a DNA sequencing technology developed based on PCR and gene chips. NGS has the characteristics of high throughput and short read length, but it is necessary to build a library of biological nucleic acids before conducting NGS.

[0004] For a long time, the supply of upstream gene sequencing equipment and consumables in China has been basically monopolized by foreign-funded enterprises, and the key NGS technology is in the hands of European and American manufacturers including Illumina. The entire NGS liquid biopsy industry is heavily dependent on Illumina's sequencers and supporting reagents, resulting in high testing costs. Therefore, the development of domestic sequencing-related equipment is still facing tremendous pressure.

[0005] Gene sequencing has a wide detection range and high efficiency. mNGS is the mainstream technology in infectious disease detection. At present, the gene sequencing platform has reached the third generation, but the accuracy of the third generation sequencing is still not high enough and the cost is relatively high, so NGS is still the mainstream. The technologies currently used in mNGS are represented by Illumina's Hiseq technology and ABI's Solid technology.

[0006] Currently, there is a lack of miniaturized, automated and rapid biological nucleic acid library construction instruments on the market.

[0007] In order to address the difficulties of biological library construction, which requires large equipment, complex operations, and high professional and technical requirements, we have developed an instant, convenient, and easy-to-operate biological nucleic acid automated library construction instrument through the cross-integration of microelectronics, functional materials, analytical chemistry and other disciplines with bioengineering to meet the country's major needs for the rapid analysis of large amounts of biological information.

[0008] In addition, during the nucleic acid amplification process, the wettability of the inner surface of the sample well significantly affects the adhesion behavior of the sample reagent. First, when the inner surface of the sample well is highly hydrophilic, the sample or product will adhere to it, causing the loss of the sample or product. Especially in the magnetic bead purification process, repeated elution will cause more products to remain on the hydrophilic inner wall of the sample well, and ultimately significantly reduce the amount of product actually built in the library. Secondly, when the bottom plate of the sample well is highly hydrophobic, bubbles will be generated during the sample loading process, and it is not conducive to the mixing of reagents, thereby affecting nucleic acid amplification. Therefore, how to improve the super-hydrophobic properties of the inner wall of the sample well and enhance the hydrophilic properties of the bottom surface is the key to achieving efficient nucleic acid amplification, purification and even library construction. Summary of the invention

[0009] Based on the dense micro-protrusion structure and nano-scale papillae on the surface of the lotus leaf, the surface is also covered with a layer of biological wax. The characteristics of the combination of the micro-nano composite structure on the surface of the lotus leaf and the biological wax can make the lotus leaf have excellent super-hydrophobic and self-cleaning properties. The present invention incorporates bionic elements, constructs the micro-nano structure of the inner wall of the sample addition hole, reduces the surface energy of the inner wall of the sample addition hole, prepares a well plate with a super-hydrophobic and low-adhesion inner wall, and places a hydrophilic silicon wafer under the well plate, thereby providing a bionic super-hydrophobic-hydrophilic integrated well plate and a high-throughput automated nucleic acid library construction instrument based on the well plate, which is simple to operate and has extremely low sample loss.

[0010] The present invention adopts the following technical solution:

[0011] A bionic super-hydrophobic-hydrophilic integrated orifice plate, wherein the sample adding holes of the orifice plate are through holes, the side walls of the through holes are super-hydrophobic, and the bottom of the through holes is a hydrophilic silicon wafer.

[0012] Preferably, the method for preparing the super hydrophobic-hydrophilic integrated orifice plate comprises the following steps:

[0013] (1) Preparation of laser-induced graphene (LIG)

[0014] The polyimide film is cut and ultrasonically cleaned with anhydrous ethanol and deionized water for 5 to 20 minutes respectively, the pattern to be engraved is introduced into the laser engraving machine, the laser power and the laser scanning speed are set, and the laser-induced graphene powder is prepared on the polyimide film in one step for later use;

