CN205803638U - A kind of high-flux sequence build storehouse instrument - Google Patents

A kind of high-flux sequence build storehouse instrument Download PDF

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CN205803638U
CN205803638U CN201620383191.9U CN201620383191U CN205803638U CN 205803638 U CN205803638 U CN 205803638U CN 201620383191 U CN201620383191 U CN 201620383191U CN 205803638 U CN205803638 U CN 205803638U
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reagent
oil
sample
reaction
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李星军
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Abstract

This utility model provides a kind of biochemical micro-reaction system, including the first oil-based liquid, the second oil-based liquid, water base gene samples and reaction reagent, wherein, the density of described first oil-based liquid is more than described second oil-based liquid, described water base gene samples and reaction reagent are embedded between described first oil-based liquid, the second oil-based liquid composition sandwich structure, described water base gene samples and reaction reagent are with described first oil-based liquid, the second oil-based liquid is the most miscible and does not reacts, and described first oil-based liquid, the second oil-based liquid are immiscible.The micro-reaction system of described biochemistry can the control completely of each independent sample and separation in biochemical reaction process, it is to avoid cross-contamination, also can save a large amount of consumptive material.What this utility model additionally provided high-flux sequence based on this biochemical micro-reaction system builds storehouse instrument.

Description

A kind of high-flux sequence build storehouse instrument
Technical field
This utility model belongs to biology field, particularly to the micro-reaction system of a kind of biochemistry and high-flux sequence Build storehouse instrument.
Background technology
On the market of current DNA sequencer, main flow is second filial generation high-flux sequence platform, mainly includes Illumina2000/2500/4000,454 sequenators of Roche Holding Ag, the SOLID sequenator of ABI company, accurate at cdna sample Before standby upper machine checks order, it is required for preparing sequencing library.Build storehouse (prepared by library) process to relate to cdna sample addition, add The process procedures such as buffer and enzyme reagent mixed liquor, PCR (polymerase chain reaction), magnetic beads for purifying, flow process is complicated and is used Liquid system is huge, occurs sample mixing and sample contamination risk high.
For solving above-mentioned difficulties, scheme is prepared in existing full-automatic library the following two kinds: one is based on traditional library Preparation method, reagent and sample system all keep constant, use liquor removing workstation by preparation process automatization, specifically with PerkinElmer Janus NGS Express is representative, and shortcoming is that single operation can only realize flux peak 24 parts, and expends A large amount of disposable plastic consumptive materials and reagent.Two is to be encapsulated into sealed reagent box based on library is prepared reagent in advance, uses and limits Property restriction endonuclease DNA molecular is sheared, digital micro-fluid control the big droplet of nanoliter level, DNA sample is loaded into automatically one Secondary property library card (Library card), typical technology is that Illumina NeoPrep is desk-top builds storehouse instrument.This technology from Dynamicization degree is high, streamlining, but sample flux prepared by library little (16 parts), and it is only applicable to Illumina order-checking platform.
In existing banking process, in order to ensure the target sample of rather high concentration, common sample volume scope is 200 μ L, reagent volume is more than 200 μ L, and relatively large volume of reaction volume can cause high reagent cost, also needs to make consuming in a large number simultaneously Medical disposable material (as rifle is first-class).
Utility model content
In view of this, this utility model first aspect provides a kind of novel micro-reaction system of water in oil biochemistry, and base Build storehouse instrument in what this biochemical micro-reaction system provided a kind of high-flux sequence, the volume of reaction system can be reduced, reduce sample Product consumption, it is ensured that the control completely of each independent sample and separation in biochemical reaction process, it is to avoid cross-contamination, also can save big Amount consumptive material.
First aspect, this utility model provides a kind of biochemical micro-reaction system, for prepare order-checking nucleic acid library and Nucleic acid amplification, the micro-reaction system of described biochemistry includes the first oil-based liquid, the second oil-based liquid, water base gene samples and reaction examination Agent, wherein, the density of described first oil-based liquid is more than described second oil-based liquid, described water base gene samples and reaction reagent It is embedded between described first oil-based liquid, the second oil-based liquid composition sandwich structure, described water base gene samples and reaction Reagent with described first oil-based liquid, the second oil-based liquid is the most miscible and does not reacts, described first oil-based liquid, Two oil-based liquids are immiscible.
Preferably, described gene samples is DNA or the aqueous solution of RNA sample of fragmentation；Described reaction reagent is base Because of the nucleic acid amplification of sample, order-checking nucleic acid library preparation in conventional reagent, described reaction reagent include enzyme, buffer molten One or more in liquid, bronsted lowry acids and bases bronsted lowry.
Preferably, the density of described first oil-based liquid is more than pure water density, and its density is 1.01-2.0g/cm3.Enter one Step is preferably 1.01-1.2g/cm3、1.6-1.95g/cm3。
Preferably, the density of described second oil-based liquid is less than pure water density, and its density is 0.09-0.99g/cm3。
The micro-reaction system of biochemistry that this utility model first aspect provides, uses two kinds of immiscible oiliness that density is different Liquid embeds gene samples and the reaction reagent of trace, the first oil-based liquid under, the second oil-based liquid, can be by not upper Reagent and gene samples with proportion are fully embedded between two kinds of oil-based liquids, form double water in oil " sandwich " structure, make Gene samples and being responded of reaction reagent complete in the micro-reaction system of the biochemistry of " Water-In-Oil " at this, to reduce reaction The purpose of the volume of system.
Second aspect, what this utility model provided a kind of high-flux sequence builds storehouse instrument, described high-flux sequence build storehouse Instrument, including board and the drive mechanism that is arranged on board, described board is additionally provided with controller, constant flow pump, purification plate, examination Agent district, reaction orifice plate；Described reagent area includes multiple reagent trough, the plurality of reagent trough in order to hold the first oil-based liquid, Two oil-based liquids, water base gene samples, magnetic beads for purifying liquid, eluent and multiple reaction reagent；Described reaction orifice plate includes array The reacting hole position of distribution；
Connecting in described drive mechanism and have micro sample-adding device and purification plate, described controller controls described drive mechanism band Dynamic described purification plate and described micro sample-adding device move in board, and control the absorption of described micro sample-adding device, discharge examination Agent；
Described purification plate includes that purification plate body, array are arranged on the pipe insert hole on purification plate body and multiple capillary Pipeline, described capillary channel through described pipe insert hole and is respectively provided with first above and below described purification plate body Elongated end and the second elongated end, at described first elongated end, described purification plate body is also placed with multiple magnet, shape The magnetic force district of described capillary channel, described first elongated end is become to be connected with described constant flow pump pipeline；Described second elongated end is used for Insert in reagent trough and described reacting hole position under the drive of described drive mechanism；
Described controller controls described constant flow pump and draws the first oil-based liquid, the second oil-based liquid from reagent area successively and note Enter the reacting hole position to reaction orifice plate, and control the described micro sample-adding device water base gene samples of absorption and reaction reagent and note Entering to described reacting hole position, form biochemical micro-reaction system, the micro-reaction system of described biochemistry is water base gene samples and reaction examination Agent is embedded between described first oil-based liquid, the second oil-based liquid the sandwich structure constituted, wherein, described first oiliness liquid The density of body is more than described second oil-based liquid, described water base gene samples and reaction reagent and described first oil-based liquid, the Two oil-based liquids are the most miscible and do not react, and described first oil-based liquid, the second oil-based liquid are immiscible.
Preferably, after the micro-reaction system of described biochemistry is reacted, under the control of described controller, described trace adds Sampling device is drawn described magnetic beads for purifying liquid and is injected into described reacting hole position, and described purification plate is under the drive of described drive mechanism The mobile top to described reaction orifice plate is so that the second elongated end of described capillary channel inserts in described reacting hole position；Described Under the control of controller, the liquid in reacting hole position is drawn by described constant flow pump and is carried out cdna sample enrichment to described magnetic force district, And after described enrichment, described constant flow pump sucks eluent to described magnetic force district and carries out eluting, and discharges gene after purification Sample.
For nucleic acid amplification, described reaction is pcr amplification reaction, and the cdna sample after eluting is pcr amplification product.
Preparing for nucleic acid library, described reaction includes end reparation reaction, joint coupled reaction successively, tag sequence Reaction, pcr amplification reaction, but it is not limited to this.Can come successively according to reaction prepared by feasible library arbitrary in prior art Carry out.According to the reaction carried out needed for often walking, add selective response reagent accordingly to prepare biochemical micro-reaction system.Often enter Row single step reaction, is required to carry out a purification to collect cdna sample after purification.Gene sample after back reaction purification This, as one of the raw material of next step micro-reaction system of biochemistry reacted.
This utility model second aspect provide build in the instrument of storehouse, based on the micro-reaction system of water in oil biochemistry with fit mutually The purification plate joined is purified, and gene samples and reagent etc. are embedded in oil, and contact with described capillary channel is all oil, hair Thin pipe can reuse, it is not necessary to uses multiple 96 hole PCR plate, the first-class medical disposable material of liquid-transfering gun, without on getting rid of The troublesome operation such as eluent are added after clear liquid.
