NUCLEIC ACIDS EXTRACTION SYSTEM AND METHOD BASED ON 3D-PRINTED MICRODEVICE

Abstract

The present invention discloses a nucleic acids extraction system and method based on a 3D-printed microdevice, and belongs to the technical field of nucleic acids extraction. The nucleic acids extraction system is a monomer 3D-printed microdevice or a 3D-printed microdevice prepared by 3D printing technologies, the monomer 3D-printed microdevice comprises a nucleic acids binding region and a handle region, and the 3D-printed microdevice is composed of more than two monomer 3D-printed microdevices through a joining region; and the nucleic acids binding region is made of photosensitive resin or thermoplastic. An extraction method for the nucleic acids extraction system based on a 3D-printed microdevice is used to bind, clean and elute nucleic acids by moving the monomer 3D-printed microdevice or the 3D-printed microdevice among a solution containing target nucleic acids, a washing buffer and an elution buffer.

Claims

1. A nucleic acids extraction system based on a 3D-printed microdevice, wherein the nucleic acids extraction system based on a 3D-printed microdevice is a monomer 3D-printed microdevice or a 3D-printed microdevice prepared by 3D printing technologies; the monomer 3D-printed microdevice comprises a nucleic acids binding region and a handle region, the 3D-printed microdevice is composed of more than two monomer 3D-printed microdevices and a joining region used for connecting monomer 3D-printed microdevices, and the handle region ends of the monomer 3D-printed microdevices, which are far away from the nucleic acids binding region, are connected in parallel to the joining region; the nucleic acids binding region of the monomer 3D-printed microdevice comprises type a, type b, type c, type d, type e and type f, wherein the type a is in a shape of cone, and the center position of the bottom surface of the cone is combined with the handle region; the type b is in a shape of conoid, the vertex of the cone is smoothed, and the center position of the bottom surface of the cone is combined with the handle region; the type c is in a shape of cylinder and hemispheroid, the diameter of the cylinder is the same as that of the hemispheroid, the center position of the bottom surface of one end of the cylinder is combined with the handle region, and the bottom surface of the other end is combined with the maximum diameter surface of the hemispheroid; the type d is in a shape of hemispheroid, and the center position of the maximum diameter surface of the hemispheroid is combined with the handle region; the type e is in a shape of cylinder and cone, the diameter of the cylinder is the same as that of the bottom surface of the cone, the center position of one end of the cylinder is combined with the handle region, and the other end is combined with the bottom surface of the cone; and the type f is in a shape of cylinder and conoid, the diameter of the cylinder is the same as that of the bottom surface of the conoid, the center position of one end of the cylinder is combined with the handle region, the other end is combined with the bottom surface of the conoid, and the vertex of the conoid is smoothed.

2. The nucleic acids extraction system based on a 3D-printed microdevice according to claim 1, wherein the nucleic acids binding region has a smooth or rough surface, with or without a microstructure; the microstructure comprises one or a combination of more than two of thread structure, groove structure, porous structure, porous channel structure, coarse grain and convex structure; the number of the thread structure or groove structure is zero or at least one; the coarse grain is in any shape; the porous structure, the porous channel structure and the convex structure are in nanometer size or micron size; the groove structure and the convex structure may be in a shape of cylinder, cone, platform and spheroid or in an irregular shape; and the microstructure is distributed in any position of the nucleic acids binding region, and is combined and distributed in any way when the number thereof is more than one.

3. The nucleic acids extraction system based on a 3D-printed microdevice according to claim 1, wherein the handle region is in a shape of cylinder or prism; the handle region is made of photosensitive resin or thermoplastic, the nucleic acids binding region is made of photosensitive resin or thermoplastic, and the material of the handle region is the same as or different from that of the nucleic acids binding region; and the joining region, the handle region and the nucleic acids binding region are prepared integrally or separately through 3D printing technologies, and assembled by bonding or buckling when being prepared separately.

