CN114100714B - Nucleic acid or polypeptide high-throughput synthesis chip and application thereof - Google Patents
Nucleic acid or polypeptide high-throughput synthesis chip and application thereof Download PDFInfo
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- CN114100714B CN114100714B CN202111386263.7A CN202111386263A CN114100714B CN 114100714 B CN114100714 B CN 114100714B CN 202111386263 A CN202111386263 A CN 202111386263A CN 114100714 B CN114100714 B CN 114100714B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
Abstract
The invention provides a nucleic acid or polypeptide high-flux synthesis chip and application thereof. The synthesis chip comprises a body with a plate-shaped structure, wherein a plurality of micropores are vertically formed downwards on the body, the micropores comprise a straight hole for sample adding liquid drops at the upper layer and a fancy hole for synthesis reaction at the lower layer, and a circulating micro-channel is arranged in the fancy hole. Through the accurate control to chip structure parameter, it can improve the load capacity on the unit area effectively, can realize the demand of the simultaneous synthesis of multiple sequence polypeptide or nucleic acid on same substrate to can prevent effectively that solution from overflowing from the top of hole, can avoid the problem of mutual pollution between the different point positions in the synthetic process.
Description
Technical Field
The invention relates to the field of biosynthesis, in particular to a nucleic acid or polypeptide high-throughput synthesis chip and application thereof.
Background
With the rapid development of biology, nucleic acids and polypeptides are widely applied in many fields, and the number of chemically synthesized oligonucleotides in the world is hundreds of thousands every day, so that the oligonucleotides are widely applied to biomedical hotspots such as gene therapy, gene chips and the like. Solid phase chemical synthesis is the main means of chemical synthesis of DNA, and the current general DNA solid phase synthesis strategy in the industry is a solid phase phosphoramidite method, wherein the tail end of nucleotide is connected to a solid phase carrier with active groups, a DNA synthesis unit is introduced, the chemical synthesis reaction is carried out on the nucleotide and the active groups on the solid phase carrier one by one, impurities and byproducts are left in a mobile phase and are removed by cleaning, and finally a target DNA product is cracked.
In the latest research progress at home and abroad, research on a new generation of DNA synthesis technology and instruments based on microfluidic chips, inkjet printing technology and the like becomes a new research and development method. The DNA synthesizer based on the microfluidic chip integrates a trace amount of reagent into the microfluidic chip for reaction, so that the cost is reduced, but the modification of a solid phase carrier of the DNA synthesizer mostly adopts a photoetching technology, so that the process is complex. A large number of synthesis units are designed on the basis of the nucleic acid or polypeptide synthesis chip for ink-jet printing, different reaction raw materials are selectively input into each synthesis unit through an ink-jet printing head, and the in-situ custom synthesis of high-flux nucleic acid or polypeptide can be flexibly realized. However, how to explore the chip design and processing mode to better control the flow rate, prevent the liquid overflow, increase the unit area loading and the solution utilization efficiency, etc. is still a key issue worth to be researched to realize high-throughput DNA synthesis.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the above problems in the prior art, the present invention provides a nucleic acid or polypeptide high-throughput synthesis chip capable of controlling flow rate, preventing liquid overflow, and effectively increasing the unit area loading and solution utilization efficiency.
Another object of the present invention is to provide the use of the chip for high-throughput synthesis of oligonucleotides based on the nucleic acid or polypeptide.
Means for solving the problems
The invention provides the following technical scheme:
[1] a chip for high-throughput synthesis of nucleic acids or polypeptides, wherein,
the synthesis chip comprises a body with a plate-shaped structure, a plurality of micropores are vertically and downwards formed in the body, the micropores comprise a through hole on the upper layer for sample adding liquid drops and a fancy hole on the lower layer, the fancy hole is coaxial with the through hole and has the same aperture as the through hole, the through hole vertically extends into the body from an opening on the upper surface of the body, a circulating micro channel is arranged in the fancy hole, and the micro channel vertically extends to the through hole from an opening on the lower surface of the body; the ratio of the aperture of the straight-through hole to the depth of the straight-through hole is marked as A, the number of the liquid drops dripped from a single straight-through hole in a single time is marked as n, the A is more than or equal to 1/4n and less than or equal to 1/n, and n is an integer not less than 1.
[2] The nucleic acid or polypeptide high-throughput synthesis chip according to [1], wherein,
the volume of the cavity of the fancy holes and the surface area of the cavity are marked as B, and B is more than or equal to 0.4 mu m and less than or equal to 10 mu m.
[3] The nucleic acid or polypeptide high-throughput synthesis chip according to [1] or [2], wherein,
the ratio of the cavity cross-sectional area to the solid cross-sectional area of the fancy holes in the top view direction is marked as C,0.3 and C < -0.7.
[4] The nucleic acid or polypeptide high-throughput synthesis chip according to [3], wherein,
the ratio of the aperture of the straight-through hole to the depth of the fancy hole is marked as D, and D is more than or equal to 27C/40n and less than or equal to 5C/6n.