Preferably, described purification plate body is configured to three layers, in described purification plate, on it be arranged in parallel with interval Plate, middle plate and lower plate, described pipe insert hole is arranged in the described upper plate overlapped, middle plate, lower plate, described capillary channel Through the pipe insert hole in upper plate, middle plate, lower plate and outside extending respectively to the upper plate of described purification plate body, lower plate, institute Stating the first elongated end and extend described upper plate, described second elongated end extends described lower plate, described upper plate is also placed with many Individual magnet.
Preferably, described purification plate also includes the enclosure for being surrounded the side of purification plate, described enclosure Height less than the height of purification plate body, described enclosure is connected with described driving structure.
Preferably, described magnet is permanent magnet or electric magnet, and being connected between described magnet with described controller has control line Road, after sucking eluent extremely described magnetic force district, the magnetic force of described magnet can control magnetic force by power-off and disappear or by permanent magnetism Outside the scope of magnet removal capillary channel, to carry out eluting cdna sample.
Preferably, described controller is programmable logic controller (PLC) (PLC), and described controller is also equipped with high-resolution and touches Display screen.
Preferably, described reagent trough includes room temperature reagent trough, refrigeration reagent trough, and the bottom of described refrigeration reagent trough is provided with Peltier cooling piece, to maintain enzyme reagent to be in active temperature.Storage condition according to required biological reagent selects phase The reagent trough answered.
Preferably, described reaction orifice plate being additionally provided with temperature controller, described temperature controller is for controlling the temperature of reaction system Temperature needed for reaction.Described temperature controller includes temperature sensor and to the heat riser of described reaction orifice plate heating with to institute State the heat sink of reaction orifice plate refrigeration.
Preferably, described drive mechanism include the first workbench arranged in the first direction, arrange in a second direction Two workbench, the 3rd workbench and the gripper components arranged along third direction, wherein, described first direction is horizontal direction, institute State second direction and be perpendicular to described first direction, described third direction and described first, second direction, described second workbench edge First direction is slidably connected with described first workbench, and described 3rd workbench slides with described second workbench in a second direction Connecting, described gripper components is slidably connected along third direction with described second workbench, and described gripper components connects trace Sample adding device and purification plate；
It is provided with the first servomotor in described first workbench, in described second workbench, is provided with the second servomotor, institute Be provided with the 3rd servomotor in stating the 3rd workbench, described controller respectively with described first servomotor, the second servomotor With the 3rd servomotor electrical connection；Described first servomotor drives described second workbench to move in the first direction by screw rod Dynamic, described second servomotor drives described 3rd workbench to move in a second direction by screw rod, described 3rd servomotor Described gripper components is driven to move along third direction by screw rod.
Preferably, described gripper components is mechanical hand.
Preferably, described first workbench includes the first guide rail being fixed on described first workbench and is slidably connected at The first slide block on described first guide rail；Described second workbench includes the second guide rail and is slidably connected on described second guide rail The second slide block, described second guide rail is fixed on described first slide block；Described 3rd workbench includes the 3rd guide rail and slip Being connected to the 3rd slide block on described 3rd guide rail, described 3rd guide rail is fixed on described second slide block, described gripper components It is fixed on described 3rd slide block.
In present embodiment, described second workbench along described first guide rail by described first slide block and described first work Station is slidably connected, and described 3rd workbench is slided with described second workbench along described second guide rail by described second slide block Connecting, described gripper components is slidably connected with described 3rd workbench along described 3rd guide rail by described 3rd slide block.Described First servomotor drives described second workbench to do side-to-side movement in the first direction by screw rod, and described second servomotor leads to Crossing screw rod drives described 3rd workbench to seesaw in a second direction, and described 3rd servomotor drives described by screw rod Gripper components moves up and down along third direction, and described 4th servomotor drives described trace under the control of described controller Sample adding device is drawn, is discharged reagent.
Preferably, described micro sample-adding device includes the first microsyringe and the 4th servomotor, described first trace Piston screw in syringe connects the outfan of described 4th servomotor, and the input of described 4th servomotor is with described Controller electrically connects, and described 4th servomotor drives described first micro-under the control of described controller by piston screw Amount syringe is drawn, is discharged reagent.
Preferably, when described first microsyringe discharges reagent to described reacting hole position, the injection of described syringe Head inserts the second oil-based liquid position (i.e. light oil position) of the micro-reaction system of described biochemistry, is then then exhausted from reagent.This Sample can promote to be easy to form double Water-In-Oil structure.
It is further preferred that the inner surface of described first microsyringe scribbles Teflon coating.Can ensure that syringe Interior no liquid remains.
In another embodiment of the present utility model, described micro sample-adding device is micro liquid jetting system, institute State micro liquid jetting system and include air supply source, digital voltage regulator, cleanout fluid reservoir bottle, micro-electromagnetic valve, mini sprinkler, atmospheric pressure mould Block, ramjet duct, micro-nozzle road, air pressure conveying branch pipe(tube), air pressure conveying main pipeline, cleanout fluid intake line, wherein, described gas Modular pressure includes the screw mandrel moving cell that motor, the second syringe are connected, described second injection with described motor Piston rod in device connects described screw mandrel moving cell, and the outlet of described second syringe connects described air pressure conveying supervisor Road；Described digital voltage regulator pipeline is connected between described cleanout fluid reservoir bottle and described air supply source, and described digital voltage regulator is used In regulating described air supply source with ramjet duct described in constant pressure feed；Described controller is respectively connecting to by controlling circuit Described motor and micro-electromagnetic valve；
It is connected between described mini sprinkler with described atmospheric pressure module and has micro-nozzle road, air pressure conveying branch pipe(tube) and air pressure conveying Main pipeline；Being connected between described mini sprinkler with described rinsing bottle and have micro-nozzle road and ramjet duct, described micro-electromagnetic valve is arranged on For controlling the spray sample amount of described mini sprinkler on described ramjet duct, the one end in described micro-nozzle road connects described mini sprinkler；Institute Stating to be connected between atmospheric pressure module with described rinsing bottle has air pressure to carry main pipeline, cleanout fluid intake line；Wherein, described micro-spray Pipeline, ramjet duct, described air pressure carry and are provided with the first three-way valve between the port that branch pipe(tube) closes on mutually, described micro-nozzle road, It is provided with the second three-way valve between the port that air pressure conveying branch pipe(tube) and air pressure conveying main pipeline close on mutually；
When suck reagent time, regulate described first three-way valve, the second three-way valve make described micro-nozzle road, air pressure carry arm Road connects with air pressure conveying main pipeline, and the drop-down described piston rod of described motor sucks reagent to described micro-nozzle road；Work as spray When going out the reagent sucked, regulating described first three-way valve and make described micro-nozzle road connect with described ramjet duct, described numeral is adjusted Depressor supplies described ramjet duct pressure, and described micro-electromagnetic valve is connected, and controls described mini sprinkler spray sample；When detergent line, Regulating described second three-way valve makes described cleanout fluid intake line connect with described air pressure conveying main pipeline, described motor Drop-down described piston rod makes cleanout fluid fill described air pressure conveying main pipeline；Then described first three-way valve, the second threeway are regulated Valve makes described micro-nozzle road, air pressure conveying branch pipe(tube) again connect with air pressure conveying main pipeline, above pushes away described piston rod and makes cleaning After waste liquid discharge through described mini sprinkler.
Preferably, when sucking reagent, described micro-nozzle road, air pressure conveying branch pipe(tube) connects with air pressure conveying main pipeline After, use motor drop-down described piston rod elder generation suction part air to described micro-nozzle road.Avoid when discharging reagent, institute State the aperture of micro-electromagnetic valve bigger time, cleanout fluid is entered described micro-nozzle road by strong pressurizing and reagent to be discharged mixes. First suction part air, after discharging reagent, then can discharge air, then the trace cleanout fluid brought into is expelled to waste liquid Groove.
In this utility model, when described micro-electromagnetic valve is connected, described micro liquid jetting system can realize unsettled spray Go out the reagent of trace, it is to avoid mini sprinkler contacts oil droplet in inserting described reacting hole position, prevents cross-contamination, clean simple.Additionally, The pipeline of all contact reagent also can be cleaned by described micro liquid jetting system automatically.
In this utility model, being provided with two kinds of sample loading alternative of trace reagent, one is the first microsyringe and the 4th The small throughput sample-adding that servomotor matches, the second is the micro liquid jetting system of high flux sample-adding.First kind of way is fitted Together in the pending less situation of cdna sample, micro liquid jetting system is applicable to the situation that sample size is more.It is applicable to Maximum sample flux is 96 parts.First microsyringe and the 4th servomotor are connected to described driving removably and tie In the gripper components (the 3rd work platforms) of structure.Described micro liquid jetting system drives described in being connected to the most removably In the gripper components of dynamic structure.
This utility model second aspect builds storehouse instrument described in providing, based on micro sample-adding device, constant flow pump and can be at XYZ The drive mechanism of axle motion prepares double micro-reaction system of water in oil biochemistry accurately, can be substantially reduced reaction system Volume, reduces sample and the usage amount of reagent.And by the suitable purification plate of reaction system micro-with described biochemistry to reaction after System be purified, purify processed after gene samples.Build storehouse instrument described in can passing through and complete simple nucleic acid amplification And prepared by the nucleic acid library being responsible for, save medical disposable material, simple to operate.