[0015] (2) Preparation of biomimetic super-hydrophobic low-adhesion substrate

[0016] Weigh PDMS polymer and curing agent (RTV615 silicone rubber compound), mix and stir the PDMS polymer and curing agent in a weight ratio of 5:1 to 20:1, add SiO2 (silicon dioxide) microspheres with a particle size of 30nm-30μm, PTFE (polytetrafluoroethylene) micropowder, and laser-induced graphene (LIG) powder, place the mixed mixture in a vacuum barrel, and evacuate for about 0.5 to 2h to discharge bubbles in the mixture; inject the prepared mixture into a silicon-based mold using a syringe, and the size of the silicon-based mold can be changed according to the size of the orifice plate; bake the silicon-based mold containing the mixture at 70-80°C for 1.5-4h, and cure it at room temperature for 10-25min, peel the cured mixture from the silicon-based mold, and a bionic super-hydrophobic low-adhesion substrate can be obtained;

[0017] (3) Punching

[0018] A laser engraving machine is used to punch holes on the prepared biomimetic super-hydrophobic low-adhesion substrate to form sample holes, wherein the sample holes are through holes;

[0019] Draw the required pattern in the computer software, set the laser engraving parameters, and load the drawn document. Place the bionic super-hydrophobic low-adhesion substrate on the wooden board, put it into the laser, adjust the distance between the laser port and the bionic super-hydrophobic low-adhesion substrate, turn on the laser, and punch the hole, which is a through hole.

[0020] (4) Integration of silicon wafer and biomimetic super-hydrophobic low-adhesion orifice plate

[0021] The silicon wafer is cut into rectangles with the same size as the bionic super-hydrophobic low-adhesion orifice plate, and the silicon wafer is tightly attached to the back of the bionic super-hydrophobic low-adhesion orifice plate by utilizing the viscosity of PDMS to obtain a bionic super-hydrophobic-hydrophilic integrated orifice plate.

[0022] PDMS has a certain degree of stickiness and can be bonded to glass, metal, silicon and other substrates, but it is not easy to bond to wooden materials such as wood boards. However, the bonding between PDMS and silicon wafers is not permanent. If permanent bonding is required, the PDMS substrate needs to be plasma treated.

[0023] The function of the silicon wafer is to better store and conduct heat to ensure temperature stability during PCR amplification. In addition, the hydrophilicity of the silicon wafer also enhances the fusion reaction between samples and avoids the generation of bubbles during the sample addition process.

[0024] Preferably, in step (1), the polyimide film has a thickness of 50 μm to 2 mm, a length of 3 cm to 50 cm, and a width of 0.5 cm to 40 cm, and the length and width can be adjusted according to the amount of graphene required;

[0025] The wavelength used by the laser engraving machine is 460nm, the laser power is 1w~10w, and the laser scanning speed is 10mm / s~120mm / s;

[0026] The particle size of the prepared laser-induced graphene powder is 3nm to 500μm.

[0027] Preferably, in step (2), the particle size of the PTFE (polytetrafluoroethylene) powder is 1 nm to 500 nm;

[0028] The weight ratio of PDMS polymer, SiO2 microspheres, PTFE micropowder and laser-induced graphene powder is preferably 20:2:1:1, which can be flexibly processed according to the geometric structure and quality of the prepared PDMS.

[0029] In the present invention, SiO2 microspheres and LIG powders construct a micro-nano composite structure on the substrate surface, while PTFE micropowders greatly reduce the surface energy of the substrate. PDMS acts as a binder to firmly adsorb SiO2 microspheres, PTFE micropowders and LIG powders. The coupling of physical modification and chemical modification gives the substrate excellent super-hydrophobic and low-adhesion properties like lotus leaves. The contact angle, rolling angle and droplet morphology of water droplets at any five positions on the substrate surface.

[0030] A high-throughput automated nucleic acid library building instrument comprises a temperature control module, a three-dimensional automatic liquid transfer module and the above-mentioned bionic super-hydrophobic-hydrophilic integrated well plate.

