CN102962015B - DNA (Deoxyribose Nucleic Acid) or RNA (Ribose Nucleic Acid) synthesizer with fixed nanometer material microballoons serving as base - Google Patents

Abstract

The invention provides a DNA (Deoxyribose Nucleic Acid) or RNA (Ribose Nucleic Acid) synthesizer with fixed nanometer material microballoons serving as a base. The DNA or RNA synthesizer comprises a liquid supply device and a substrate, wherein a through hole array is formed on the substrate, and the nanometer material microballoons are fixed in each hole; the nanometer material microballoons are subjected to surface chemical modification and then serve as the base materials for synthesizing DNA or RNA; deoxyribonucleotide monomer solution or ribonucleotide monomer solution and a reagent are added into each hole in which the microballoons are fixed by the liquid supply device; and the DNA or the RNA is synthesized on the surfaces of the microballoons. The DNA or RNA synthesizer combines the advantages of a microfluidics synthetic method and a microarray synthetic method, so that the DNA or RNA synthesizer has the characteristics of proper synthetic amount, high throughput, and no cross contamination of the microfluidics synthetic method, also has the characteristics of being simple synthesis technology and high synthesis efficiency of the microarray synthetic method; and the DNA or RNA synthesizer is expected to be applied to the circumstance of synthesizing specificity short chain oligonucleotides, such as the work of synthesizing primers, probes, and long chains by short chains.

Description

With the nano material microballoon of fixing, make DNA or the RNA synthesizer of substrate

Technical field

The present invention relates to the synthetic technology of DNA/RNA, specifically, relate to a kind of DNA or RNA synthesizer of doing substrate with the nano material microballoon of fixing.

Background technology

In recent years, genetic engineering receives much concern because of its application prospect on medical diagnosis on disease, treatment field.In routine techniques means, long-chain DNA is spliced by polymeric enzyme reaction by the short chain oligonucleotides of chemical synthesis.Therefore, the synthetic accuracy of short chain oligonucleotides, cost, efficiency have directly determined quality and the production cost of long-chain DNA.

The minimum output of commercial synthesizer is 10nmol magnitude, and this has exceeded the demand in most laboratory, and the short chain oligonucleotides of synthetic pmol magnitude is devoted in much research, be able to meet primary demand.Microfluidic methods and microarray method are the Main Means of synthetic pmol magnitude short chain oligonucleotides, and research is also carried out based on this mostly.Wherein, Quake group is the representative of research microfluid synthetic method, at " A microfluidic oligonucleotide synthesizer " Vol.38|21February 2010, in Nucleic Acids Research, fully demonstrated the advantage of microfluid synthetic DNA, as moderate in synthetic quantity (being generally nmol magnitude), without cross pollution, can to realize high flux synthetic etc.But this method also exists problems not yet to solve, as complicated operation, structure or raw material is expensive, material solution consumption is superfluous, cause cost to rise and decrease in efficiency etc. because of the introducing of Micropump and micro-valve.Jingdong Tian group is the representative of research microarray synthetic method, at " Accurate multiplex gene synthesis from programmable DNA microchips " Vol.432|23/30 December, in Nature, elaborated the advantage of microarray method synthetic DNA, as high in integral density, high flux, precision is high, combined coefficient is high.But the method also exists some problems, as too little in output, be not even enough to cause PCR reaction; Independent certain specific oligonucleotides difficulty of processing is very large etc.Therefore, develop a kind of synthetic quantity and be pmol magnitude, high accuracy, high efficiency, DNA synthesizer cheaply, to meet laboratory requirement, become problem demanding prompt solution.

Summary of the invention

The object of this invention is to provide a kind of DNA or RNA synthesizer of doing substrate with the nano material microballoon of fixing.

In order to realize the object of the invention, a kind of DNA or RNA synthesizer of doing substrate with the nano material microballoon of fixing of the present invention, it comprises liquid feed device and substrate; Wherein, substrate is provided with via-hole array, nano material microballoon is fixed in each hole, described nano material microballoon after surface chemical modification as DNA or the synthetic base material of RNA, deoxyribonucleotide or ribonucleotide monomer solution and reaction reagent are joined by liquid feed device in each hole that is fixed with microballoon, at microsphere surface, complete DNA or RNA is synthetic.

Described nano material microballoon is silicon dioxide microsphere, or is suitable for the microballoon made at the material of its surperficial synthetic DNA or RNA, such as polystyrene microsphere etc.

Preferably the method by sintering is fixed on nano material microballoon in each hole of via-hole array.