4. The nucleic acids extraction system based on a 3D-printed microdevice according to claim 1, wherein the nucleic acids binding region is loaded or not loaded with functional groups or particle materials, wherein the functional groups comprise amino groups, carboxyl groups and hydroxyl groups; the particle materials comprise inorganic particle materials or metallic particle materials, and the inorganic particle materials comprise silicon dioxide, titanium dioxide, manganese dioxide, ferroferric oxide, graphene oxide and mica; and the metallic particle materials comprise gold, silver and iron.

5. The nucleic acids extraction system based on a 3D-printed microdevice according to claim 1, wherein the nucleic acids extraction system based on a 3D-printed microdevice is used in conjunction with a centrifugal tube, and when the nucleic acids extraction system based on a 3D-printed microdevice is placed in a centrifugal tube, the height of the part of the handle region exposed from the centrifugal tube is not less than 3 mm.

6. A nucleic acids extraction method for the nucleic acids extraction system based on a 3D-printed microdevice according to claim 1, comprising the following steps: (1) inserting the nucleic acids binding region of a monomer 3D-printed microdevice or a 3D-printed microdevice into a solution containing target nucleic acids for nucleic acids binding; (2) moving the handle region or the joining region of the monomer 3D-printed microdevice or the 3D-printed microdevice completing step (1) by hand or a machine to make the nucleic acids binding region inserted into a washing buffer for nucleic acids cleaning; (3) taking out and drying the monomer 3D-printed microdevice or the 3D-printed microdevice completing step (2); (4) moving the handle region or the joining region of the monomer 3D-printed microdevice or the 3D-printed microdevice completing step (3) by hand or a machine to make the nucleic acids binding region placed into an elution buffer for nucleic acid elution, and the obtained elution buffer is the target nucleic acids extraction solution.

7. The nucleic acids extraction method according to claim 6, wherein the specific steps of nucleic acids binding in step (1) are as follows: inserting the nucleic acids binding region of a monomer 3D-printed microdevice or a 3D-printed microdevice into a mixed solution containing target nucleic acids, and adding or not adding an auxiliary binding solvent, wherein the auxiliary binding solvent comprises one or a mixed solution of isopropyl alcohol and absolute ethyl alcohol, and the volume of the auxiliary binding solvent is 0.6-0.8 time that of the lysate; and the binding time is 5 s-24 h; the nucleic acids cleaning in step (2) is carried out for 1-5 times; and the cleaning time is 2 s-1 min each time; the drying in step (3) can be carried out at room temperature or under the heating condition, and the drying time is 1 min-24 h; the elution buffer in step (4) is a solution capable of separating nucleic acids bound to the monomer 3D-printed microdevice or the 3D-printed microdevice, comprising water, PBS buffer, TE buffer and downstream PCR reaction liquid; and the elution time is 5 s-5 min.

8. The nucleic acids extraction method according to claim 6, wherein the target nucleic acids in step (1) comprise one or a mixture of more than two of RNA, genomic DNA and plasmid DNA.

9. The nucleic acids extraction method according to claim 6, wherein the lysate is CTAB lysate, NaHCO.sub.3 lysate, Chelex lysate, proteinase K lysate, SDS lysate or Trizol lysate; the lysate is added or not added with RNA digestive enzyme or DNA digestive enzyme; and the lysis time is 1 min-24 h, and the lysis temperature is −20-100° C.

10. The nucleic acids extraction method according to claim 9, wherein the SDS lysate comprises 0.5 wt %-20 wt % of SDS, wherein proteinase K is added at 0-30 μg/mL.

Description

DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is a structural schematic diagram of a preferred monomer 3D-printed microdevice of the present invention.

[0037] FIG. 2 is a structural schematic diagram of a monomer 3D-printed microdevice prepared in embodiment 1 of the present invention. FIG. 2(a) to FIG. 2(h) respectively show monomer 3D-printed microdevices 1-8.

[0038] FIG. 3 is a structural schematic diagram of a special-shaped 3D-printed microdevice of embodiment 4 of the present invention.

[0039] FIG. 4 is an agarose gel electrophoretogram of embodiment 4 of the present invention, wherein lane 1 is negative control, lanes 2-9 are 8 samples, and M is DL2000 marker.