Advantage of the present utility model will partly illustrate, and a part is aobvious and easy according to description See, or can be known by the enforcement of this utility model embodiment.
Accompanying drawing explanation
The structural representation building storehouse instrument of the high-flux sequence that Fig. 1 provides for this utility model embodiment；
Fig. 2 is the enlarged drawing of purification plate 4 in Fig. 1；
Fig. 3 (A) is the structural representation of micro sample-adding device in this utility model one preferred embodiment, and wherein, (B) is (A) enlarged drawing of the first microsyringe 30 of micro sample-adding device 3 in；
Fig. 4 is the structural representation of micro sample-adding device 3B in another preferred embodiment of this utility model, and wherein, (A) is When inhaling reagent；(B) reagent sucked for ejection；(C) when being that cleanout fluid is drawn in detergent line.
Detailed description of the invention
The following stated is preferred implementation of the present utility model, it is noted that for the ordinary skill of the art For personnel, on the premise of without departing from this utility model principle, it is also possible to make some improvements and modifications, these improve and profit Decorations are also considered as protection domain of the present utility model.
Below in conjunction with the accompanying drawing in this utility model embodiment, to the technical scheme in this utility model embodiment It is clearly and completely described.
First aspect, this utility model provides a kind of biochemical micro-reaction system, for prepare order-checking nucleic acid library and Nucleic acid amplification, the micro-reaction system of described biochemistry includes the first oil-based liquid, the second oil-based liquid, water base gene samples and reaction examination Agent, wherein, the density of described first oil-based liquid is more than described second oil-based liquid, described water base gene samples and reaction reagent It is embedded between described first oil-based liquid, the second oil-based liquid composition sandwich structure, described water base gene samples and reaction Reagent with described first oil-based liquid, the second oil-based liquid is the most miscible and does not reacts, described first oil-based liquid, Two oil-based liquids are immiscible.
Preferably, described water base gene samples be DNA or RNA sample through fragmentation process obtain, i.e. DNA fragmentation or RNA fragment.
(as prepared by rapid DNA sample high flux library) in another embodiment of the present utility model, described water Base gene samples is DNA or RNA sample, and fragmentation processes and can also complete in double micro-reaction systems of water in oil biochemistry.
Described reaction reagent is the nucleic acid amplification (including PCR, dPCR, qPCR, TMA, bDNA, LCR etc.) of gene samples, surveys The nucleic acid library of sequence prepare (as end reparation, add joint, the sequence that tags, hybrid capture, PCR amplification) in commonly used various Reagent, includes but not limited to various enzyme, buffer solution, acid, alkali, saline solution.Can expand according to the required nucleic acid carried out Increase or respectively walking in nucleic acid library preparation reacts corresponding selective response reagent.
The density of water base gene samples and reaction reagent generally between described first oil-based liquid, the second oil-based liquid close Between degree.The density of the most water base gene samples and reaction reagent is usually 0.9-1.2g/cm3。
Preferably, the density of described first oil-based liquid is more than pure water density, and its density is 1.01-2.0g/cm3.Enter one Step is preferably 1.01-1.2g/cm3、1.6-1.95g/cm3。
Preferably, the density of described second oil-based liquid is less than pure water density, and its density is 0.09-0.99g/cm3。
Preferably, described first oil-based liquid, described second oil-based liquid are nonpolar or low pole silicone oil material.
Preferably, described first oil-based liquid includes FC40 electronics fluorination liquid (fluorocarbon oil) or perfluorinate amine oil.
Preferably, described second oil-based liquid includes benzyl polysiloxanes (methyl-silicone oil) or methyl-silicone oil and poly-mountain The mixture of pear ester additive, wherein, described Polysorbate additive is preferably the one in sorbester p17, sorbester p38 and polysorbas20 Or multiple, the volume of described Polysorbate additive is the 0.01%-8% of described methyl-silicone oil volume, to reach nonpolar flat Weighing apparatus (hydrophile-lipophile balance value (HLB value) of additive is 4-7, preferably 5.
Preferably, described first oil-based liquid is 1:(2-4 with the volume ratio of described second oil-based liquid).
It is further preferred that the volume of described first oil-based liquid is 15 μ L.
It is further preferred that the volume of described second oil-based liquid is 40 μ L.
Preferably, described first oil-based liquid, described second oil-based liquid consumption with the volume of DNA sample and equal proportion Increase.
The micro-reaction system of biochemistry that this utility model first aspect provides, uses two kinds of immiscible oiliness that density is different Liquid embeds gene samples and the reaction reagent of trace, the first oil-based liquid under, the second oil-based liquid, can be by not upper Reagent and gene samples with proportion are fully embedded between two kinds of oil-based liquids, form double water in oil " sandwich " structure, make Gene samples and being responded of reaction reagent complete in the micro-reaction system of the biochemistry of " Water-In-Oil " at this, reduce reaction system Volume.Such as, by 15 μ L, DNA sample consumption initial in tradition banking process can be reduced≤1.5 μ L, reaction reagent is by ＞ 200 μ L are kept to be reduced to≤20 μ L (usually 5 μ L).
Seeing Fig. 1-Fig. 3, the structure building storehouse instrument for a kind of high-flux sequence of this utility model second aspect offer is shown It is intended to.The storehouse instrument of building of described high-flux sequence includes board 100 and the drive mechanism 1 being arranged on board 100, described board Controller (not shown), constant flow pump 2, micro sample-adding device 3, purification plate 4, reagent area 5, reaction orifice plate 6 it is additionally provided with on 100；Institute Stating reagent area 5 and include multiple reagent trough 51, the plurality of reagent trough 51 is in order to hold the first oil-based liquid, the second oiliness liquid respectively Body, water base gene samples, magnetic beads for purifying liquid, eluent and multiple reaction reagent；Described reaction orifice plate 6 includes the anti-of array distribution Ying Kongwei 61, described reacting hole position 61 provides foundation and the reacting environment of biochemical micro-reaction system；
Connecting in described drive mechanism 11 and have micro sample-adding device 33 and purification plate 44, described controller controls described driving Mechanism 1 drives described purification plate 4 and described micro sample-adding device 3 to move in board 100, and controls described micro sample-adding device 3 absorptions, discharge reagent；
Described purification plate 4 includes pipe insert hole 42 and that purification plate body 41, array are arranged on purification plate body 41 Multiple capillary channels 43, described capillary channel 43 through described pipe insert hole 42 and above described purification plate body 41 and Lower section is respectively provided with the first elongated end 431 and the second elongated end 432 (not shown), at close described first elongated end 432, Also being placed with multiple magnet 44 on described purification plate body 41, form the magnetic force district of described capillary channel 43, described first extends End 431 is connected with described constant flow pump 2 pipeline；Described second elongated end 432 is for inserting examination under the drive of described drive mechanism 1 In agent groove 51 and described reacting hole position 61；
Described controller controls described constant flow pump 2 and draws the first oil-based liquid, the second oil-based liquid successively also from reagent area 5 Be injected into reaction orifice plate 6 reacting hole position 61, and control described micro sample-adding device 3 draw water base gene samples and reaction examination Agent is also injected into described reacting hole position 61, forms biochemical micro-reaction system, and the micro-reaction system of described biochemistry is water base gene samples With reaction reagent is embedded between described first oil-based liquid, the second oil-based liquid the sandwich structure constituted, wherein, described the The density of one oil-based liquid is more than described second oil-based liquid, described water base gene samples and reaction reagent and described first oil Property liquid, the second oil-based liquid is the most miscible and does not reacts, and described first oil-based liquid, the second oil-based liquid are the most mutual Molten.
Further, after the micro-reaction system of described biochemistry is reacted, under the control of described controller, described trace Sample adding device 3 is drawn described magnetic beads for purifying liquid and is injected into described reacting hole position 61, and described purification plate 4 is in described drive mechanism 1 Drive the lower mobile top to described reaction orifice plate 6 so that the second elongated end of described capillary channel inserts described reacting hole position In 61；Under the control of described controller, the liquid in reacting hole position 61 is drawn by described constant flow pump 2 and is carried out to described magnetic force district Cdna sample is enriched with, and after described enrichment, described constant flow pump 2 sucks eluent to described magnetic force district and carries out eluting, side by side Go out cdna sample after purification.
Preferably, after sucking eluent, remove described magnet 44 and make described magnetic force district disappear, in order to DNA is from being inhaled Elute on attached magnetic bead；Carry out described for recovery magnetic force district the most again being enriched with magnetic bead composition, discharge gene sample after purification This.
Wherein, described reaction reagent is reagent conventional in the nucleic acid library preparation of the nucleic acid amplification of gene samples, order-checking, Described reaction reagent includes one or more in enzyme, buffer solution, acid, alkali and saline solution.
For nucleic acid amplification, described reaction is pcr amplification reaction, and the cdna sample after eluting is pcr amplification product.
Preparing for nucleic acid library, described reaction includes end reparation reaction, joint coupled reaction successively, tag sequence Row, pcr amplification reaction, but it is not limited to this.Can enter successively according to reaction prepared by feasible library arbitrary in prior art OK.