[5] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of claims 1 to 4, wherein,
the thickness of the body is not more than 10cm; further preferably, the thickness of the body is less than 1cm.
[6] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of claims 1 to 5, wherein,
the inner wall of the micro-channel cavity of the fancy hole is chemically treated; further, the inner wall surface is provided with a linker arm having a hydroxyl group, an amino group, or a carboxyl group protected with a protecting group or without a protecting group by the chemical treatment.
[7] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of the technical aspects of [1] to [6], wherein,
the inner wall of the through hole and the upper surface of the fancy hole connected with the through hole are subjected to smoothing treatment and hydrophobic treatment.
[8] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of claims 1 to 7, wherein,
the pattern holes have a middle-divided shape or a vortex shape in the plan view direction of the microchannel.
[9] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of claims 1 to 8, wherein,
the basic material of the body is silicon material or glass, and further the basic material of the body is silicon material.
[10] The nucleic acid or polypeptide high-throughput synthesis chip according to any one of the technical schemes [1] to [9] is applied to oligonucleotide high-throughput synthesis.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention designs the nucleic acid or polypeptide high-flux synthesis chip which can slow down the flow rate and increase the surface area, and the loading capacity on the unit area can be effectively improved by accurately controlling the structural parameters of the chip. The synthesis chip can meet the requirement of simultaneous synthesis of multiple sequence polypeptides or nucleic acids on the same base material, can effectively prevent solution from overflowing from the upper part of the hole, and can avoid the problem of mutual pollution between different point positions in the synthesis process. The synthesis chip is particularly suitable for high-throughput synthesis of oligonucleotides, and further provides the oligonucleotide high-throughput synthesis chip.
The synthesis chip is suitable for a high-flux DNA synthesizer, the base and the activating agent are sequentially dripped into the holes for the linking reaction, and the in-situ synthesis mode avoids the complex production flow of the traditional detection product. In some embodiments of the present invention, the solution of bases and activators is realized by micro-ink-jet technology, and the solution is dripped into the holes in the form of small droplets, and the residence and effective reaction of the droplets of bases and activators in the patterned holes can be realized by the chip structure design of the present invention, and simultaneously, high solution use efficiency and patterned hole flow channel use rate are obtained.
In addition, the micropore size of the synthesis chip can be defined according to the actual, and compared with the traditional synthesis column mode, the method realizes the synthesis of oligonucleotides with different micro-scale.
The above description does not disclose all embodiments of the present invention and all advantages of the present invention.
Drawings
FIG. 1: the nucleic acid or polypeptide high-flux synthesis chip micropore sectional view of the invention.
FIG. 2 is a schematic diagram: top-view of a patterned well of a nucleic acid or polypeptide high throughput synthesis chip according to a first embodiment of the invention.
FIG. 3: top-view of a patterned well of a nucleic acid or polypeptide high throughput synthesis chip according to the second embodiment of the invention.
FIG. 4 is a schematic view of: the nucleic acid or polypeptide high-throughput synthesis chip of the first or second embodiment of the invention comprises a matrix of equally spaced microwells.
FIG. 5 is a schematic view of: the nucleic acid or polypeptide high-throughput synthesis chip of the first or second embodiment of the invention comprises a staggered array of microwells.
FIG. 6: the nucleic acid or polypeptide high-throughput synthesis chip of the first or second embodiment of the invention comprises a circular array of microwells.
FIG. 7 is a schematic view of: the synthesis procedure of example 1 of the present invention is schematically illustrated.
Description of the reference numerals
100. Noumenon
101. Micro-pores
10. Straight-through hole
20. Fancy hole
30. Micro flow channel
D1 Diameter of the through hole
H1 Depth of through hole
H2 Depth of the fancy hole
Radius of R1 fancy hole outer ring
R2 radius of inner circle of flower-shaped hole
L1 flower type hole micro-channel width
L2 fancy hole micro-channel spacing
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention. All documents described in this specification are incorporated herein by reference.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
In the present specification, a numerical range represented by "a value to B value" or "a value to B value" means a range including the end points of the numerical values a and B.
In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
In the present specification, "high throughput" refers to large scale and/or rapid execution of the synthetic methods of the present specification, and in the present specification refers specifically to the ability to obtain large quantities of nucleic acids or polypeptides at once.
In the present specification, "nucleic acid" includes oligonucleotides and polynucleotides.