[0040] In the figures: 1 nucleic acids binding region; 2 handle region; and 3 joining region.

DETAILED DESCRIPTION

[0041] The contents of the present invention are further described below in combination with the technical solution. It should be pointed out that the following descriptions are all illustrative and are intended to provide further description of the present invention. Unless otherwise specified, all scientific and technical terms used in the present invention have the same meanings as those generally understood by those skilled in the art of the present invention.

[0042] The materials and the actual and experimental equipment involved in embodiments of the present invention are in line with commercially available products in the related technical fields of chemical industry and biology.

[0043] Primers involved in the embodiments of the present invention are synthesized by a commissioned bioengineering company, and the specific primer information is as follows:

TABLE-US-00001 SEQ ID NO: 1: 5′-actgggataatacgatagaag-3′ SEQ ID NO: 2: 5′-gtgcgttaggattagttatgt-3′

Embodiment 1

[0044] 3D MAX software is adopted to design, draw and set the structure. Photosensitive resin, polyacrylic acid (PAA), as a raw material, is subjected to photocuring reaction by the DLP 3D printing technology at wavelength of 400-800 nm to prepare a monomer 3D-printed microdevice, and the structural schematic diagram is shown in FIG. 2(a) to FIG. 2(h), wherein the handle regions of monomer 3D-printed microdevices shown in FIG. 2(a) to FIG. 2(f) are cylindrical, and have diameters of 3 mm, 3 mm, 5 mm, 4 mm, 4 mm and 6 mm and heights of 1.5 cm, 3.0 cm, 12.0 cm, 2.0 cm, 4.0 cm and 12 cm; and the handle regions of monomer 3D-printed microdevices shown in FIG. 2(g) to FIG. 2(h) are respectively in a shape of square cylinder, the handle region of a monomer 3D-printed microdevice shown in FIG. 2(g) is 5 mm in length and width and 6.0 cm in height, and the handle region of the monomer 3D-printed microdevice shown in FIG. 2(h) is 6 mm in length and width and 12.0 cm in height. The nucleic acids binding regions of monomer 3D-printed microdevices shown in FIG. 2(a) to FIG. 2(h) have diameters of 0.38 cm, 0.4 cm, 1.2 cm, 0.6 cm, 0.6 cm, 2.0 cm, 1.0 cm and 2.0 cm and heights of 0.8 cm, 1.0 cm, 2.0 cm, 1.0 cm, 1.0 cm, 2.5, cm, 1.0 cm and 2.5 cm. The nucleic acids binding regions of monomer 3D-printed microdevices shown in FIG. 2(a), FIG. 2(c) and FIG. 2(d)-FIG. 2(g) respectively have a helical structure, which has 8 layers, 4 layers, 6 layers, 10 layers, 3 layers and 5 layers respectively; The nucleic acids binding regions of monomer 3D-printed microdevices shown in FIG. 2(b) to FIG. 2(f) respectively have a groove structure, which comprises 6 grooves, 8 grooves, 3 grooves, 4 grooves and 20 grooves; and the nucleic acids binding region of a monomer 3D-printed microdevice shown in FIG. 2(h) has a porous structure.

[0045] Results show that monomer 3D-printed microdevices shown in FIG. 2(a)-FIG. 2(h) can be placed into a 0.2 ml conical centrifuge tube, a 0.5 ml conical centrifuge tube, a 15 ml conical centrifuge tube, a 1.5 ml conical centrifuge tube, a 2.0 ml conical centrifuge tube, a 50 ml round-bottom centrifuge tube, a 5 ml round-bottom centrifuge tube, and a 50 ml conical centrifuge tube respectively.

[0046] In the embodiment, PAA is used as the raw material, which is easy to obtain, and the 3D printing process is simple and fast, which can significantly reduce the economic cost; and microdevices with different sizes and different shapes can be obtained by setting parameters according to different requirements of downstream biomolecular technology application, and are used in conjunction with centrifugal tubes of different types and specifications, so as to flexibly meet application requirements, with high adjustment flexibility.