According to the reaction carried out needed for often walking, corresponding reaction reagent is selected to prepare biochemistry from reagent area 5 interpolation micro-instead Answer system.Often carry out single step reaction, be required to use purification plate to carry out a purification to collect cdna sample after purification.Previous Cdna sample after step reaction purification, as one of the raw material of next step micro-reaction system of biochemistry reacted.Such as, end is carried out After repairing reaction, the purified cdna sample obtaining end reparation；Then cdna sample end repaired and joint enzyme, Jian Ku Required sequence label, buffer, acid, alkali, salt etc. are prepared as the micro-reaction system of another biochemistry and carry out adding joint reaction, purified Obtain the cdna sample of tape label sequence；Afterwards the cdna sample of tape label sequence is carried out PCR amplification, through expansion after purification Volume increase thing constitutes described high-throughput sequencing library.Wherein, described sequence label by the basic base random combine of ATCG tetra-kinds, and Described sequence label is different, to make a distinction different DNA samples.
Preferably, described controller is programmable logic controller (PLC) (PLC), and described controller is also equipped with high-resolution touch Display screen.In this utility model, described controller and " electrical connection " of miscellaneous part, refer to that miscellaneous part is by controlling circuit It is connected with controller.
In this utility model, the liquid that described constant flow pump 2 is applied not only to draw in the reacting hole position 61 after adding magnetic bead mixes Compound, is additionally operable to draw the first oil-based liquid, the second oil-based liquid, eluent.Similarly, constant flow pump 2 is being used to draw first During the liquid such as oil-based liquid, the second oil-based liquid, eluent, described purification plate 4 needs to be moved to holding by described drive mechanism 1 Corresponding reagent trough 51 position of above material, makes the second elongated end 432 of described capillary channel 43 insert corresponding reagent groove In 51.Wherein, the first elongated end of multiple capillary channels is connected with described constant flow pump 2 pipeline, can be to draw from described constant flow pump 2 Going out a house steward, the first elongated end 431 of house steward and each capillary channel is connected with the lid of pipe hole by one, it is also possible to It is that the first elongated end 431 of multiple capillary channel is directly connected on the pump head of constant flow pump 2.
In this utility model, described micro sample-adding device 3 draws water base gene samples and reaction reagent, magnetic bead.Often discharge A kind of material, is required to be carried out described micro sample-adding device 3, in order to carry out the absorption of lower a kind of material, it is to avoid exempt from Fork pollutes.Preferably, described reagent area 5 also includes waste liquid tank 52, cleanout fluid storage tank (not shown) etc..Fig. 1 also may be used at 53 Using as reagent trough, it is mainly used in depositing the different genes sample needing to add each reacting hole position and sequence label temporarily.
Preferably, described reagent trough 51 includes room temperature reagent trough, refrigeration reagent trough, and the bottom of described refrigeration reagent trough is arranged There is Peltier cooling piece, to maintain enzyme reagent to be in active temperature.Storage condition according to required biological reagent selects Corresponding reagent trough.
Preferably, described reaction orifice plate 6 being additionally provided with temperature controller, described temperature controller is for controlling the temperature of reaction system Temperature needed for reaction.Described temperature controller include temperature sensor and give described reaction orifice plate 6 heating heat riser and to The heat sink of described reaction orifice plate 6 refrigeration.Described temperature controller electrically connects with described controller, preferably by serial communication mode Realize.It is understood that can include multiple reaction orifice plate on described board 100, each reaction orifice plate can be identical, also Can be different.
In this utility model embodiment, described reaction orifice plate 6 is the temperature control reaction cavity in PCR instrument.Described reaction orifice plate 6 Including 96 reacting hole positions 61.
Preferably, described eluent is placed in 96 orifice plates consistent with the reacting hole position 61 of described reaction orifice plate 6, example As being placed in Fig. 1 at the 53 of reagent area 5.
Preferably, in prepared by nucleic acid library, sequence label used is also disposed in and the reacting hole of described reaction orifice plate 6 In 61 another consistent 96 orifice plates of position (the 53 of reagent area 5).Described sequence label is for carrying out label to testing gene sample Distinguish.Hold district and the described reaction orifice plate 6 of described eluent and described sequence label are spaced setting in described board 100.
It is further preferred that described purification plate body 41 is configured at least two-layer, in order to described capillary channel 43 is more preferable Be fixed in described purification plate 4.Such as, purification plate body 41 is configured to three layers, and its interval Horizon row arranges upper plate, middle plate And lower plate, described pipe insert hole 42 is arranged in the described upper plate overlapped, middle plate and lower plate；Described capillary channel 43 is worn Cross the pipe insert hole 42 in upper plate, middle plate and lower plate and described purification plate body 41 upper plate, lower plate be respectively provided with One elongated end 431 and the second elongated end 432, wherein, the one end extending described upper plate is the first elongated end 431, extends institute The one end stating lower plate is the second elongated end 432.At close described first elongated end 431, the upper plate of described purification plate body 41 On be also placed with multiple magnet 44, outside be placed with magnet capillary channel part constitute described capillary channel 43 magnetic force district. It is understood that array is provided with multiple pipe insert hole 42 on the purification plate body 41 of every layer, the plurality of magnet 44 is put Put at the interval of pipe insert hole 42 on described upper plate.Described magnet 44 is placed to save space the most in a vertical manner. Preferably, the height of described magnet 44 is 3-6mm.Described magnet 44 can be permanent magnet or electric magnet, as long as can realize carrying For magnetic action power.It is connected between described magnet 44 and described controller and has control circuit, sucking eluent to institute After stating magnetic force district, the magnetic force of described magnet 44 can control magnetic force by power-off and disappears or permanent magnetism magnet is removed capillary channel 43 Scope outer (such as removing 5mm), in order to DNA eluting.
It is understood that there is identical magneticaction district in every capillary road.
Preferably, when described magnet 44 is permanent magnet, spring can be set in the bottom of magnet 44, be set by controller Put outside the flexible scope that described magnet 44 is ejected described capillary channel 43 controlling spring.
Preferably, when described magnet 44 is electric magnet, can be connected between described magnet 44 and described controller and have Control circuit, sucking eluent to behind described magnetic force district, by power-off control the magnetic force of magnet 44 disappear (even if capillary channel Magnetic force district disappear).
And after DNA eluting, the magnetic force of described magnet 44 can be recovered or moved back to (i.e. recover institute on purification plate State magnetic force district, in order to avoid magnetic bead is discharged with gene samples), it is simple to enrichment magnetic bead composition, and discharge the aqueous base after eluting because of Sample.
Preferably, described magnetic beads for purifying liquid includes that magnetic bead and volumetric concentration are the ethanol of 70%-85%, described magnetic bead and wine The volume ratio of essence is preferably 1:(0-2).Magnetic beads for purifying liquid is in order to arrest cdna sample (such as DNA).It is used herein as magnetic bead For common magnetic bead, surface does not has the affine enzyme of streptomycin, and the most described magnetic bead can be from Beckman company AMpure magnetic bead.
Preferably, the volume that adds of described magnetic bead is (1-2) of aqueous phase reagent times in system to be purified.
Preferably, described eluent includes TE buffer or pure water.
Preferably, suck before described eluent at described constant flow pump, use the described constant flow pump suction body volume concentrations to be The alcoholic solution of 70%-85% is to described magnetic force district, in order to wash the impurity such as the protein of magnetic bead surfaces adhesion.It is preferably body Volume concentrations is the alcoholic solution of 75%, and the volume of ethanol is react micro-reaction system cumulative volume 3-5 times, preferably cleans twice. Preferably stand 5 minutes at magnetic bead binding domain.Described constant flow pump is used to suck described eluent (TE buffer or pure the most again Water) carry out eluting to described magnetic force district.
Further, described purification plate 4 also includes that the side of purification plate 4 is surrounded by enclosure 45, described enclosure 45 Come, so that each regular arrangement of capillary channel 43.Described enclosure 45 is located at the surrounding of the latter half of purification plate 4, Ji Jiangsuo State the middle plate of purification plate body 41, being surrounded of lower plate.The highly preferred of described enclosure 45 is less than purification plate body 41 Height.The connection of described purification plate 4 and described driving structure is realized by capturing the one side of described enclosure 45.
What this utility model second aspect provided builds in the instrument of storehouse, when purification, by raw for " the double Water-In-Oil " after addition magnetic bead Changing micro-reaction system and suck the magnetic force district of capillary channel 44, the cdna sample (such as DNA) in capillary channel 44 is through magnetic force district Time by enrichment with magnetic bead, and discharge from Water-In-Oil structure, and relayed at capillary channel 44 by other compositions of double oil embeddings Continuous flow forward；Resorb eluent afterwards to magnetic force district, remove and remove magnetic force district and (disappeared by the magnetic force of Electromagnetic Control magnet Or permanent magnetism magnet is removed pipeline scope), make the magnetic bead composition containing DNA be suspended in eluent, by the DNA eluting of enrichment, The DNA discharging enrichment obtains cdna sample after purification.Preferably, when discharging liquid, first receive the cdna sample of eluting to water Or in buffer solution, as the follow-up raw material preparing biochemical micro-reaction system, and by embedding remaining in capillary channel 44 other The Water-In-Oil structure of composition is exhausted directly in the waste liquid tank 52 of reagent area 5.Due to a capillary road contact is for same Oil in the water-in-oil system of sample, can wait whole flow process to clean pipeline again after terminating.