[0031] Preferably, the temperature control module includes a stage, a cover plate, a source meter and a heating plate (polyimide film and conductive wire), and the stage and the cover plate are both provided with heating plates, which can independently heat and cool down;

[0032] The stage is used to place the well plate. After the temperature control module is buckled, the cover plate is placed on the upper surface of the well plate for amplification heating to ensure the temperature inside the well plate and prevent the solvent in the well plate from condensing or volatilizing.

[0033] The source meter is used to provide current for heating the heating plate. By adjusting the voltage through the source meter, the heating temperature of the heating plate can be controlled, and the temperature can be increased and decreased, and the temperature can be stabilized.

[0034] Preferably, the three-dimensional automatic pipetting module comprises a housing, a motor housing, a three-dimensional motion component, a base plate, a mounting seat and a pipette, the base plate is a supporting base plate of the entire three-dimensional pipetting module, the pipette is installed in the housing, and a power switch and a power jack are provided on the motor housing;

[0035] The pipette realizes the automatic installation of the pipette tip through the Z-axis movement of the three-dimensional motion component, and the linear thrust motor drives the movement of the piston to complete the continuous actions of aspirating, discharging and removing the pipette tip; the mounting seat is connected to the three-dimensional motion component and can move forward, backward, left and right. The pipette tip is used to aspirate and discharge liquid. The pipette tip of the pipette is placed in the tip box on the mounting seat in advance. After the library construction starts, the tip box is moved to the bottom of the pipette through the three-dimensional motion component, and the pipette moves down to realize the automatic installation of the tip;

[0036] The three-dimensional motion assembly of the present invention adopts a Cartesian coordinate system design, and linear guides, drive motors, synchronous pulleys and limiters are installed in the X-axis and Y-axis directions, and a pipette is installed in the Z-axis direction, and the slider drives the X-axis and Y-axis to move in association, realizing synchronous belt transmission, that is, the pipette can realize three-dimensional motion of the X, Y and Z axes. The three-dimensional motion assembly moves, driving the gun tip box to move directly below the pipette, and the pipette pushes the piston up and down through the linear thrust motor to complete the continuous actions of aspirating, discharging and removing the gun tip.

[0037] The function of the pipette is to aspirate and discharge liquids and control the precise amount of samples and reagents for library construction. The pipette adopts the air displacement principle to design piston pipetting. The piston is made of zirconium oxide and is fixed to the piston holder by a bushing. A permanent magnet is installed at the end of the motor shaft and connected to the piston rod. The linear thrust motor drives the movement of the piston to complete the aspiration and discharge of liquids.

[0038] The linear thrust motor is located above the pipette and is connected to the pipette.

[0039] The pipette has a built-in micro pump, and the micro pump is equipped with a liquid level monitoring sensor and a pressure sensor, which can monitor the liquid level and pressure in real time.

[0040] The software can realize digital control and interface function display of the three-dimensional automatic pipetting module.

[0041] Preferably, the operation steps of the high-throughput automated nucleic acid library construction instrument are as follows:

[0042] S1: After cleaning and drying the bionic super-hydrophobic low-adhesion substrate, place it on a silicon wafer and press it to make it tightly connected to obtain a bionic super-hydrophobic-hydrophilic integrated hole plate;

[0043] S2: placing the bionic super-hydrophobic-hydrophilic integrated well plate on the stage of the temperature control module, and placing the temperature control module on the mounting seat of the three-dimensional automatic pipetting module;

[0044] S3: Setting the relevant parameters of the three-dimensional automatic pipetting module to realize the automatic loading of reaction reagents and nucleic acid samples required for library construction;

[0045] S4: Cover the cover of the temperature control module, set the temperature of the stage and the cover respectively, and implement the nucleic acid shearing reaction and PCR amplification program;

[0046] S5: Add magnetic bead suspension to the loading holes of the bionic super-hydrophobic-hydrophilic integrated well plate to adsorb the PCR amplification product, attach it to the magnetic mounting base to separate the magnetic beads, set the parameters of the three-dimensional automatic pipetting module to realize automatic loading of the elution solvent, and finally achieve product purification.