Reference in the specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "some specific/preferred aspects," "other specific/preferred aspects," or the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
The term "comprises" and any variations thereof in the description and claims of the invention are intended to cover non-exclusive inclusions. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements but may alternatively include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
< chip for high throughput synthesis of nucleic acid or polypeptide >
The invention provides a nucleic acid or polypeptide high-flux synthesis chip, which comprises a body with a plate-shaped structure, wherein a plurality of micropores are vertically formed downwards on the body, and each micropore comprises a straight-through hole at the upper layer for sample adding liquid drops and a fancy hole at the lower layer, which is coaxial with the straight-through hole and has the same aperture. In the present invention, the fancy hole corresponds to a through hole which is not a through hole penetrating the body in the present invention, and the through hole of the present invention means a hole extending perpendicularly from an opening in the upper surface of the body into the body, and the cavity of the hole is a liquid flow path as a whole, and is hollow and circular as viewed in a plan view of the through hole. The fancy hole is different from the straight hole, a cavity of the fancy hole is provided with a flowing micro-channel, the micro-channel vertically extends to the straight hole from an opening on the lower surface of the body, and the micro-channel has fancy flow channel patterns when being observed from a top view of the fancy hole.
In the invention, the first layer of straight through holes are mainly used for sample introduction, namely, liquid drops smaller than the diameter of the straight through holes are directly dropped into the holes, and the second layer of fancy holes are mainly used for increasing the surface area in the holes, thereby increasing the carrying capacity on the unit area of the chip. After the solution is dripped into the micropores, due to the action of capillary force, the solution passes through the first layer straight-through hole and then gradually enters the microchannels of the second layer fancy holes, through the precise design of chip structure parameters, after the liquid drops fall one by one, the liquid cannot flow out from the bottom end of the second layer, unless the liquid pressure or the air pressure is increased above the first layer, the liquid cannot flow out from the bottom end of the second layer, and thus, the liquid drops of different solutions can be retained in the second layer fancy holes and are mixed to react to complete the synthesis of nucleic acid or polypeptide.
FIG. 1 is a sectional view of a microwell of the nucleic acid or polypeptide high throughput synthesis chip of the present embodiment. The nucleic acid or polypeptide high-throughput synthesis chip comprises a plate-shaped body 100, which can be elliptical, square, rectangular, polygonal or circular in shape. The micro-hole comprises a straight-through hole 10 on the upper layer for sample adding liquid drops and a fancy hole 20 on the lower layer, which is coaxial with the straight-through hole 10 and has the same aperture. The through-holes 10 extend vertically into the body 100 from the upper surface opening of the body 100, the patterned holes 20 have micro channels 30 therein for the liquid to flow through, and the micro channels 30 extend vertically from the lower surface opening of the body 100 to the through-holes 10. The entire flow channels of the patterned holes 20 need to be communicated, and the microchannels 30 need to be distributed in the holes as uniformly as possible, and in some embodiments of the present invention, the widths and the intervals of the microchannels are as uniform as possible, so as to ensure that all the inner walls of the patterned holes 20 can have liquid flowing through the droplets when the droplets pass through the patterned holes 20.
The size of the body can be designed as required. In some embodiments of the invention, the cross-section of the body is rectangular and has dimensions of 50 to 100mm x 15 to 40mm, and further has dimensions of 60 to 85mm x 20 to 35mm. The present inventors have found that when the overall thickness of the chip body is not more than 10cm, no solution flows out from the bottom of the microwell when the density and surface tension coefficient are calculated based on the reagents commonly used in nucleic acid synthesis such as acetonitrile, glutaronitrile, but when it is actually used, the thickness of the body is preferably not more than 1cm, more preferably less than 0.5cm, and further preferably not more than 0.1cm, for the purpose of miniaturization of the chip.
For the base material or base material of the body, a chemically and thermally resistant material is preferred to support chemical or biochemical reactions, such as oligonucleotide synthesis reaction processes. In some cases, the body material comprises a flexible material. Flexible materials include, but are not limited to, modified nylon, unmodified nylon, nitrocellulose, polypropylene, and the like. In some cases, the body material comprises a rigid material. Rigid materials include, but are not limited to, glass, silicon dioxide, silicon nitride, plastics (e.g., polydimethylsiloxane (PDMS), polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, mixtures thereof, and the like), and metals (e.g., gold, platinum, and the like). The body material may be made from a combination of the foregoing materials or any other suitable material known in the art. In some embodiments of the invention, the bulk material may be a silicon material or glass, which is preferred in view of the relative ease with which silicon can be processed to form complex structures.