96 orifice plates, by directly reagent and gene samples being positioned in 96 orifice plates, are then positioned over by traditional purification process On bulk magnet, the DNA captured by magnetic bead becomes branch to assemble the bottom to 96 orifice plates due to magneticaction, and Aspirate supernatant is by hole After other reagent is discharged in plate, obtain comprising the magnetic bead of DNA sample.
In this utility model, it is purified based on the micro-reaction system of water in oil biochemistry and the most suitable purification plate 4, Gene samples and reagent etc. are embedded in oil, and contact with described capillary channel is all oil, and capillary channel can reuse, Without using multiple 96 hole PCR plate, the first-class medical disposable material of liquid-transfering gun, add eluent etc. without after getting rid of supernatant Troublesome operation.
Further, the pipe insert hole 42 (including aperture and number) on described purification plate 4 is configured to and described reaction The reacting hole position 61 of orifice plate 6 is consistent.The number of the pipe insert hole on every layer of purification plate body 41 and described reacting hole position 61, Arrange consistent, in order to the second elongated end 432 of capillary channel 43 is inserted in the reacting hole position 61 after adding magnetic bead.Described purification On plate 4, the number of capillary channel 43 determines according to the number of gene samples.
Preferably, the material of described capillary channel 43 is fluoride (such as politef), does not adhere to oily matter, protects Card is not adhered to by Water-In-Oil structure.
Further, participating in Fig. 1, described drive mechanism 1 includes the first workbench 10 arranged in the first direction, along second Second workbench 20 of direction setting, the 3rd workbench 30 along third direction setting, gripper components 40 (not shown), wherein, Described first direction is horizontal direction, and described second direction is perpendicular to described first direction, described third direction with described first, Second direction, described second workbench 20 is slidably connected with described first workbench 10 in the first direction, described 3rd workbench 30 are slidably connected with described second workbench 20 in a second direction, and described gripper components 40 is along third direction and described 3rd work Platform 30 is slidably connected, and described gripper components 40 connects micro sample-adding device 3 and purification plate 4；
It is provided with the first servomotor 101 in described first workbench 10, in described second workbench 20, is provided with the second servo Motor 102, is provided with the 3rd servomotor 103 in described 3rd workbench 30, described controller is electric with described first servo respectively Machine the 101, second servomotor 102 and the 3rd servomotor 103 electrically connect；
Described first servomotor 101 drives described second workbench 20 to move along the first direction by screw rod, and described Two servomotors 102 drive described 3rd workbench 30 to move in a second direction by screw rod, and described 3rd servomotor 103 leads to Crossing screw rod drives described gripper components 40 to move along third direction.
Preferably, described gripper components 40 is mechanical hand.
Preferably, described second workbench 20 is connected with described first workbench 10 with cantilever structure.The most permissible Save the working place in board.
In this utility model, being additionally provided with pillar 200 in described board 100, described pillar 200 supports described drive mechanism 1. It is understood that the position of described drive mechanism 1 is higher than described constant flow pump 2, reaction orifice plate 6, reagent area 5.Described first work Station 10 is fixed on described pillar 200.Described pillar is fixed by screws on board, described first workbench 10 also by Screw is fixed on described pillar 200.
In this utility model, described drive mechanism 1 is positioned at described constant flow pump 2, reaction orifice plate 6, the rear of reagent area 5, institute State drive mechanism 1 drive under the effect of described controller described purification plate 4 and described micro sample-adding device 3 reaction orifice plate 6, Move in the region of reagent area 5.Described first direction is side-to-side movement direction (alternatively referred to as X-direction), described second Direction is the direction that seesaws (alternatively referred to as Y direction), and described third direction is (alternatively referred to as Z axis side, up and down motion direction To).
Preferably, described purification plate 4 and described micro sample-adding device 3 can fix connection simultaneously, it is also possible to be with removable The mode unloaded is arranged in the gripper components 40 of described driving structure 1.In present embodiment, purification plate 4 and micro sample-adding device 3 Being to be fixedly connected in the gripper components 40 of drive mechanism 1, preferably both are to be connected to capture in the way of " back-to-back " simultaneously On parts 40.Avoiding parts operationally, another parts can stop its mobile route.Gripper components 40 preferably captures institute State the sidewall of purification plate 1.
In another embodiment of the present utility model, under the effect of described controller, can use at needs When needs use purification plate 4 or described micro sample-adding device 3, capture respectively from board during by these 2 parts.
Specifically, described first workbench 10 includes the first guide rail and the company of slip being fixed on described first workbench 10 It is connected on the first slide block on described first guide rail；Described second workbench 20 includes the second guide rail and is slidably connected at described second The second slide block 23 on guide rail, described second guide rail is fixed on described first slide block；Described 3rd workbench 30 includes the 3rd Guide rail and the 3rd slide block 34 being slidably connected on described 3rd guide rail, described 3rd guide rail is fixed on described second slide block 23 On, described gripper components 40 is fixed on described 3rd slide block 34.
In present embodiment, described second workbench 20 passes through described first slide block and described first along described first guide rail Workbench 10 is slidably connected, described 3rd workbench 30 along described second guide rail by described second slide block 23 and described second work Station 20 is slidably connected, and described gripper components 40 passes through described 3rd slide block 34 and described 3rd workbench along described 3rd guide rail 30 are slidably connected.Described first servomotor 101 drives described second workbench 20 to do left and right fortune in the first direction by screw rod Dynamic, described second servomotor 102 drives described 3rd workbench 30 to seesaw in a second direction by screw rod, and described the Three servomotors 103 drive described gripper components 40 to move up and down along third direction by screw rod.
In present embodiment, in described first workbench 10, it is provided with first servomotor the 101, first screw rod, described first It is connected by shaft coupling between servomotor 101 and described first screw rod.Similarly, it is provided with in described second workbench 20 Two servomotor the 102, second screw rods, are connected by shaft coupling between described second servomotor 102 and described second screw rod.
Fig. 3 is the enlarged drawing of the described micro sample-adding device 3 building storehouse instrument in Fig. 1.At an embodiment of the present utility model In, participate in Fig. 3, described micro sample-adding device 3 includes the first microsyringe 30 and the 4th servomotor 104, described first micro- Piston screw 301 in amount syringe 30 connects the outfan of described 4th servomotor 104, described 4th servomotor 104 Input electrically connect with described controller, described 4th servomotor 104 under the control of described controller by piston spiral shell Bar 301 drives described first microsyringe 30 to draw, discharge reagent.
Further, as it is shown on figure 3, described first microsyringe 30 includes piston screw 301, injection head 302, sample introduction Nut 305 fixed by device piston 303, injector sleeve 304 and injector.Nut 305 fixed by injector can be fixed on the described 4th On servomotor 104.
Preferably, when described first microsyringe discharges reagent to reacting hole position 61, the injection head of described syringe 302 the second oil-based liquid positions (i.e. light oil position) inserting the micro-reaction system of described biochemistry, are then then exhausted from reagent.This Sample waterborne liquid (including water base cdna sample and reaction reagent) can be embedded in described first oil-based liquid, the second oiliness liquid automatically Constituting sandwich structure between body, described first oil-based liquid is below, described second oil-based liquid above, two kinds of oil-based liquids Immiscible.After described first microsyringe discharges reagent, draw clear from the cleanout fluid storage tank of described reagent area 5 Washing liquid, is carried out described first microsyringe, in order to carry out the absorption of lower a kind of reagent.In described cleanout fluid storage tank Cleanout fluid be ethanol or liquor natrii hypochloritis.
It is further preferred that the inner surface of described first microsyringe 30 scribbles Teflon coating.Can ensure that injection No liquid residual in device.
Described 4th servomotor 104 can accurately control the motion of described first microsyringe 30, and kinematic accuracy can Reach 5 μm.The range of described first microsyringe 30 can as little as 2 μ L.Under the cooperation of described 4th servomotor 104, described The liquid absorption of the first microsyringe 30 can minimum 0.4 μ L, the uniformity of imbibition can be controlled in 5% precision.
It is further preferred that the number of described first microsyringe 30 can be one or more.Such as 1,6,8 Individual, it is also possible to simultaneous with 1 and multiple syringes (i.e. simultaneous with 1 and 8 microsyringes in Fig. 1).When can Select with the number according to required sample, can sample introduction singly, it would however also be possible to employ multiple syringes are line upon line Sample introduction.
In another embodiment of the present utility model, participating in Fig. 4, described micro sample-adding device 3 sprays for micro liquid Penetrating system 3B, described micro liquid jetting system 3B includes air supply source 31, digital voltage regulator 32, cleanout fluid reservoir bottle 33, micro-electricity Magnet valve 34, mini sprinkler 35, atmospheric pressure module 36, ramjet duct p1, micro-nozzle road p2, air pressure conveying branch pipe(tube) p3, air pressure conveying Main pipeline p4, cleanout fluid intake line p5, wherein, described atmospheric pressure module 36 includes motor and described motor even The screw mandrel moving cell (not shown) connect and the second syringe 360, the piston rod in described second syringe 360 connects described silk Bar moving cell, the outlet of described second syringe 360 connects has described air pressure to carry main pipeline p4.Specifically, described second Syringe 360 includes piston rod 361, piston 362, and described motor promotes piston rod 361 to carry by screw mandrel moving cell The piston 362 of dynamic described second syringe.