Embodiment 2

[0047] A part of silkworm chrysalis tissues are taken and placed in a 1.5 mL EP tube. 200 μL of proteinase K lysate (100 mmol of Tris-HCl, 25 mmol of EDTA, 500 mmol of NaCl and 1% of SDS) and 5 μL of proteinase K solution are added into the EP tube, oscillated and blended to lyse the extracted sample tissues, and placed at room temperature for 30 min, 150 μL of absolute ethyl alcohol is added, the monomer 3D-printed microdevice shown in FIG. 2(d) is placed into the EP tube to make the nucleic acids binding region of the monomer 3D-printed microdevice inserted into the solution, the handle region is held by hand or a machine, the 3D-printed microdevice is slightly shaken to blend the solution, the monomer 3D-printed microdevice is transferred to an EP tube with 200 μL of washing buffer after 10 s to make the nucleic acids binding region of the monomer 3D-printed microdevice inserted into the washing buffer, then the handle region is held by hand or a machine, and the monomer 3D-printed microdevice is slightly shaken for 5 s, and then transferred to the washing buffer repeatedly two times; and the monomer 3D-printed microdevice is taken out from the washing buffer, the residual droplets on the monomer 3D-printed microdevice are shaken off, the monomer 3D-printed microdevice is dried in a 37° C. blast incubator for 1 min, and the nucleic acids binding region of the monomer 3D-printed microdevice is placed into a 1.5 mL EP tube with 50 μL of elution buffer and slightly shaken for 30 s for elution.

[0048] 3 μL of elution buffer is taken and tested with an ultraviolet spectrophotometer three times, and for the extracted nucleic acid samples, 260/230 is 1.610-1.728, 260/280 is 1.817-1.902, and the concentration is 42.32-48.274 ng/μL.

[0049] Results show that the DNA samples extracted by the nucleic acids extraction system based on a 3D-printed microdevice of the present invention from the lysate of animal tissue samples have better quality and almost no pollution of protein, salt, polysaccharide, etc.

[0050] The embodiment illustrates that when the nucleic acids extraction system based on a 3D-printed microdevice is used for nucleic acids extraction, nucleic acid binding and cleaning can be completed in 25 s without pipetting, centrifugation, filtration and other operations, which is convenient and rapid with good extraction quality.

Embodiment 3

[0051] 4.5 ml of Escherichia coli culture solution is taken and added into a 5 ml EP tube, and centrifuged to remove supernatant, 500 ml of sterile resuspension (50 mmol/L glucose, 25 mmol/L Tris with PH=8.0, and 10 mmol/L EDTA with PH=8.0) is added to suspend thalli, then 1 mL of lysate (0.2 M of NaOH and 1% of SDS) is added, the centrifugal tube is turned upside down five times for 2 min to blend the solution until the solution is thick but clear, then 750 μL of neutralization buffer (5 mol/L potassium acetate and 5 mol/L glacial acetic acid) is added, the centrifugal tube is immediately turned upside down several times until flocculent precipitation appears in the solution, the solution is centrifuged at 12000 r/min for 10 min, and 2 mL of supernatant is drawn carefully and transferred to another 5 mL centrifugal tube to obtain a solution containing plasmid DNA.

[0052] 1500 μL of absolute ethyl alcohol is added to the solution containing plasmid DNA, the monomer 3D-printed microdevice shown in FIG. 2(g) is placed into the solution to make the nucleic acids binding region of the monomer 3D-printed microdevice inserted into the solution, the handle region is held by hand, the monomer 3D-printed microdevice is slightly shaken to blend the solution, and then transferred to an EP tube with 2000 μL of washing buffer (75% alcohol) after 25-30 s to make the nucleic acids binding region inserted into the washing buffer, then the handle region is held by hand, and the monomer 3D-printed microdevice is slightly shaken for 10-15 s, and then transferred to the washing buffer repeatedly 2-3 times; and the monomer 3D-printed microdevice is taken out from the washing buffer, the residual droplets on the monomer 3D-printed microdevice are shaken off, the monomer 3D-printed microdevice is dried at room temperature for 15 min, and the nucleic acids binding region of the monomer 3D-printed microdevice is placed into 100 μL of elution buffer and slightly shaken for 30 s for elution.