The present inventors have conducted intensive studies with respect to the design of the through hole. How to prevent the liquid from overflowing from the top of the through hole and how to reduce the risk of contamination of the neighboring hole sites, the design of the relative dimensions of the aperture (i.e., diameter) and depth of the through hole is critical, corresponding to fig. 1, i.e., the relative dimensions of D1 and H1 need to be controlled. In some preferred embodiments of the present invention, the chip of the present invention is suitable for pl-scale inkjet printing devices, and each solution preferably ejects 1 drop of liquid at one dot (i.e. one through hole) from the viewpoint of production efficiency, and since the solution has a certain compatibility with the head of the micro-jet ink generation system, a plurality of drops sometimes occur. The inventor of the invention finds that, if the ratio of the aperture of the through hole (D1 in figure 1) to the depth of the through hole (H1 in figure 1) is A, on the premise that the diameter of the liquid drop is equal to the aperture of the through hole, the number of the liquid drops dropped from a single through hole at a time is n, n is an integer not less than 1 (e.g. n is an integer from 1 to 5), under an ideal limit condition, 2n liquid drops of two solutions respectively pass through in sequence, under an imperfect limit condition, 2n liquid drops of the two solutions pass through simultaneously, and in order to ensure that the liquid just does not overflow and ensure that the occupancy rate of the cavity of the first layer of the through hole is not less than 50%, the inventor finds that the ratio A of the aperture of the through hole to the depth is required to satisfy 1/4n ≤ A ≤ 1/n, the overflow of the liquid from the upper part of the through hole can be effectively prevented, and the pollution risk of the overflow part to the adjacent hole site can be reduced. In some preferred embodiments of the present invention, n is preferably 1, i.e., 0.25. Ltoreq. A.ltoreq.1, in order to improve the production efficiency, further, in order to ensure a high utilization rate (50% or more) of the first-layer through-hole cavity and reduce the possibility of solution overflow when 2 drops of two solutions are dropped continuously, preferably 0.35. Ltoreq. A.ltoreq.0.75, further 0.40. Ltoreq. A.ltoreq.0.70. In other embodiments of the present invention, n is 2, i.e., 0.125. Ltoreq. A.ltoreq.0.50, and further, in order to ensure a high utilization rate (e.g., more than 50%) of the first layer of the through-hole chamber and reduce the possibility of overflow of the solution when 2 drops of two solutions are continuously dropped, preferably 0.20. Ltoreq. A.ltoreq.0.50, and further 0.30. Ltoreq. A.ltoreq.0.50. In some embodiments of the invention, the diameter of the through-hole is equal to or slightly larger than the diameter of the droplet, the diameter of the through-hole is between about 0.001mm and about 1mm, further the diameter of the through-hole is between about 0.01mm and about 0.5mm, and still further about 0.03mm and about 0.1mm.
In order to allow the liquid droplets to pass through the first layer of through holes completely and quickly and reduce the residue of the solution in the through holes, in some preferred embodiments of the present invention, the inner walls of the cavities of the through holes and the upper surfaces of the patterned holes connected to the through holes are preferably subjected to smoothing treatment and hydrophobic treatment. Further, in some more preferred embodiments of the present invention, the contact angle of the nucleic acid or polypeptide synthesis solution on the above surface is made to exceed 150 ° and the sliding angle is made to be less than 20 ° by the smoothing treatment and the hydrophobic treatment.
The reaction between the droplets of the synthesis solution is mainly performed in the fancy wells of the present invention. In order to increase the loading per unit area on the chip, the ratio of the cavity volume of the patterned holes to the surface area of the cavities is denoted as B, preferably 0.4 μm B10 μm, further 1 μm B6 μm, and further 1.11 μm B <3 μm. In some embodiments of the present invention, the volume cross-sectional area to solid cross-sectional area ratio in the top view direction of the tessellated apertures of the present invention is denoted as C, and in order to further increase the capacity per unit area of chips, preferably 0.3 yarn C-yards 0.7 further, 0.35 yarn C-yards 0.60, further, 0.40 yarn C-yards 0.50. In some more preferred embodiments of the invention, the smooth surface of the inner wall of the micro flow channel cavity of the second layer of patterned holes is chemically treated to form a nano-scale rough surface, thereby increasing the loading capacity, considering that the rough surface has a larger effective area than the smooth surface. Since the chip of the present invention is used for synthesis of nucleic acids or polypeptides, further, the inner wall surface is provided with a linker arm having a hydroxyl group, an amino group or a carboxyl group protected with a protecting group or without a protecting group as a starting point for oligonucleotide synthesis extension by chemical treatment.
In some embodiments of the present invention, in order to realize efficient inkjet printing and at least 90% of the second layer of patterned-hole flow channel space is utilized or 90% of the solution is in the second layer of patterned-hole flow channel space, if the ratio of the aperture diameter of the through hole (i.e. D1 in FIG. 1) to the depth of the patterned hole (i.e. H2 in FIG. 1) is denoted as D, the inventors have studied that D needs to satisfy 27C/40n ≦ D ≦ 5C/6n, n as defined before in the present invention. The main area of synthesis is in the micro-channel of the second layer of the fancy holes, in order to make full use of the surface space of the chip, the smaller the size of each point is, the better (namely, the smaller the aperture of the through hole is, the better), the optimal condition is that the aperture size of the through hole is the same as the diameter of the liquid drop when the comprehensive consideration is given, and under the condition that the space in the flow channel of the second layer of the fancy holes is just fully used, when the number of the liquid drops of each liquid drop at the same point is n, the volume of the flow channel of the second layer of the fancy holes is equal to the volume of 2n liquid drops. If at least 90% of the flow channel spaces of the second layer of the pattern holes are utilized or 90% of the solution is in the flow channel spaces of the second layer of the pattern holes, a preferred range of the ratio D of the hole diameter of the through holes to the depth of the pattern holes can be correspondingly deduced.