Preferably, the signal of telecommunication of described micro-electromagnetic valve 34 is connected with described controller by interface board, to enter spray sample amount Row controls.
Preferably, described air supply source 31 includes, but are not limited to helium, nitrogen.It is preferably the helium tank equipped with helium.
Described digital voltage regulator 32 pipeline is connected between described cleanout fluid reservoir bottle 33 and described air supply source 31.Specifically Ground, described cleanout fluid reservoir bottle 33 includes intake interface I1, first interface O1 and the second interface O1, described intake interface I1 and institute The outfan stating digital voltage regulator 32 connects, and described first interface O1 is communicated in the micro-electromagnetic valve 34 on described ramjet duct p1, Described second interface O1 is connected to the suction side of described cleanout fluid suction line.The input of described digital voltage regulator 32 is with described Air supply source 31 is connected by a gas path pipe, the outfan of described digital voltage regulator 32 and the air inlet of described cleanout fluid reservoir bottle 33 Interface I1 connects.
Described digital voltage regulator 32 is for regulating described air supply source 31 with ramjet duct p1 described in constant pressure feed；Institute State controller and be respectively connecting to described motor and micro-electromagnetic valve 34 by controlling circuit.
It is connected between described mini sprinkler 35 with described atmospheric pressure module and has micro-nozzle road p2, air pressure conveying branch pipe(tube) p3 gentle Pressure conveying main pipeline p4；It is connected between described mini sprinkler 35 and described rinsing bottle and has micro-nozzle road p2 and ramjet duct p1, described Micro-electromagnetic valve 34 is arranged on described ramjet duct p1 the spray sample amount for controlling described mini sprinkler 35, described micro-nozzle road p2's One end connects described mini sprinkler 35；It is connected between described atmospheric pressure module and described rinsing bottle and has air pressure conveying main pipeline p4, clear Washing liquid intake line p5；Wherein, the end that described micro-nozzle road p2, ramjet duct p1, described air pressure conveying branch pipe(tube) p3 phase are closed on It is provided with the first three-way valve V, described micro-nozzle road p2, air pressure conveying branch pipe(tube) p3 between Kou to close on air pressure conveying main pipeline p4 phase Port between be provided with the second three-way valve U.
Specifically, described first three-way valve V includes the first valve port V1, the second valve port V2, the 3rd valve port V3, described first valve Mouth V1 connects the first interface O1 in described washer bottle, and (the connection pipeline between described first valve port V1 and described washer bottle is constituted Described ramjet duct p1), described second valve port V2 connects in described mini sprinkler 35 (described second valve port V2 and described mini sprinkler 35 Connection pipeline constitute described micro-nozzle road p2), described 3rd valve port V3 connect in described air pressure conveying branch pipe(tube) p3 first End.Described second three-way valve U includes that the first port U1, the second port U2, the 3rd port U3, described first port U1 connect in institute State the second end (the 3rd of the first port U1 of described second three-way valve U and described first three-way valve V of air pressure conveying branch pipe(tube) p3 Connection pipeline between valve port V3 constitutes described air pressure conveying branch pipe(tube) p3), described second port U2 connects in described atmospheric pressure (the connection pipeline between described second port U2 and described atmospheric pressure module constitutes described gas in the outlet of the second syringe of module Pressure conveying main pipeline p4), described 3rd port U3 connects the second interface O1 (described 3rd end in described cleanout fluid reservoir bottle 33 Connection pipeline between mouth U3 and described second interface O1 constitutes described cleanout fluid intake line p5).
When needs suck reagent from reagent trough 51 (A see Fig. 4), regulate described first three-way valve V, the second three-way valve U Make described micro-nozzle road p2, air pressure conveying branch pipe(tube) p3 and air pressure conveying main pipeline p4 connect (that is, make the first port U1, second Port U2 is connected, and the second valve port V2, the 3rd valve port V3 are connected), the drop-down described piston rod 361 of described motor sucks examination Agent extremely described micro-nozzle road p2.
When the reagent that ejection sucks (B in see Fig. 4), regulate described first three-way valve V and make described micro-nozzle road p2 and institute Stating ramjet duct p1 and connect (that is, making the first valve port V1, the second valve port V2 be connected), described digital voltage regulator 32 supplies described punching Pressure pipeline p1 pressure, described micro-electromagnetic valve 34 connects, and controls described mini sprinkler 35 and spray sample.
When detergent line, it is necessary first to draw cleanout fluid (see C in Fig. 4), regulate described second three-way valve U make described clearly Washing liquid intake line p5 connects (that is, making) with described air pressure conveying main pipeline p4, the drop-down described piston rod 361 of described motor Cleanout fluid is made to fill described air pressure conveying main pipeline p4；Then regulate described first three-way valve V, the second three-way valve U makes described micro- Jet pipe road p2, air pressure conveying branch pipe(tube) p3 connects (schematic diagram is with A in Fig. 4) again with air pressure conveying main pipeline p4, above pushes away described Waste liquid after piston rod 361 makes cleaning is discharged through described mini sprinkler 35.
Further, when sucking reagent, described micro-nozzle road p2, air pressure conveying branch pipe(tube) p3 and air pressure conveying main pipeline After p4 connection, use the drop-down described piston rod 361 of motor first suction part air extremely described micro-nozzle road p2.Avoid row When going out reagent, when the aperture of described micro-electromagnetic valve 34 is bigger, cleanout fluid is entered described micro-nozzle road p2 and to be discharged by strong pressurizing Reagent mixes.First suction part air, after discharging reagent, then can discharge air, the trace then will brought into Cleanout fluid is expelled to waste liquid tank.
In this utility model, when described micro-electromagnetic valve 34 is connected, described micro liquid jetting system 3B can realize hanging The reagent of empty ejection trace is in described reacting hole position 61, and owing to spray sample pressure is relatively big, and the reagent press-in that can will draw Between one oil-based liquid and the second oil-based liquid, without by the light oil position in position, mini sprinkler 35 insertion reaction hole 61, can To avoid mini sprinkler 35 to contact oil droplet in inserting described reacting hole position 61, prevent cross-contamination, clean simple.Additionally, it is described micro- The pipeline of all contact reagent also can be cleaned by quantity of fluid spraying system 3B automatically.The speed of the injection reagent of described mini sprinkler Degree can reach more than 10 per second, and the volume of the reagent of injection can be less than 0.1 μ L, it is possible to achieve quick, high flux sample preparation.
In present embodiment, when sucking reagent or sucking cleanout fluid when detergent line, drop-down piston 362, make described gas The manifold volume of pressure conveying main pipeline p4 diminishes, and produces negative pressure, sucks reagent；When cleanout fluid is discharged in detergent line, above push away work Plug 362, makes the manifold volume of described air pressure conveying main pipeline p4 become big, produces malleation, discharge cleanout fluid.
Preferably, described mini sprinkler 35 and the micro-electromagnetic valve 34 connected by pipeline (include micro-nozzle road p2, part punching press Pipeline p1) it is fixedly connected in the gripper components 40 of described drive mechanism 1.
For saving space, remaining part (such as by cleanout fluid reservoir bottle 33 and air supply source 31) can be placed in described high flux Outside the cabin building storehouse instrument of order-checking.Aperture can be set at the sidewall in described cabin, pipeline is passed cabin by aperture.Its In, described board 100 entirety building storehouse instrument is surrounded by cabin, forms a chamber.
Described micro liquid jetting system 3B can cooperate with drive mechanism 1, by institute under the control of described controller Stating drive mechanism 1 drives described mini sprinkler 35 according to pre-set programs shift position, prepares the sample array of setting.
In this utility model, being provided with two kinds of sample loading alternative of trace reagent, one is the first microsyringe and the 4th The small throughput sample-adding that servomotor matches, the second is the micro liquid jetting system 3B of high flux sample-adding.First kind of way Being suitable for the situation of pending cdna sample negligible amounts, micro liquid jetting system 3B is applicable to the feelings that sample size is more Condition.Be applicable to single running maximum sample flux is 96 parts.First microsyringe and the 4th servomotor are with dismountable side Formula is connected in the gripper components 40 of described driving structure.Described micro liquid jetting system 3B connects the most removably In the gripper components 40 of described driving structure.Can experimental need, the micro sample-adding mode required for switching.
This utility model second aspect builds storehouse instrument described in providing, based on micro sample-adding device, constant flow pump and can be at XYZ The drive mechanism of axle motion and prepare double micro-reaction system of water in oil biochemistry accurately, and by reaction micro-with described biochemistry Reacted system is purified by the suitable purification plate of system, purifies the gene samples after being processed.Institute can be passed through State and build storehouse instrument and complete simple nucleic acid amplification and prepared by responsible nucleic acid library, save medical disposable material, simple to operate.
High-flux sequence described in this utility model second aspect build storehouse instrument can be used for preparation order-checking nucleic acid library and In nucleic acid amplification.