[0047] Preferably, in step S3, the relevant parameters include volume parameters and position parameters. The volume parameters include the pipetting volume, the speed of aspiration and discharge, the number of mixing times and the mixing volume before aspiration / discharge, and the waiting time after the step is completed; the position parameters include the Z-axis (up and down), the X-axis (left and right), and the number of columns to support the single-column loading and withdrawal of the pipette head and the aspiration and discharge of the liquid.

[0048] Preferably, in step S4, the temperatures of the stage and the cover are:

[0049] Nucleic acid shearing procedure: the cover plate temperature was set to 100°C for preheating; the stage temperature was 55°C for 5 minutes and then the temperature was reduced to 10°C;

[0050] Nucleic acid amplification program: the cover plate temperature was set to 100°C for preheating; stage temperature: ① 72°C for 3 min; ② 95°C for 30 s; ③ 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, 12 cycles; ④ 72°C for 5 min; ⑤ the temperature dropped to 10°C.

[0051] The beneficial effects of the present invention are:

[0052] 1. Currently, most popular 96-well plates are made of hydrophilic or moderately hydrophobic materials. There are problems of reagent and product loss during nucleic acid amplification or reagent reaction. Based on the principle of bionics, the present invention designs a bionic super-hydrophobic low-adhesion well plate with a surface micro-nano composite structure and ultra-low surface energy. The inner wall of the well plate is super-hydrophobic and low-adhesion, which greatly reduces the adsorption and loss of samples and reagents; the bottom of the well plate is integrated with a silicon chip with high thermal conductivity, good thermal stability and good hydrophilicity, which fully guarantees the temperature control during PCR amplification. At the same time, the hydrophilic property also strengthens the fusion reaction between samples and reagents, reduces bubbles, greatly reduces the adsorption and loss of reagents and products on the inner wall during nucleic acid amplification and purification, and significantly increases the product amount of actual library construction.

[0053] 2. A silicon wafer is placed at the bottom of the well plate of the present invention to construct a super hydrophobic-hydrophilic integrated system. The good thermal conductivity of the silicon wafer can fully guarantee the temperature control during the PCR amplification process and improve the temperature stability of the PCR amplification. The good hydrophilicity of the silicon wafer can strengthen the fusion reaction between reagents, improve the uniformity of liquid temperature, and reduce the generation of bubbles during the sample addition process.

[0054] 3. The holes on the well plate are flat through holes. Compared with the pointed holes of the 96-well plate, the hole capacity is larger and suitable for nucleic acid library construction with large amplification sample requirements.

[0055] 4. The high-throughput automated nucleic acid library construction instrument of the present invention is simple to operate and has extremely low sample loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0057] Figure 1 The morphology, contact angle and rolling angle of the droplet on the super-hydrophobic low-adhesion substrate;

[0058] Figure 2 This is the structural dimension diagram of the super hydrophobic low adhesion orifice plate;

[0059] Figure 3 This is a three-dimensional image of the super-hydrophobic low-adhesion orifice plate structure;

[0060] Figure 4 The integrated structural dimensions of the silicon wafer and the super-hydrophobic low-adhesion orifice plate;

[0061] Figure 5 It is a structural schematic diagram of the temperature control module;

[0062] Figure 6 is a schematic diagram of the structure of the pipette;

[0063] Figure 7 It is a structural schematic diagram of a three-dimensional automatic liquid transfer module;

[0064] Among them, 1-bionic super-hydrophobic low-adhesion substrate, 2-silicon wafer, 3-stage, 4-cover plate, 5-source meter, 6-housing, 7-motor housing, 8-three-dimensional motion component, 9-base plate, 10-mounting seat, 11-pipette, 12-power switch, 13-power jack. DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the drawings in the implementation of this specification, but are not limited to this. Anything not fully described in the present invention shall be based on the conventional technology in the art.

[0066] Example 1

[0067] A bionic super-hydrophobic-hydrophilic integrated orifice plate, wherein the sample adding holes of the orifice plate are through holes, the side walls of the through holes are super-hydrophobic, and the bottom of the through holes is a hydrophilic silicon chip 2.