The arrangement shape of the micro flow channels in the plan view of the pattern holes is not particularly limited in the present invention. The design parameters B, C and D of the fancy holes can be met, the whole flow channels of the fancy holes are communicated, the micro-flow channels are uniformly distributed in the holes as much as possible, and the sizes of the micro-flow channels are consistent as much as possible. In some embodiments of the invention, the width of the microchannels and the spacing between the microchannels are in the range of 0.001mm to 0.01mm, further in the range of 0.003mm to 0.008mm, and even more preferably the width of the microchannels and the spacing between the microchannels are the same. In some embodiments of the invention, the patterned holes have a vortex shape or a bisectional shape in a top view direction of the microchannel, as shown in fig. 2 and 3, respectively. FIG. 2 shows a top view of a patterned well of a nucleic acid or polypeptide high throughput synthesis chip according to a first embodiment of the present invention. In fig. 2, the micro flow channel has a spiral shape in the plan view direction of the patterned holes 20, the radius R1 of the outer circle of the patterned holes 20 is 0.03mm, the radius R2 of the inner circle of the patterned holes 20 is 0.005mm, the width L1 of the patterned holes 30 is 0.005mm, and the pitch L2 of the patterned holes 30 is 0.005mm, and it is found by calculation that the perimeter of the patterned holes is 527.28 μm, and the pattern holes are formed in the micro flow channelThe cross-sectional area of the cavity or the micro-channel in the top view direction is 1290.37 mu m 2 . FIG. 3 shows a top view of a patterned well of a nucleic acid or polypeptide high throughput synthesis chip according to a second embodiment of the invention. In FIG. 3, the micro flow channel has a middle-divided shape in the top view direction of the flower type orifice 20, the radius R1 of the outer circle of the flower type orifice 20 is 0.03mm, the radius R2 of the inner circle of the flower type orifice 20 is 0.025mm, the width L1 of the flower type orifice micro flow channel 30 is 0.005mm, and the distance L2 between the flower type orifice micro flow channels 30 is 0.005mm, and it can be found by calculation that the perimeter of the flower type orifice micro flow channel is 468.94 μm, and the cross-sectional area of the cavity or the micro flow channel in the top view direction is 1148.22 μm 2 。
In order to facilitate the synthesis reaction, the upper surface of the chip body is processed into latticed micropores (namely 101 in figure 1), and the arrangement mode of the micropores, the sizes of the micropores and the intervals of the micropores can be set in a user-defined mode according to actual requirements. FIGS. 4 to 6 show the array of microwells (101) in the nucleic acid or polypeptide high-throughput synthesis chip according to the first or second embodiment of the present invention, which are arranged in an equidistant manner, staggered manner, and arranged in a circular pattern, respectively. In some embodiments of the present invention, the pitch between the micro wells (101) is 10 to 500 μm, and further 12 to 100 μm, and when one chip has a size of 76.2mm × 25.4mm, one chip may have 0.7 to 2000 ten thousand micro wells, and further, 19 to 1950 ten thousand micro wells. In some embodiments of the present invention, the size of the synthesized chip is 76.2mm × 25.4mm, and the surface of the synthesized chip has 483 to 1934 ten thousand micropores.
< preparation method of nucleic acid or polypeptide high throughput Synthesis chip >
The nucleic acid or polypeptide high-flux synthesis chip can be prepared by combining the conventional preparation method in the field with the design parameters, can be prepared by adopting an MEMS micro-nano processing method if the chip body material is a silicon chip, and can be used for preparing micropores in a 3D printing or injection molding mode if the chip body material is a plastic.
In some embodiments of the present invention, the synthesis chip may be prepared by a photolithography micro-nano processing method, and may be prepared by the following steps: 1) Cleaning and drying the silicon wafer, for example, cleaning by a wet method, and dehydrating and baking (for example, heating the silicon wafer for 1 to 2 minutes at a temperature of between 150 and 250 ℃ by a hot plate and protecting the silicon wafer by nitrogen gas); 2) Priming, such as hot plate priming or spin priming using a gas phase to form a base film; 3) Coating photoresist, such as static or dynamic coating, spin coating, and solvent evaporation; 4) Soft baking, for example, heating by a vacuum hot plate at 85-120 ℃ for 30-60 seconds; 5) Removing the accumulation of the photoresist on the front surface and the back surface of the edge of the silicon wafer; 6) Alignment, such as laser auto-alignment or author alignment mark, to ensure alignment between the pattern and the pattern already on the silicon wafer; 7) Exposing; 8) Postbaking, for example, heating with a hot plate at 110-130 ℃ for 1 minute; 9) Developing, namely, the whole box of silicon wafer can be used for immersion developing or continuous spraying/automatic rotation developing; 10 Hard baking, for example, heating with a hot plate at 100-130 deg.C for 1-2 minutes; 11 Internal surface modification of the holes, such as treating the interior of the tessellated holes with chemical treatments to surfaces with connecting arms.