Application case 1: prepared by the high flux library of common DNA sample
Use and build storehouse instrument, using 12 weeks pregnant woman blood plasma cfDNA samples as gene samples, use Life company shown in Fig. 1 Ion Plus Fragment Library Kit test kit prepares high-throughput sequencing library, and concrete operations flow process is as follows:
(1) end reparation, sets up biochemical micro-reaction system 1:
Constant flow pump 5 is used to draw the first oil-based liquid, the second oil-based liquid successively to the reacting hole position 61 reacting orifice plate 6 In, described reaction orifice plate 6 is the temperature control cavity of PCR instrument, and wherein, the first oil-based liquid (heavy oil) is specially fluorocarbon oil FC-40, Taking 15 μ L, the second oil-based liquid (light oil) is that the mixing of benzyl polysiloxanes PD5 and Polysorbate additive (sorbester p17) is molten Liquid, wherein, the volume of sorbester p17 is the 5% of PD5 volume, and the cumulative volume of the second oil-based liquid is 40 μ L, and oil-based liquid adds fashionable Softly add along tube wall, it is to avoid produce bubble；
Use micro sample-adding device to be injected in reaction orifice plate 6 by following reagent successively, form double water in oil biochemistry micro-instead Answer system 1:
Reagent Volume (ul)
DNA 1
5X End Repair Buffer 0.3
End Repair Enzyme 0.2
Total 1.5
Arranging temperature by controller makes the micro-reaction system of above-mentioned biochemistry 1 react 30min at 20 DEG C, wherein, and can be first DNA is injected in two kinds of oil-based liquids, other reagent first can be mixed and reinject afterwards in reaction orifice plate.
(2) purification after end is repaired:
In reacting hole position, inject Beckman company AMpure XP magnetic bead 2.7 μ L (1.8 times of water system volume), stand 10min, then uses constant flow pump that the liquid in reacting hole position sucks the magnetic force district of capillary channel in purification plate and is enriched with, rich After collection 5min, then use constant flow pump to suck magnetic bead cleanout fluid (75% ethanol) 20 μ L, stand 1min, be repeated 1 times, to clean Fall the impurity such as protein that magnetic bead surfaces adheres to, be further continued for using constant flow pump to suck eluent (the TE buffer of specially 3 μ L) To the magnetic force district of each capillary channel, remove Magnet and make DNA fully be dissolved in eluent, after standing 5min, re-use Magnet Be placed back into capillary channel constitute magnetic force district be enriched with magnetic bead composition, use constant flow pump discharge eluting after liquid, obtain 3 μ L ends The TE solution of the DNA sample repaired.
(3) Adapter connects (joint connection), sets up biochemical micro-reaction system 2:
Use constant flow pump draw successively above-mentioned first oil-based liquid of 15 μ L, 40 μ L the second oil-based liquid to reacting orifice plate In the reacting hole position 61 of 6；The DNA sample using micro sample-adding device to be repaired by the end of above-mentioned 3 μ L injects reaction orifice plate 6 In, it is also injected into following reagent (containing the sequence that tags) simultaneously the most again reacting in orifice plate 6, sets up biochemical micro-reaction system 2:
Arranging temperature by controller makes the micro-reaction system of above-mentioned biochemistry 2 react 30min at 20 DEG C,
(4) purification after joint connects:
The same above-mentioned steps of purification process (2), difference is that the AMpure XP magnetic bead volume added is that above-mentioned biochemistry is micro- In reaction system 2 1.5 times of water system reagent (1.7+3), i.e. 7 μ L, carry out eluting with the TE buffer of 3 μ L, obtain 3 μ L after purification and connect The TE solution of the DNA sample that head connects.
(5) PCR reaction, sets up biochemical micro-reaction system 3:
Use constant flow pump draw successively above-mentioned first oil-based liquid of 15 μ L, 40 μ L the second oil-based liquid to reacting orifice plate In the reacting hole position 61 of 6；The DNA sample using micro sample-adding device to be connected by the joint of above-mentioned 3 μ L injects reaction orifice plate 6 In, it is also injected into following reagent the most again reacting in orifice plate 6, sets up biochemical micro-reaction system 3:
Subsequently above-mentioned system 3 is carried out PCR reaction by following procedure:
(6) purification after PCR:
The same above-mentioned steps of purification process (2), difference is that the AMpure XP magnetic bead volume added is that above-mentioned biochemistry is micro- In reaction system 31 times of water system reagent (1.9+3), i.e. 5 μ L.Carry out eluting with the TE buffer of 3 μ L, collect the gene after eluting Sample, storehouse is built in the order-checking completing to obtain pregnant woman blood plasma cfDNA sample.
Application Example 2:(is applicable to rapid DNA sample high flux library and prepares)
Use and build storehouse instrument shown in Fig. 1, using neonate monogenic disease DNA sample as gene samples, use enzyme action to interrupt Method prepares high-throughput sequencing library, and concrete operations flow process is as follows:
(1) biochemical micro-reaction system 1 is set up:
Constant flow pump 5 is used to draw the first oil-based liquid, described second oil-based liquid successively to the reacting hole position reacting orifice plate 6 In 61, described reaction orifice plate 6 is the temperature control cavity of PCR instrument, and wherein, the first oil-based liquid (heavy oil) is specially fluorocarbon oil FC- 40, take 15 μ L, the second oil-based liquid (light oil) is benzyl polysiloxanes PD5 and the mixing of Polysorbate additive (sorbester p17) Solution, wherein, the volume of sorbester p17 is the 5% of PD5 volume, and the cumulative volume of the second oil-based liquid is 40 μ L, and oil-based liquid adds Time softly add along tube wall, it is to avoid produce bubble；
Interrupt reaction: first (concentration is 3.6ng/ μ L, real by the DNA sample of 1.4 μ L to use the micro sample-adding device 3 in Fig. 3 Testing DNA sample concentration is to use Qubit BR test kit to measure) inject the light oil position in reacting hole position 61, light oil can be certainly The water base DNA sample of dynamic embedding, is then based on transposase dna library kits TruePrepTMDNA Library Prep Kit joins The water system reagent that DNA processed interrupts, is sequentially added into tagmentation buffer 0.4ul in the light oil layer of reacting hole position 61, Tagmentation enzyme 0.2ul also ensures that it forms the double micro-reaction system of water in oil biochemistry 1 (aqueous phase 1.4+0.6 altogether =2 μ L).10min is hatched at 55 DEG C.Wherein, interrupt enzyme individually to add with buffer (being placed in freezing in reagent trough) in batches Entering in reacting hole position, volume summation is 0.6 μ L；
(2) rear purification is interrupted:
After interrupting, in reacting hole position, inject Beckman company AMpure XP magnetic bead 0.9 μ L, stand 10min, then adopt With constant flow pump, the liquid in reacting hole position is sucked the magnetic force district of capillary channel in purification plate to be enriched with, after enrichment 5min, Then use constant flow pump to suck magnetic bead cleanout fluid (75% ethanol) 20 μ L, stand 1min, be repeated 1 times, to wash magnetic bead surfaces The impurity such as the protein adhered to, are further continued for using constant flow pump to suck eluent (specially pure water 5 μ L) to each capillary channel Magnetic force district, removes Magnet and makes DNA fully be dissolved in eluent, after standing 5min, re-uses Magnet enrichment magnetic bead composition, uses Constant flow pump discharges the liquid after eluting, obtains comprising the recovery solution of DNA composition.
(3) biochemical micro-reaction system 2 is set up:
Above-mentioned 5 μ L are comprised the recovery solution first oil-based liquid with above-mentioned 18 μ L of DNA composition, second oiliness of 45 μ L Liquid again pulls up micro-reaction system, be implanted sequentially the most again PCR reaction system reaction reagent (PCR enzyme, buffer, Acid, alkali, saline solution etc.) to the light oil position of reacting hole position 61, the liquid of DNA sample with rear addition can be merged by light oil automatically, Forming double micro-reaction system of water in oil biochemistry 2, wherein, PCR reaction reagent is for mix in advance, and another rising adds reacting hole position In, the cumulative volume of PCR reaction reagent is 16 μ L, and the test kit of PCR reaction system is TruePrepTM DNA Library Prep Kit (Vazyme#TD501-TD503), is specifically made up of following reagent:
Reagent Volume (μ L)
5x TruePrep Amplify Buffer 10
10mM each dNTP 1
TruePrep Amplify Enzyme 1
Primer1 1
Primer2 1
N5 1
N7 1
Total 16
(4) reaction system 2 micro-to above-mentioned biochemistry sets circulating temperature and completes PCR amplification, obtains PCR primer, wherein, specifically Condition is as follows:
(4) purification after PCR:
PCR terminate after the same above-mentioned steps of purification (2), simply the addition volume of magnetic bead is 6 μ L.Collect the gene after eluting Sample, i.e. obtains the sequencing library of neonate monogenic disease DNA.