Preferred described substrate is silicon chip.Take silicon chip as substrate, adopt low-pressure chemical vapor phase deposition to form inside and outside two-layer mask layer altogether, wherein, internal layer is earth silicon mask layer, and skin is silicon nitride mask layer, makes substrate.

Described liquid feed device is preferably used ink-jet printer, and in different print cartridges, loads respectively four kinds of deoxyribonucleotides or ribonucleotide monomer solution and reaction reagent, and different solution and reagent are corresponding to corresponding rgb value in printer tinting system.

Contain after chemical modification-NH-CO-group of described nano material microsphere surface, and at outermost end formation-OH group.

Preferred described through hole is for falling wedge shape.

The present invention further provides the method that adopts above-mentioned synthesizer synthetic DNA or RNA: deoxyribonucleotide or ribonucleotide monomer solution and reaction reagent are joined in on-chip via-hole array by liquid feed device, in the through hole of substrate, there is fixing microballoon, at microsphere surface, complete DNA or RNA synthetic, after reaction finishes, rinse or soak substrate, reclaiming synthetic DNA or RNA.

A kind of DNA or RNA synthesizer of doing substrate with the nano material microballoon of fixing of the present invention, combine the advantage of microfluid synthetic method and microarray synthetic method, both had that microfluid synthetic method synthetic quantity is moderate, high flux, without the feature of cross pollution, there is again the feature that microarray synthetic method synthesis technique is simple, combined coefficient is high, be expected to be applied to the occasion of synthetic specificity short chain oligonucleotides, such as primer, probe with formed the synthetic work etc. of long-chain by short chain.Compared with prior art, advantage is:

(1) adopt the nano material microballoon being fixed in hole or cavity to do the substrate of synthetic DNA/RNA, high flux synthesizes different types of DNA/RNA simultaneously on the one hand, and its output is moderate; Guaranteeing that on the basis of reaction chamber integration density, the physical isolation between cavity also can be avoided cross pollution on the other hand.

(2) adopt ink-jet printer that trace reagent required in synthetic reaction is delivered to and in corresponding reaction chamber, carries out synthetic reaction, can avoid because introducing the problem that reagent circulating rate is little, cost is high due to Micropump, micro-valve, and there is the advantages such as flux is high, simple to operate, efficiency is high.

Accompanying drawing explanation

Fig. 1 is the preparation flow schematic diagram of via-hole array on substrate and substrate in preferred embodiment of the present invention.

Fig. 2 carries out the schematic flow sheet of chemical modification to silicon dioxide microsphere surface in preferred embodiment of the present invention.

DNA chemical synthesis schematic diagram in Fig. 3 preferred embodiment of the present invention.

DNA or RNA synthesizer operation principle schematic diagram in Fig. 4 preferred embodiment of the present invention.

The specific embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.

Embodiment makes DNA or the RNA synthesizer of substrate with the nano material microballoon of fixing

1.1 take silicon chip is that substrate adopts the method for film growth, photoetching, dry etching and wet etching to make silicon array formula through hole

Operating procedure is as follows:

1) take that thickness is 400 μ m two to throw silicon chips 1 be substrate, adopt the two-layer mask layer of the two-sided growth of low-pressure chemical vapor phase deposition (internal layer is earth silicon mask layer 2, thickness 100nm, skin is silicon nitride mask layer 3, thickness 100nm), as Figure 1A;

2) the required figure of front photoetching making array through hole, back-protective, wet etching or dry etch step 1) middle mask layer of growing, then take photoresist as mask, adopt reactive ion etching technology etch silicon nitride mask layer, use again hydrofluoric acid solution corrode silicon dioxide mask layer, as Figure 1B;

3) use potassium hydroxide solution corrosion of silicon to silicon chip break-through, as Fig. 1 C;

4) adopt back side reactive ion etching technology to remove silicon nitride mask layer and earth silicon mask layer, form the array through hole of falling wedge shape 4, and chamfering is 53 °.When the bottom opening length of side is 120 μ m, its volume is 78.5nL, as Fig. 1 D, obtains silicon array formula through hole.