< application of chip for high-throughput synthesis of nucleic acid or polypeptide >
The invention further provides the application of the nucleic acid or polypeptide high-throughput synthesis chip in nucleic acid or polypeptide synthesis, in particular the application of oligonucleotide high-throughput synthesis. In some embodiments of the invention, the chip of the invention is suitable for high-throughput DNA synthesizers, and DNA synthesis can be achieved by using the conventional solid-phase phosphoramidite triester method. Further, by dropping base and activator solutions into the through holes of the chip of the present invention by micro-inkjet technology, the size and number of droplets can be set as desired, and the two solutions mainly complete the synthesis reaction in the patterned holes. Compared with the traditional synthesis column mode, the method realizes the synthesis of the oligonucleotides with different micro-scale levels by adopting the chip of the invention. Due to the special design of the structure, the effective area of unit area is increased, the synthetic loading capacity is greatly improved, the problem of mutual pollution among different point positions in the synthetic process can be effectively avoided, and high solution use efficiency and the use rate of the fancy hole flow channel are obtained.
In some embodiments of the invention, the synthesis chip of the invention may be used for nucleic acid synthesis. The chemical synthesis of DNA consists of a plurality of reaction cycles, wherein a single cycle mainly comprises four steps of deprotection, coupling, capping and oxidation, the end of a protected DNA chain on a synthetic carrier is cyclically deprotected, a new protected nucleotide monomer is connected, and the length of the DNA chain is increased by one. In the synthesis process, redundant raw materials and impurities are remained in the solution, and the product is remained on the chip, so that the purification of each step of reaction is not required, and the method is particularly suitable for the automatic synthesis of long-chain biomacromolecules. In the first cycle: respectively spotting the basic group solution and the activator solution to corresponding hole sites through a pl-level liquid drop distribution system to wait for coupling reaction; washing the porous site with acetonitrile and positive pressure; a capping solution and positive pressure impact are used for realizing capping reaction; washing the porous site with acetonitrile and positive pressure; oxidizing solution and positive pressure flushing are used for realizing oxidation reaction; washing the porous site with acetonitrile and positive pressure; using deprotection solution and positive pressure impact to realize deprotection reaction; the porous sites were rinsed with acetonitrile and positive pressure. Nth cycle (N ≠ 1): using deprotection solution and positive pressure impact to realize deprotection reaction; washing the porous site with acetonitrile and positive pressure; respectively spotting the basic group solution and the activating agent solution to corresponding hole sites of the chip through a pl-grade liquid drop distribution system to wait for coupling reaction; washing the porous site with acetonitrile and positive pressure; realizing a capping reaction by using a capping solution and positive pressure flushing; washing the porous site with acetonitrile and positive pressure; oxidizing solution and positive pressure flushing are used for realizing oxidation reaction; the porous sites were flushed with acetonitrile and positive pressure. The production efficiency of the chip synthesized by the method is far higher than that of the traditional synthesizer.
After the synthesis is finished, two post-treatment modes are provided according to different applications. One is direct use and does not need ammonolysis for harvesting, and the other is ammonolysis, and the length of synthesis is from 15 bases to 350 bases. The device can realize the synthesis of oligonucleotides with different lengths and different sequences on the same plate, really realizes high-flux synthesis, and can help the related application of oligonucleotide pool. Pools of oligonucleotides are widely used as a pool of probes, as hybridization probes are often used in genetic disease screening services to capture fragments of interest for sequencing. To achieve coverage of the entire human exome, it is often necessary to synthesize millions of oligonucleotide probes. It is desirable to construct probe libraries of this size in a high throughput synthetic manner. In addition, it will be easier to customize the probe library specifically for certain regions of interest. Pools of oligonucleotides are also commonly used in the construction of libraries for various functional screens, such as CRISPR gRNA libraries targeted to customized gene sets, gene mutant libraries for functional screening, antibody libraries, and the like. With the popularity of high-throughput synthesis, these approaches will alternatively become "routine approaches" to verify and exploit biological functions. The gene is synthesized at a very low cost, providing the possibility for a DNA data storage function.
The invention is further illustrated, but not limited, by the following examples.
Examples
1. Design parameter study of through hole
A picoliter type microarray inkjet printing biosynthesis platform of RD-MAS200 produced by Shanghai Rui Degree opto-electronic technology Limited is adopted, 1 drop or 2 drops of liquid are sprayed on each solution of each point, the diameter of each drop is set to be 60 mu m, the aperture (D1) of a chip through hole is set to be 60 mu m, the depth (H1) of the chip through hole is shown in table 1, solution overflow under the conditions of ideal limit and non-ideal limit and the utilization rate of a first layer through hole (cavity) are respectively calculated, comprehensive evaluation is given, and specific results are shown in table 1.