[0068] The preparation method of the super hydrophobic-hydrophilic integrated orifice plate comprises the following steps:

[0069] (1) Preparation of laser-induced graphene (LIG)

[0070] Cut the polyimide film, use anhydrous ethanol and deionized water to ultrasonically clean for 5 to 20 minutes respectively, introduce the pattern to be engraved into the laser engraving machine, the wavelength used by the laser engraving machine is 460nm, the laser power is set to 1w to 10w, the laser scanning speed is 10mm / s to 120mm / s, and the laser induced graphene powder is prepared on the polyimide film in one step, and the particle size of the laser induced graphene powder is 3nm to 500μm. ;

[0071] In this embodiment 1, the thickness of the polyimide film is 50 μm to 2 mm, the length is 3 cm to 50 cm, and the width is 0.5 cm to 40 cm. The length and width can be adjusted according to the amount of graphene required;

[0072] (2) Preparation of biomimetic super-hydrophobic low-adhesion substrate 1

[0073] Weigh a PDMS polymer and a curing agent (RTV615 silicone rubber compound), mix and stir the PDMS polymer and the curing agent in a weight ratio of 5:1 to 20:1, add a number of SiO2 (silicon dioxide) microspheres with a particle size of 30nm-30μm, PTFE (polytetrafluoroethylene) micropowder, and laser-induced graphene (LIG) powder, the weight ratio of the PDMS polymer, SiO2 microspheres, PTFE micropowder and laser-induced graphene powder is 20:2:1:1, place the mixed mixture in a vacuum barrel, and evacuate for about 0.5 to 2h, the purpose is to discharge the bubbles in the mixture; inject the prepared mixture into a silicon-based mold using a syringe, and the size of the silicon-based mold can be changed according to the size of the orifice plate; bake the silicon-based mold containing the mixture at 70-80°C for 1.5-4h, and cure it at room temperature for 10-25min, peel the cured mixture from the silicon-based mold, and a bionic super-hydrophobic low-adhesion substrate can be obtained;

[0074] The particle size of PTFE (polytetrafluoroethylene) powder is 1nm to 500nm.

[0075] (3) Punching

[0076] A laser engraving machine is used to punch holes on the prepared biomimetic super-hydrophobic low-adhesion substrate to form sample holes, wherein the sample holes are through holes;

[0077] Draw the desired pattern in the computer software, set the laser engraving parameters, and load the drawn document. Place the bionic super-hydrophobic low-adhesion substrate on the wooden board, put it into the laser, adjust the distance between the laser port and the bionic super-hydrophobic low-adhesion substrate, turn on the laser, and punch holes. The holes are through holes. The structure and size of the bionic super-hydrophobic low-adhesion hole plate are as follows: Figure 2 , Figure 3 As shown, Figure 2 Dimensions are in mm.

[0078] (4) Integration of silicon wafer and biomimetic super-hydrophobic low-adhesion orifice plate

[0079] The silicon wafer is cut into rectangles with the same size as the bionic super-hydrophobic low-adhesion orifice plate, and the silicon wafer is tightly attached to the back of the bionic super-hydrophobic low-adhesion orifice plate by utilizing the viscosity of PDMS to obtain a bionic super-hydrophobic-hydrophilic integrated orifice plate.

[0080] PDMS has a certain degree of stickiness and can be bonded to glass, metal, silicon and other substrates, but it is not easy to bond to wooden materials such as wood boards. However, the bonding between PDMS and silicon wafers is not permanent. If permanent bonding is required, the PDMS substrate needs to be plasma treated.

[0081] The function of the silicon wafer is to better store and conduct heat to ensure temperature stability during PCR amplification. In addition, the hydrophilicity of the silicon wafer also enhances the fusion reaction between samples and avoids the generation of bubbles during the sample addition process.

[0082] The integrated structure and size of silicon wafer and bionic super-hydrophobic low-adhesion orifice plate are as follows Figure 4 shown.