[0053] The extracted plasmid DNA is tested with an ultraviolet spectrophotometer three times, and for the plasmid DNA, 260/230 is 1.723-1.789, 260/280 is 1.857-1.931, and the concentration is 178.30-181.464 ng/μL.

[0054] Results show that the plasmid DNA samples extracted by the monomer 3D-printed microdevice of the present invention from bacteria have high quality and no pollution. The plasmid DNA separation process does not need pipetting, centrifugation, filtration and other operations, which is convenient and rapid with good extraction quality.

Embodiment 4

[0055] (1) Preparation of Special-Shaped 3D-Printed Microdevice (Composed of Eight Monomer 3D-Printed Microdevices Connected by a Joining Region)

[0056] 3D MAX software is adopted to design, draw and set the structure. Photosensitive resin (PAA), as a raw material, is subjected to photocuring reaction by the DLP 3D printing technology at wavelength of 400-800 nm to prepare a 3D-printed microdevice having a structure shown in FIG. 3, which has the nucleic acids binding region with the height of 9 mm and the diameter of 3.8 mm and the handle region with the height of 21 mm and can be placed in a 8-tube strip for use.

[0057] (2) Preparation of Target Nucleic Acid Solution

[0058] A frozen clam is taken, a tissue block weighing about 500 mg thereof is directly put into a mortar sterilized by high temperature and high pressure, added with liquid nitrogen and ground rapidly, after the tissue is softened, a small amount of liquid nitrogen is added, the tissue block is ground again, and the above operation is repeated three times. Then 150-200 mg of tissue samples is taken, added with 2 ml of Trizol and fully blended with an electric homogenizer for 1-2 min. Centrifugation is conducted at 12000 r/min for 5 min, precipitation is discarded, 400 μL of chloroform is added, the centrifugal tube is covered tightly, and the mixture is shaken by hand for 15 s and placed at room temperature for 10 min. Then, the mixture is centrifuged at 4° C. at 12000 g for 15 min, and the aqueous phase is transferred to a new EP tube to obtain a solution containing RNA.

[0059] (3) High Throughput and Rapid RNA Separation by Special-Shaped Microdevice

[0060] 80 μL of RNA solution is taken and added into each reaction hole of a 8-tube strip, a special-shaped microdevice is placed into each reaction hole to make the nucleic acids binding region completely inserted into the solution, 55 μL of isopropyl alcohol is added, the special-shaped microdevice is slightly shaken to blend the solution, and after 5-10 s, transferred to an EP tube with 200 μL of washing buffer (75% alcohol), and the special-shaped microdevice is slightly shaken for 5-10 s, and then transferred to the washing buffer repeatedly one time; and the special-shaped microdevice is taken out from the washing buffer, the residual droplets on the special-shaped microdevice are shaken off, and the special-shaped microdevice is dried at room temperature for 5 min, placed into 40 μL of elution buffer and slightly shaken for 30 s for elution. The RNA concentration of each elution buffer is tested with an ultraviolet spectrophotometer between 41 ng/μL and 52 ng/μL, A260/230 is 1.703-1.920, and 260/280 is 1.974-2.133.

[0061] (4) Downstream Application of Extracted RNA

[0062] 7 μL of liquid is taken from each reaction hole of the 8-tube strip, cDNA is prepared through a reverse transcription kit (Takara, Code No. RR047A) according to operating instructions, and 1 μL of product is respectively taken as an PCR amplification template to prepare PCR reaction liquid: every 25 μL of system contains 5 μL of primer (10 pM) shown in SEQ ID NO:1 and SEQ ID NO:2, 1 μL of template, 0.25 μL of ExTaq, 2.5 μL of 10×ExBuffer, 2 μL of dNTPs and 17.25 μL of deionized water, and sterile distilled water is used as a negative reference template. The PCR condition is 4 min at 95° C.; 35 cycles are performed at 95° C. for 30 s, 55° C. for 30 s and 72° C. for 60 s, and extension is performed at 72° C. for 5 min. The obtained PCR products are subjected to gel electrophoresis with 2% agarose, and the results are observed under UV light after the electrophoresis, as shown in FIG. 4.