Application Example 3
Using from the DNA of deafness patient sample as gene samples, based on Agena1 company Complete Pro Reagent Set test kit carries out kind of a high-throughput multi PCR mass spectral analysis, comprises the following steps:
1, constant flow pump 5 is used to draw the first oil-based liquid, described second oil-based liquid successively to the reacting hole reacting orifice plate 6 In position 61, described reaction orifice plate 6 is the temperature control cavity of PCR instrument, and wherein, the first oil-based liquid (heavy oil) is specially fluorocarbon oil FC-40, takes 15 μ L, and the second oil-based liquid (light oil) is specially benzyl polysiloxanes PD5 and Polysorbate additive (span 80) mixed solution, wherein, the volume of sorbester p17 is the 5% of PD5 volume, and the cumulative volume of the second oil-based liquid is 40 μ L；
The micro sample-adding device 3B in Fig. 3 is used first the DNA sample of 1 μ L to be injected the light oil position in reacting hole position 61, Light oil can embed water base DNA sample automatically；It is implanted sequentially the light oil of Pre-PCR reaction reagent extremely described reacting hole position 61 the most again Position, the liquid of DNA sample with rear addition can be merged by light oil automatically, the double micro-reaction system of water in oil biochemistry of formation, wherein, The water-based system being embedded includes the various materials of volumes below:
2, subsequently above-mentioned system is carried out Pre-PCR reaction by following procedure:
Pre-PCR response procedures:
3, adding SAP digestion reagent in the microbody system of above-mentioned Pre-PCR reaction, SAP digestion reagent specifically includes Below:
Reagent Volume (μ L)
H2O (HPLC level) 1.53
SAP Buffer 0.17
SAP enzyme(1.7U/μL) 0.30
Total 2
SAP digestion reagent needed for each Kong Weizhong, can be first to be blended in by the SAP digestion reagent that many parts required Micro sample-adding device is used to draw hole position aequum and inject (lower same) in reaction orifice plate the most again, then according to below SAP response procedures reacts:
37℃ for 40mins
85℃ for 5mins
12℃ forever
4, in above-mentioned SAP reacted microbody system, add extension reagent, specifically include following:
Reagent Volume (μ L)
H2O (HPLC level) 0.619
EX Buffer Plus(10x) 0.2
EX Termination mix 0.2
iPLEX enzyme 0.041
EX Extend Primer mix 0.94
Total 2
5, the reaction system after above-mentioned extension is carried out magnetic beads for purifying:
The 75% ethanol work being injected simultaneously into Beckman company AMpure XP magnetic bead 9 μ L and 18 μ L is injected in reacting hole position For magnetic beads for purifying liquid, stand 10min, then use constant flow pump that the liquid in reacting hole position sucks capillary channel in purification plate Magnetic force district is enriched with, and after enrichment 5min, (water of specially pure water 20 μ L or TE delay then to use constant flow pump to suck eluent Rush liquid) to the magnetic force district of each capillary channel, remove Magnet and make DNA fully be dissolved in eluent, after standing 5min, re-use Magnet enrichment magnetic bead composition, the liquid after using constant flow pump to discharge eluting, obtain comprising the recovery solution of DNA composition, the most multiple Pcr amplification product, analyzes followed by mass spectrum loading.
The above is preferred implementation of the present utility model, it is noted that for the ordinary skill of the art For personnel, on the premise of without departing from this utility model principle, it is also possible to make some improvements and modifications, these improve and profit Decorations are also considered as protection domain of the present utility model.

Claims (8)

1. a high-flux sequence build storehouse instrument, it is characterised in that include board and the drive mechanism being arranged on board, institute State and on board, be additionally provided with controller, constant flow pump, purification plate, reagent area and reaction orifice plate；Described reagent area includes multiple reagent trough, The plurality of reagent trough is in order to hold the first oil-based liquid, the second oil-based liquid, water base gene samples, magnetic beads for purifying liquid, eluting Liquid and multiple reaction reagent；Described reaction orifice plate includes the reacting hole position of array distribution；Connect in described drive mechanism and have trace Sample adding device and purification plate, described controller controls described drive mechanism and drives described purification plate and described micro sample-adding device to exist Move in board, and control the absorption of described micro sample-adding device, discharge reagent；Described purification plate includes purification plate body, array Being arranged on the pipe insert hole on purification plate body and multiple capillary channel, described capillary channel is through described pipe insert hole also Above and below described purification plate body, it is respectively provided with the first elongated end and the second elongated end, is extending near described first At end, described purification plate body is also placed with multiple magnet, forms the magnetic force district of described capillary channel, described first elongated end It is connected with described constant flow pump pipeline；Described second elongated end is for inserting reagent trough and described under the drive of described drive mechanism In reacting hole position.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described micro sample-adding device is micro liquid jetting system, Described micro liquid jetting system includes air supply source, digital voltage regulator, cleanout fluid reservoir bottle, micro-electromagnetic valve, mini sprinkler, atmospheric pressure Module, ramjet duct, micro-nozzle road, air pressure conveying branch pipe(tube), air pressure conveying main pipeline, cleanout fluid intake line, wherein, described Atmospheric pressure module includes the screw mandrel moving cell that motor, the second syringe are connected, described second note with described motor Piston rod in emitter connects described screw mandrel moving cell, and the outlet of described second syringe connects described air pressure conveying supervisor Road；Described digital voltage regulator pipeline is connected between described cleanout fluid reservoir bottle and described air supply source, and described digital voltage regulator is used In regulating described air supply source with ramjet duct described in constant pressure feed；Described controller is respectively connecting to by controlling circuit Described motor and micro-electromagnetic valve；
It is connected between described mini sprinkler with described atmospheric pressure module and has micro-nozzle road, air pressure conveying branch pipe(tube) and air pressure conveying supervisor Road；Being connected between described mini sprinkler with described rinsing bottle and have micro-nozzle road and ramjet duct, described micro-electromagnetic valve is arranged on described For controlling the spray sample amount of described mini sprinkler on ramjet duct, the one end in described micro-nozzle road connects described mini sprinkler；Described gas It is connected between modular pressure with described rinsing bottle and has air pressure to carry main pipeline, cleanout fluid intake line；Wherein, described micro-nozzle It is provided with the first three-way valve, described micro-nozzle road, gas between the port that road, ramjet duct, described air pressure conveying branch pipe(tube) closes on mutually It is provided with the second three-way valve between the port that pressure conveying branch pipe(tube) and air pressure conveying main pipeline close on mutually；
When suck reagent time, regulate described first three-way valve, the second three-way valve make described micro-nozzle road, air pressure conveying branch pipe(tube) and Air pressure conveying main pipeline connection, the drop-down described piston rod of described motor sucks reagent to described micro-nozzle road；When ejection is inhaled During the reagent entered, regulate described first three-way valve and make described micro-nozzle road connect with described ramjet duct, described digital voltage regulator Supplying described ramjet duct pressure, described micro-electromagnetic valve is connected, and controls described mini sprinkler spray sample；When detergent line, regulation Described second three-way valve makes described cleanout fluid intake line connect with described air pressure conveying main pipeline, the drop-down institute of described motor Stating piston rod makes cleanout fluid fill described air pressure conveying main pipeline；Then regulate described first three-way valve, the second three-way valve makes institute State micro-nozzle road, air pressure conveying branch pipe(tube) connects again with air pressure conveying main pipeline, above pushes away giving up after described piston rod makes cleaning Liquid is discharged through described mini sprinkler.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described micro sample-adding device include the first microsyringe and 4th servomotor, the piston screw in described first microsyringe connects the outfan of described 4th servomotor, described The input of the 4th servomotor electrically connects with described controller, and described 4th servomotor is logical under the control of described controller Crossing piston screw drives described first microsyringe to draw, discharge reagent.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described purification plate also includes for being wrapped the side of purification plate The enclosure fenced up, the height of described enclosure is less than the height of purification plate body, described enclosure and described driving structure It is connected.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described magnet is permanent magnet or electric magnet, described magnet with Connecting between described controller and have control circuit, after sucking eluent extremely described magnetic force district, the magnetic force of described magnet can pass through Power-off controls magnetic force and disappears or by outside the scope of permanent magnetism magnet removal capillary channel, to carry out eluting cdna sample.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described controller is programmable logic controller (PLC), described control Device processed is also equipped with high-resolution and touches display screen.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that on described reaction orifice plate, be additionally provided with temperature controller, described temperature control Device for controlling the temperature needed for reaction by the temperature of reaction system.
Build storehouse instrument the most as claimed in claim 1, it is characterised in that described drive mechanism includes first arranged in the first direction Workbench, the second workbench arranged in a second direction, along third direction arrange the 3rd workbench and gripper components, wherein, Described first direction is horizontal direction, and described second direction is perpendicular to described first direction, described third direction with described first, Second direction, described second workbench is slidably connected with described first workbench in the first direction, and described 3rd workbench is along Two directions are slidably connected with described second workbench, and described gripper components is slided even along third direction with described second workbench Connecing, described gripper components connects micro sample-adding device and purification plate；
It is provided with the first servomotor in described first workbench, in described second workbench, is provided with the second servomotor, described Be provided with the 3rd servomotor in three workbench, described controller respectively with described first servomotor, the second servomotor and Three servomotor electrical connections；Described first servomotor drives described second workbench to move along the first direction by screw rod, institute Stating the second servomotor drives described 3rd workbench to move in a second direction by screw rod, and described 3rd servomotor passes through spiral shell Bar drives described gripper components to move along third direction.
CN201620383191.9U 2016-04-29 2016-04-29 A kind of high-flux sequence build storehouse instrument Active CN205803638U (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109439514A (en) * 2018-12-14 2019-03-08 贵州大学 A kind of support device for array microfluidic point sample microfluidic control device

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109439514A (en) * 2018-12-14 2019-03-08 贵州大学 A kind of support device for array microfluidic point sample microfluidic control device

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