[0083] In this embodiment, SiO2 microspheres and LIG powders construct a micro-nano composite structure on the substrate surface, while PTFE micropowders greatly reduce the surface energy of the substrate. PDMS acts as a binder to firmly adsorb SiO2 microspheres, PTFE micropowders and LIG powders. The coupling of physical modification and chemical modification gives the substrate excellent super-hydrophobic and low-adhesion properties like lotus leaves. The contact angle, rolling angle and droplet morphology of water droplets at any five positions on the substrate surface are shown in Figure 1. Figure 1 shown.

[0084] Example 2

[0085] A high-throughput automated nucleic acid library building instrument comprises a temperature control module, a three-dimensional automatic liquid transfer module and the bionic super-hydrophobic-hydrophilic integrated well plate of embodiment 1.

[0086] The temperature control module includes a stage 3, a cover plate 4, a source meter 5 and a heating plate (polyimide film and conductive wire). The stage 3 and the cover plate 4 are both provided with heating plates, which can independently heat and cool down.

[0087] The stage 3 is used to place the well plate. After the temperature control module is buckled, the cover plate 4 covers the upper surface of the well plate and contacts the upper surface of the well plate to amplify heating, ensure the temperature inside the well plate, and avoid condensation or volatilization of the solvent in the well plate;

[0088] The source meter 5 is used to provide current for heating the heating plate. By adjusting the voltage through the source meter, the heating temperature of the heating plate can be controlled, and the temperature can be increased or decreased, and the temperature can be stabilized. The structural design of the temperature control module is as follows: Figure 5 shown.

[0089] The three-dimensional automatic liquid transfer module comprises a housing 6, a motor housing 7, a three-dimensional motion component 8, a base plate 9, a mounting seat 10 and a pipette 11. The base plate 9 is a supporting base plate of the entire three-dimensional liquid transfer module. The pipette 11 is installed in the housing. The motor housing 7 is provided with a power switch 12 and a power socket 13.

[0090] The pipette 11 realizes the automatic installation of the pipette tip through the Z-axis movement of the three-dimensional motion component, and the linear thrust motor drives the movement of the piston to complete the continuous actions of aspirating, discharging and removing the tip; the mounting seat is connected to the three-dimensional motion component and can move forward, backward, left and right. The pipette tip is used to aspirate and discharge liquid. The pipette tip of the pipette is placed in the tip box on the mounting seat in advance. After the library construction starts, the tip box is moved to the bottom of the pipette through the three-dimensional motion component, and the pipette moves down to realize the automatic installation of the tip;

[0091] The three-dimensional motion assembly of the present invention adopts a Cartesian coordinate system design, and linear guides, drive motors, synchronous pulleys and limiters are installed in the X-axis and Y-axis directions, and a pipette is installed in the Z-axis direction, and the slider drives the X-axis and Y-axis to move in association, realizing synchronous belt transmission, that is, the pipette can realize three-dimensional motion of the X, Y and Z axes. The three-dimensional motion assembly moves, driving the gun tip box to move directly below the pipette, and the pipette pushes the piston up and down through the linear thrust motor to complete the continuous actions of aspirating, discharging and removing the gun tip.

[0092] The function of the pipette is to aspirate and discharge liquids and control the precise amount of samples and reagents used in library construction. The pipette adopts the air displacement principle to design piston pipetting. The piston is made of zirconium oxide and is fixed to the piston frame through a bushing. The motor shaft end is equipped with a permanent magnet connected to the piston rod. The linear thrust motor drives the movement of the piston to complete the aspiration and discharge of liquids. The structure of the pipette is a mature technology in this field, such as Figure 6 shown.

[0093] The linear thrust motor is located above the pipette and is connected to the pipette.

[0094] The pipette 11 has a built-in micro pump, and the micro pump is equipped with a liquid level monitoring sensor and a pressure sensor, which can monitor the liquid level and pressure in real time.

[0095] The software can realize the digital control and interface function display of the three-dimensional automatic pipetting module. The structural diagram of the three-dimensional automatic pipetting module is as follows Figure 7 shown.