TABLE 1 design parameters study of through-holes
TABLE 2 description and calculation formulas for the parameters in TABLE 1
2. Design parameter study of fancy holes
A picoliter-grade microarray inkjet printing biosynthesis platform of RD-MAS200 type produced by Shanghai Rui degree optoelectronic technology Limited is adopted, 1 drop of liquid is sprayed to each solution at each point, namely n =1, the diameter of the liquid drop is set to be 60 μm, the aperture (D1) of a chip straight-through hole is set to be 60 μm, the depth (H1) of the chip straight-through hole is set to be 90 μm, the depth (H2) of a chip pattern hole is shown in Table 3, the width (L1) of a chip pattern hole micro-channel is 5 μm, the utilization rate of pattern holes in a second layer and the utilization rate of the solution are respectively calculated, and comprehensive evaluation is given, and specific results are shown in Table 3.
The chip pattern holes used have a vortex shape or a midsplit shape in the top view direction of the micro flow channel as shown in fig. 2 and 3, respectively. The perimeter of the vortex flow channel shown in figure 2 is 527.28 μm and the cross-sectional area of the cavity or channel in the plan view direction is 1290.37 μm 2 . The perimeter of the middle flow channel micro flow channel shown in FIG. 3 is 468.94 μm, and the cross-sectional area of the cavity or micro flow channel in the top view direction is 1148.22 μm 2 。
TABLE 3 study of design parameters of the fancy holes
TABLE 4 description and calculation formulas for the parameters in TABLE 3
Example 1
The method comprises the following steps of preparing by utilizing a photoetching micro-nano process and adopting a synthesis chip with the following structural parameters: the size of the chip body is 76.2mm multiplied by 25.4mm, the aperture (D1) of the chip through hole is 60 μm, the depth (H1) of the chip through hole is 90 μm, the depth (H2) of the chip pattern hole is 180 μm, the width (L1) and the interval (L2) of the chip pattern hole micro-channel are both 5 μm, and the micro-channel has a vortex shape in the top view direction of the pattern hole. The perimeter of the vortex flow channel micro-channel is 527.28 μm, and the cross-sectional area of the cavity or the micro-channel in the top view direction is 1290.37 μm 2 . It was calculated that A was 0.667, B was 2.45 μm, and C was 0.456,D was 0.33. The dot pitch of the microwells was varied to prepare a plurality of different chips, and the production efficiency was evaluated separately.
The used equipment is RD-MAS200 type pico-liter micro-array ink-jet printing biosynthesis platform produced by Shanghai Rui degree photoelectricity technology Limited. The goal was to synthesize oligonucleotides, set each microwell site to eject 1 drop of liquid per solution, i.e., n =1, with the drop diameter set at 60 μm.
The synthesis steps are as follows: as shown in fig. 7.
The synthesis effect is as follows: the maximum number of synthetic layers is 200, the coupling efficiency of each layer can reach more than 99%, and the production efficiency is shown in the following table 5.
TABLE 5 evaluation of production efficiency
Comparative example 1
The efficiency of synthesis was evaluated by changing the hole number of wells using a conventional synthesizer (dr. Oligo series) and the same synthesis method as in example 1, respectively, see table 6.
Table 6 synthesis efficiency of conventional synthesizer (dr. Oligo series)
As can be seen from the comparison between Table 5 and Table 6, the synthesis chip of the present invention can significantly improve the production efficiency after the conventional synthesis columns are all reduced to one chip.
The above examples are intended only to illustrate several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as imposing any limitation on the scope of the present invention. It should be apparent that those skilled in the art can make various changes and modifications without departing from the spirit of the invention, which fall within the scope of the invention.
Claims (20)
1. A nucleic acid or polypeptide high-throughput synthesis chip,
the synthesis chip comprises a body with a plate-shaped structure, a plurality of micropores are vertically and downwardly formed in the body, the micropores comprise a through hole on the upper layer for sample adding liquid drops and a fancy hole on the lower layer, the fancy hole is coaxial with the through hole and has the same aperture as the through hole, the through hole vertically extends into the body from an opening on the upper surface of the body, a circulating micro channel is arranged in the fancy hole, and the micro channel vertically extends to the through hole from an opening on the lower surface of the body; the ratio of the aperture of the straight-through hole to the depth of the straight-through hole is marked as A, the number of liquid drops dripped from a single straight-through hole in one time is marked as n, then A is more than or equal to 1/4n and less than or equal to 1/n, and n is an integer not less than 1; the pattern holes have a vortex shape in the direction of the top view of the microchannel.
2. The nucleic acid or polypeptide high-throughput synthesis chip according to claim 1,
the ratio of the volume of the cavity of the fancy holes to the surface area of the cavity is marked as B, and B is more than or equal to 0.4 mu m and less than or equal to 10 mu m.