1.2 are fixed on silicon dioxide microsphere in the silicon of falling wedge shape through hole

Operating procedure is as follows:

5) front side of silicon wafer bonding one deck dry film 5, and photoetching forms the figure identical with the silicon of falling wedge shape via-hole array, as Fig. 1 E;

6) with pipettor, by the concentration that contains preparing, be that 10% the turbid drop of silicon dioxide microsphere enters the through hole of falling wedge shape that sticks dry film, repeatedly, complete self assembly, make to fill in the through hole of falling wedge shape silicon dioxide microsphere 6, subsequent chemistry synthetic reaction is all carried out at microsphere surface, should the through hole of falling wedge shape be reaction chamber, and as this technique of Fig. 1 F(both can guarantee that the synthetic quantity in through hole was moderate, can avoid again introducing cost rising and reagent circulating rate decline problem due to Micropump, micro-valve);

7) first silicon chip is immersed to acetone, make dry film on it occur curling and peel off, the excessive silicon dioxide microsphere 7 removing between reaction chamber, on dry film, then remove remaining dry film with potassium hydroxide, as Fig. 1 G;

8) by silicon chip in 1050 ℃ of sintering processes 12h, make silicon dioxide microsphere surface melting, realize between microballoon and microballoon and the physical property bonding of falling between wedge side wall, microballoon can be fixed in through hole, be formed with the chip 8 of array reaction chamber, to meet the requirement of rinsing residual agent in reaction chamber in subsequent chemistry compound experiment, as Fig. 1 G.

1.3 carry out DNA/RNA synthetic reaction in reaction chamber/hole

First chemical modification microballoon, makes its formation-NH-CO-group, to cut flexibly the DNA/RNA after synthesizing; Proceed chemical modification, make its outermost end formation-OH group, so that first required nucleotides reacts with it, link on it, as Fig. 2.

The present invention adopts typically " deprotection-coupling-block-oxidation " synthetic method.Concrete chemical synthesis implementation method is: in the different print cartridges of EPSON six look piezoelectric ink jet printers, pack the required reagent of synthetic reaction into, be respectively the weak acid (for example tetrazolium) of four kinds of different deoxyribonucleoside acid solutions, a kind of deprotection and the weak base (for example ammoniacal liquor) that a kind of cleaved products is used.Ink using six kinds of reagent as printer respective color is processed, and six kinds of inks all have unique rgb value.The present invention arranges with six kinds of on all four color lumps of ink rgb value as input in Microsoft PPT, by controlling position, the size of different color blocks, control printhead ejection class of liquids, position and quantity, realization is delivered to trace reagent required in synthetic reaction in the cavity of filling microballoon on chip, complete the chemical synthesis of DNA, as Fig. 3.

Basic functional principle is as Fig. 4.The chip 8 of the via-hole array of falling wedge shape and support 9 thereof etc. all maintain static, and support lower end vavuum pump interface 10 can produce negative pressure, to rinse the residual agent in chemical synthesis process; On chip, have and fill the fixedly via-hole array of falling wedge shape of silicon dioxide microsphere, all chemical syntheses are all carried out in hole; Printer passes through print cartridge and the pipeline 11 of different colours by required different liquids in experiment, be delivered to definite reaction chamber of falling wedge shape, now the direction of motion of printhead 12 is left and right directions, ejection reagent 13 is not single kind, Fig. 4 A is the front view of system works, the side view that Fig. 4 B is system works.Now the direction of motion of printhead is perpendicular to paper inwards or outward direction, and ejection reagent 14 is single kind.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (2)

1. with the nano material microballoon of fixing, make DNA or the RNA synthesizer of substrate, it is characterized in that, comprise liquid feed device and substrate; Wherein, substrate is provided with via-hole array, nano material microballoon is fixed in each hole, described nano material microballoon after surface chemical modification as DNA or the synthetic base material of RNA, deoxyribonucleotide or ribonucleotide monomer solution and reaction reagent are joined by liquid feed device in each hole that is fixed with microballoon, at microsphere surface, complete DNA or RNA is synthetic;

Described nano material microballoon is silicon dioxide microsphere or polystyrene microsphere, and the method by sintering is fixed on nano material microballoon in each hole of via-hole array;

Described substrate is silicon chip, take silicon chip as substrate, adopts low-pressure chemical vapor phase deposition to form inside and outside two-layer mask layer altogether, and wherein, internal layer is earth silicon mask layer, and skin is silicon nitride mask layer, makes substrate;

Described liquid feed device is ink-jet printer, and in different print cartridges, loads respectively four kinds of deoxyribonucleotides or ribonucleotide monomer solution and reaction reagent, and the solution in different print cartridges and reagent are corresponding to corresponding rgb value in printer tinting system;

Contain after chemical modification-NH-CO-group of described nano material microsphere surface, and at outermost end formation-OH group.

2. adopt the method for synthesizer synthetic DNA described in claim 1 or RNA, it is characterized in that, deoxyribonucleotide or ribonucleotide monomer solution and reaction reagent are joined in on-chip via-hole array by liquid feed device, at microsphere surface, complete DNA or RNA synthetic, after reaction finishes, rinse or soak substrate, reclaiming synthetic DNA or RNA.

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