[0096] Example 3

[0097] A high-throughput automated nucleic acid library construction instrument, the operation steps of which are as follows:

[0098] S1: washing and drying the bionic super-hydrophobic low-adhesion substrate 1, placing it on the silicon wafer 2, and pressing it to make it tightly connected to obtain a bionic super-hydrophobic-hydrophilic integrated hole plate;

[0099] S2: placing the bionic super-hydrophobic-hydrophilic integrated well plate on the stage 3 of the temperature control module, and placing the temperature control module on the mounting seat 10 of the three-dimensional automatic pipetting module;

[0100] S3: Set the relevant parameters of the three-dimensional automatic pipetting module to realize the automatic loading of reaction reagents and nucleic acid samples required for library construction; the relevant parameters include volume parameters and position parameters. The volume parameters include the pipetting volume, the speed of aspiration and discharge, the number of mixing times and the mixing volume before aspiration / discharge, and the waiting time after the step is completed; the position parameters include the Z axis (up and down), the X axis (left and right), and the column from which to which to support the single-column loading and withdrawal of pipette tips and aspiration and discharge.

[0101] S4: Cover the cover of the temperature control module, set the temperature of the stage 3 and the cover 4 respectively, and implement the nucleic acid shearing reaction and PCR amplification program;

[0102] Nucleic acid shearing procedure: the cover plate temperature was set to 100°C for preheating; the stage temperature was 55°C for 5 minutes and then the temperature was reduced to 10°C;

[0103] Nucleic acid amplification program: the cover plate temperature was set to 100°C for preheating; stage temperature: ① 72°C for 3 min; ② 95°C for 30 s; ③ 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, 12 cycles; ④ 72°C for 5 min; ⑤ the temperature dropped to 10°C.

[0104] S5: Add magnetic bead suspension to the loading holes of the bionic super-hydrophobic-hydrophilic integrated well plate to adsorb the PCR amplification product, attach it to the magnetic mounting base to separate the magnetic beads, set the parameters of the three-dimensional automatic pipetting module to realize automatic loading of the elution solvent, and finally achieve product purification.

[0105] The above is 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bionic super hydrophobic-hydrophilic integrated orifice plate, characterized in that: The sample loading hole of the well plate is a through hole, the side wall of the through hole is super hydrophobic, and the bottom of the through hole is a hydrophilic silicon wafer; The preparation method comprises the following steps: (1) Preparation of laser-induced graphene The polyimide film was cut and ultrasonically cleaned with anhydrous ethanol and deionized water for 5 to 20 minutes respectively, the pattern to be engraved was introduced into the laser engraving machine, the laser power and laser scanning speed were set, and the laser-induced graphene powder was prepared on the polyimide film in one step for later use; (2) Preparation of biomimetic super-hydrophobic low-adhesion substrate Weigh a PDMS polymer and a curing agent, mix and stir the PDMS polymer and the curing agent in a weight ratio of 5:1 to 20:1, add a number of SiO2 microspheres with a particle size of 30nm-30μm, PTFE micropowder, and laser-induced graphene powder, place the mixed mixture in a vacuum barrel, and evacuate for 0.5-2h; inject the prepared mixture into a silicon-based mold using a syringe; bake the silicon-based mold containing the mixture at 70-80°C for 1.5-4h, and cure it at room temperature for 10-25min, peel off the cured mixture from the silicon-based mold, and obtain a bionic super-hydrophobic low-adhesion substrate; The particle size of PTFE micropowder is 1nm~500nm; the weight ratio of PDMS polymer, SiO2 microspheres, PTFE micropowder and laser-induced graphene powder is 20:2:1:1; (3) Punching A laser engraving machine is used to punch holes on the prepared biomimetic super-hydrophobic low-adhesion substrate to form sample holes, wherein the sample holes are through holes; (4) Integration of silicon wafer and biomimetic super-hydrophobic low-adhesion orifice plate The silicon wafer is cut into rectangles with the same size as the bionic super-hydrophobic low-adhesion orifice plate, and the silicon wafer is tightly attached to the back of the bionic super-hydrophobic low-adhesion orifice plate by utilizing the viscosity of PDMS to obtain a bionic super-hydrophobic-hydrophilic integrated orifice plate.