[0063] Results show that FIG. 4 shows negative reference, marker and PCR amplification products of 8 samples from left to right, target strips appear in the 8 samples, and no target strip appears in the negative reference, indicating that 8 solutions used as templates contain target nucleic acids.

[0064] The embodiment shows that the RNA separated by the 3D-printed microdevice has good quality and less pollution and can fully meet the experimental requirements of downstream molecular technologies.

[0065] The embodiment also shows that the special-shaped microdevice is used for nucleic acids extraction, which can extract target nucleic acids at the same time with high throughput, effectively breaks through the bottleneck of low separation throughput of non-magnetic solid phase separation due to dependence on multi-step centrifugation, pipetting and other complicated operations and is conducive to realization of automation.

Embodiment 5

[0066] Photosensitive resin (PAA), as a raw material, is subjected to photocuring reaction by the DLP 3D printing technology at wavelength of 400-800 nm to prepare a cubic strip-shaped microdevice: 2 mm wide, 1 mm thick and 4 cm high, and the cubic strip-shaped microdevice has no special microstructure design.

[0067] Animal tissues (shellfish tissues) are taken, lysate (100 mmol of Tris-HCl, 25 mmol of EDTA, 500 mmol of NaCl and 1% SDS) and 5 μL of proteinase K are added, after warm bath at 55° C. for 2 h, 100 μL is respectively taken and added into two 0.5 mL centrifugal tubes which are numbered 1 # and 2 #, and 10 μL of RNA digestive enzyme is respectively added into the two centrifugal tubes and placed at room temperature for 30 min to obtain an RNA-free solution containing target DNA.

[0068] 70 μL of absolute ethyl alcohol is added into the 1 # tube, and the monomer 3D-printed microdevice shown in FIG. 2(b) and prepared in embodiment 1 is placed into the 1 # tube. 70 μL of absolute ethyl alcohol is added into the 2 # tube, and the cubic strip-shaped microdevice is placed into the 2 # tube. The microdevices are slightly shaken to blend the solutions, and after 15-20 s, respectively transferred to an EP tube with 100 μL of washing buffer (75% alcohol), and the microdevices are slightly shaken for 15-20 s, and then transferred to the washing buffers repeatedly three times; and the microdevices are respectively taken out from the washing buffers, the residual droplets on each microdevice are shaken off, and each microdevice is dried in a 37° C. blast incubator for 3 min, placed into 40 μL of elution buffer and slightly shaken for 30 s for elution.

[0069] The DNA concentration of the extracted nucleic acids is tested with an ultraviolet spectrophotometer, and for the nucleic acid sample extracted from the 1 # tube, 260/230 is 1.864, 260/280 is 1.902, and the concentration is 90.547 ng/μL. For the nucleic acid sample extracted from the 2 # tube, 260/230 is 1.616, 260/280 is 1.736, and the concentration is 21.233 ng/μL.

[0070] Results show that the embodiment can quickly and effectively separate DNA with high quality, and the separated and purified DNA has low pollution and high purity. Meanwhile, compared with other monomer 3D-printed microdevices, the monomer 3D-printed microdevice preferred in the embodiment, especially the monomer 3D-printed microdevice with microstructure, has higher nucleic acids separation ability.

[0071] The technology of the present invention is described through preferred embodiments. Related technicians can obviously modify or appropriately change and combine the system and method described herein without departing from the scope of the content and spirit of the present invention to realize the technology of the present invention. It should be pointed out that all similar replacements and changes, such as reasonable adjustment, change and combination of shape, size, surface and structure of monomer 3D-printed microdevice or 3D-printed microdevice, reasonable composition adjustment of lysate, washing buffer and elution buffer, reasonable extension and contraction of operation time, and reasonable change of operating temperature, are apparent to those skilled in the art and are considered to be included in the spirit, scope and content of the present invention.