3. The nucleic acid or polypeptide high throughput synthesis chip of claim 1 or 2,
the ratio of the cavity cross-sectional area to the solid cross-sectional area in the top view direction of the fancy opening is marked as C, and 0.3< C <0.7.
4. The nucleic acid or polypeptide high throughput synthesis chip of claim 3,
the ratio of the aperture of the straight-through hole to the depth of the fancy hole is marked as D, and D is more than or equal to 27C/40n and less than or equal to 5C/6n.
5. The chip for high throughput synthesis of nucleic acids or polypeptides according to claim 1 or 2,
the thickness of the body is not more than 10cm.
6. The nucleic acid or polypeptide high throughput synthesis chip of claim 5,
the thickness of the body is less than 1cm.
7. The nucleic acid or polypeptide high throughput synthesis chip of claim 1 or 2,
the inner wall of the micro-channel cavity of the fancy hole is chemically treated.
8. The nucleic acid or polypeptide high throughput synthesis chip of claim 7,
the inner wall of the micro-channel cavity of the fancy hole is chemically treated to enable the surface of the inner wall to be provided with connecting arms, and the connecting arms are provided with hydroxyl, amino or carboxyl which are protected by protecting groups or are not protected by the protecting groups.
9. The nucleic acid or polypeptide high throughput synthesis chip of claim 1 or 2,
the inner wall of the through hole and the upper surface of the fancy hole connected with the through hole are subjected to smoothing treatment and hydrophobic treatment.
10. The chip for high throughput synthesis of nucleic acids or polypeptides according to claim 1 or 2,
the basic material of the body is silicon material or glass.
11. The nucleic acid or polypeptide high-throughput synthesis chip according to claim 10, wherein the base material of the body is silicon material.
12. A chip for high-throughput synthesis of nucleic acids or polypeptides,
the synthesis chip comprises a body with a plate-shaped structure, a plurality of micropores are vertically and downwards formed in the body, the micropores comprise a through hole on the upper layer for sample adding liquid drops and a fancy hole on the lower layer, the fancy hole is coaxial with the through hole and has the same aperture as the through hole, the through hole vertically extends into the body from an opening on the upper surface of the body, a circulating micro channel is arranged in the fancy hole, and the micro channel vertically extends to the through hole from an opening on the lower surface of the body; the ratio of the aperture of the straight-through hole to the depth of the straight-through hole is marked as A, the number of liquid drops dripped from a single straight-through hole in one time is marked as n, then A is more than or equal to 1/4n and less than or equal to 1/n, and n is an integer from 1 to 5; the ratio of the cavity cross-sectional area to the solid cross-sectional area of the fancy opening in the top view direction is marked as C, wherein C is more than 0.3 and less than 0.7; the ratio of the aperture of the straight-through hole to the depth of the fancy hole is marked as D, and D is more than or equal to 27C/40n and less than or equal to 5C/6n; the ratio of the volume of the cavity of the fancy holes to the surface area of the cavity is marked as B, and B is more than or equal to 0.4 mu m and less than or equal to 10 mu m; the pattern holes have a shape of a central section in the direction of a plan view.
13. The nucleic acid or polypeptide high throughput synthesis chip of claim 12,
the thickness of the body is not more than 10cm.
14. The nucleic acid or polypeptide high throughput synthesis chip of claim 12,
the thickness of the body is less than 1cm.
15. The chip for high throughput synthesis of nucleic acids or polypeptides according to claim 12 or 13,
the inner wall of the micro-channel cavity of the fancy hole is chemically treated.
16. The nucleic acid or polypeptide high throughput synthesis chip of claim 15,
the inner wall of the micro-channel cavity of the fancy hole is chemically treated to enable the surface of the inner wall to be provided with connecting arms, and the connecting arms are provided with hydroxyl, amino or carboxyl which are protected by protecting groups or are not protected by the protecting groups.
17. The chip for high throughput synthesis of nucleic acids or polypeptides according to claim 12 or 13,
the inner wall of the through hole and the upper surface of the fancy hole connected with the through hole are subjected to smoothing treatment and hydrophobic treatment.
18. The nucleic acid or polypeptide high-throughput synthesis chip according to claim 12 or 13,
the basic material of the body is silicon material or glass.
19. The nucleic acid or polypeptide high-throughput synthesis chip according to claim 18, wherein the base material of the body is silicon.
20. Use of the nucleic acid or polypeptide high-throughput synthesis chip according to any one of claims 1 to 19 for high-throughput synthesis of oligonucleotides.
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| CN1637147A (en) * | 2003-11-17 | 2005-07-13 | 松下电器产业株式会社 | Nucleic acid amplification reactor and producing method thereof |
| CN102782115A (en) * | 2009-07-20 | 2012-11-14 | 西罗亚生物科技有限公司 | Microfluidic assay platforms |
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