2. The bionic super hydrophobic-hydrophilic integrated orifice plate according to claim 1, characterized in that: In step (1), the thickness of the polyimide film is 50 μm to 2 mm, the length is 3 cm to 50 cm, and the width is 0.5 cm to 40 cm; The wavelength used by the laser engraving machine is 460nm, the laser power is 1w~10w, and the laser scanning speed is 10mm / s~120mm / s; The particle size of the prepared laser-induced graphene powder is 3nm~500μm.

3. A high-throughput automated nucleic acid library construction instrument, characterized in that: It comprises a temperature control module, a three-dimensional automatic liquid transfer module and the bionic super hydrophobic-hydrophilic integrated well plate according to any one of claims 1 to 2.

4. The high-throughput automated nucleic acid library construction instrument according to claim 3, characterized in that: The temperature control module includes a stage, a cover plate, a source meter and a heating plate. The stage and the cover plate are both provided with heating plates, which can heat and cool independently. The stage is used to place the well plate. After the temperature control module is buckled, the cover plate is placed on the upper surface of the well plate for amplification heating to ensure the temperature inside the well plate and prevent the solvent in the well plate from condensing or volatilizing. The source meter is used to provide current for heating the heater.

5. The high-throughput automated nucleic acid library construction instrument according to claim 4, characterized in that: The three-dimensional automatic liquid transfer module comprises a housing, a motor housing, a three-dimensional motion component, a base plate, a mounting seat and a pipette, wherein the pipette is mounted in the housing, and a power switch and a power socket are arranged on the motor housing; The pipette realizes automatic installation of the pipette tip through the Z-axis movement of the three-dimensional motion component, and completes the continuous actions of aspirating, discharging and removing the pipette tip by pushing the piston through the linear thrust motor; the mounting seat is connected to the three-dimensional motion component, and the pipette tip of the pipette is placed in the tip box on the mounting seat in advance. After the library construction starts, the tip box is moved to the bottom of the pipette through the three-dimensional motion component, and the pipette moves down to realize automatic installation of the tip.

6. The high-throughput automated nucleic acid library construction instrument according to claim 5, characterized in that: The steps are as follows: S1: After cleaning and drying the bionic super-hydrophobic low-adhesion substrate, place it on a silicon wafer and press it to make it tightly connected to obtain a bionic super-hydrophobic-hydrophilic integrated hole plate; S2: placing the bionic super-hydrophobic-hydrophilic integrated well plate on the stage of the temperature control module, and placing the temperature control module on the mounting seat of the three-dimensional automatic pipetting module; S3: Setting the relevant parameters of the three-dimensional automatic pipetting module to realize the automatic loading of reaction reagents and nucleic acid samples required for library construction; S4: Cover the cover of the temperature control module, set the temperature of the stage and the cover respectively, and implement the nucleic acid shearing reaction and PCR amplification program; S5: Add magnetic bead suspension to the loading holes of the bionic super-hydrophobic-hydrophilic integrated well plate to adsorb the PCR amplification product, attach it to the magnetic mounting base to separate the magnetic beads, set the parameters of the three-dimensional automatic pipetting module to realize automatic loading of the elution solvent, and finally achieve product purification.

7. The high-throughput automated nucleic acid library construction instrument according to claim 6, characterized in that: In step S3, the relevant parameters include volume parameters and position parameters.

8. The high-throughput automated nucleic acid library construction instrument according to claim 7, characterized in that: In step S4, the temperature of the stage and the cover is: Nucleic acid shearing procedure: the cover plate temperature was set to 100°C for preheating; the stage temperature was 55°C for 5 minutes and then the temperature was reduced to 10°C; Nucleic acid amplification program: the cover plate temperature was set to 100°C for preheating; stage temperature: ①72°C for 3 min; ②95°C for 30 s; ③95°C for 10 s, 55°C for 30 s, 72°C for 30 s, 12 cycles; ④72°C for 5 min; ⑤the temperature dropped to 10°C.

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