CN107937265A - A kind of palm pyrosequencing system based on digital microfluidic technology - Google Patents
A kind of palm pyrosequencing system based on digital microfluidic technology Download PDFInfo
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- CN107937265A CN107937265A CN201711084328.6A CN201711084328A CN107937265A CN 107937265 A CN107937265 A CN 107937265A CN 201711084328 A CN201711084328 A CN 201711084328A CN 107937265 A CN107937265 A CN 107937265A
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Abstract
The invention discloses a kind of palm pyrosequencing system based on digital microfluidic technology, belong to digital microfluidic technical field.The digital microcurrent-controlled chip, electromagnetic module and integrated circuit connect, and integrated circuit is connected to computer with chemiluminescence acquisition module.Palm pyrosequencing system of the present invention based on digital microfluidic technology is chip base using printed circuit board, drop formation, transport, mixing and the division in sequencing procedure are realized by the regulation and control to chip top electrode power-up sequence based on dielectric electro-wetting principle on chip, it is easily manipulated and minimizes, caused optical signal can quickly be read in real time, so as to fulfill the measure of single base on chip.The present invention has the advantages that simple in structure, cost is low, easy of integration, and detection sensitivity is high, significantly reduces the cost of sequencing, can be widely applied to the fields such as the genetic test based on pyrosequencing.
Description
Technical field
The invention belongs to digital microfluidic technical field, is specially a kind of palm pyrophosphoric acid based on digital microfluidic technology
Sequencing system.
Background technology
Since the Human Genome Project in 2003 is completed, DNA sequencing technology has been achieved for huge progress.Thus send out
Many methods of exhibition have been realized in being commercialized, and possess the ability for quickly completing the sequencing of whole gene group.At this stage,
" routine business " in life science is had changed into using DNA sequencing technology completion whole gene group examining order.However,
As the application environment of DNA sequencing becomes more and more diversified, it is expected that any single microarray dataset meets all use demands
Have become impossible thing.Therefore, it is more to need to be customized platform so as to meet various to apply journey at this stage
The particular demands and use environment of sequence.So flux is lower, less expensive, and the DNA sequencing for being more convenient for quickly measure immediately is put down
Platform just becomes the hot spot of recent research work.
It is micro-fluidic to receive much concern as a kind of micro-nano fluid treatment technology in DNA sequencing field.Its cost is low, flux is high,
Analyze speed is fast, reagent consumes few advantage, is of great significance for improving the flux of DNA sequencing and reducing processing cost,
Represent following DNA sequencing and move towards micromation, integrated developing direction.Micro-fluidic chip using configurations microchannel and
The various informative additional field of force, is manipulated, handled and is controlled to micro fluid or sample on a microscopic scale, it is achieved thereby that
The part of DNA sequencing or even repertoire are integrated on one piece of microchip.However, the limitation of conventional microfluidic control chip is also
Obviously：Complicated, difficulty of processing is big；Mechanical pump, valve is needed to be used cooperatively, integrated difficulty is big；To more reagent positions
The accurate control put with the reaction time is slightly aobvious powerless.A series of this yet unresolved issue means for the integrated of DNA sequencing
Micro-fluidic chip so far difficulty conclude it is ripe.
Digital microfluidic technology (Digital microfluidics, DMF) is as a kind of emerging discretization microlayer model behaviour
Vertical means, are expected to change this present situation due to having the advantages that a series of traditional microfluidic technologies are incomparable.DMF is a kind of base
Realize that discrete droplets accurately control the new drop of (including the operation such as movement, merging, distribution and division) in microelectrode array
Manipulation technology, it realizes the manipulation to microlayer model using dielectric wetting phenomena of the drop on hydrophobizing surface.Pass through outside
The control of electric signal, DMF can drive microlayer model to be moved along pre-set path, realize big flux discretization drop
It is controllable precisely to manipulate.This maneuverability pattern has the characteristics that high concurrency and full-automation, can realize that multi-path is controllable in real time
Reaction, fully-integratedization for realizing the biochemical test basic operation such as sample preparation, reaction, separation, detection have important
Meaning, is therefore particularly suitable for high integration, high-performance, complicated micro- biochemical analysis system.
In many sequencing systems, pyrosequencing is a kind of inorganic pyrophosphate to being generated in DNA polymerisations
(PPi) method detected in real time, it, which often takes turns the fluorescence intensity of base pairing by observing, can quantitatively determine one
Whether specific nucleotide is added to chain, is sequenced so as to fulfill in synthesis.Pyrosequencing is substantially a series of to be based on battle array
The repetition liquid processing steps of row, therefore can quick, real-time, automation progress microlayer model processing with the DMF based on electrod-array
The characteristics of match very much.
Therefore to solve the above-mentioned problems, we have proposed based on printed circuit board (Printed circuit board,
PCB), micro-fluidic palm pyrosequencing techniques of Full automatic digital using pyrosequencing as reaction system.
The content of the invention
It is an object of the invention to provide a kind of palm pyrosequencing system based on digital microfluidic technology, Ke Yishi
The miniaturization of existing pyrosequencing.
Realize the technical scheme is that：A kind of palm pyrosequencing system based on digital microfluidic technology, bag
Include digital microcurrent-controlled chip (1), integrated circuit (2), electromagnetic module (3) and chemiluminescence acquisition module (4)；The numeral miniflow
Control chip (1) and electromagnetic module (3) are connected with integrated circuit (2), and chemiluminescence acquisition module (4) and electromagnetic module (3) are respectively
On the same straight line of both sides above and below digital microcurrent-controlled chip, integrated circuit (2) and chemiluminescence acquisition module (4) connect respectively
It is connected on computer；
The digital microcurrent-controlled chip (1) is bipolar plate structure, including a bottom crown (11), a top crown (12) and
Clearance layer (13), upper and lower pole plate are placed in parallel, and top crown is placed in above bottom crown and by the clearance layer (13) with level altitude
Separate, form the sandwich structure that gap includes drop；
The bottom crown includes printed circuit board (111), dielectric layer (112) and hydrophobic layer (113), printed circuit board
(111) preferably directly generated by circuit board printing technology, as the bottom crown substrate and electrode layer of digital microcurrent-controlled chip, dielectric
Layer (112) is evenly applied to printed circuit board upper surface, and hydrophobic layer (113) is in the upper surface of bottom crown；The top crown is under
It is supreme to include hydrophobic layer (121), ground connection conductive layer (122) and top crown substrate (123) successively；
There is the one layer of effigurate copper of tool (gold-plated) electrode formed by printing speed on printed circuit board (111)
Array realizes carrier as electrode layer, electrode layer as the concrete structure of chip structure, can form single channel, double as needed
Road, multichannel even high-throughout DNA sequencing structure.Typical single channel electrode layer include eight liquid storage tank electrode units (1111),
Eight groups of drop formation channel electrode arrays (1112), droplets mixing (signal generation) electrod-array area (1113), eight groups of drop lifes
It is connected respectively with eight liquid storage tank electrode units (1111) into channel electrode array (1112), and with droplets mixing (signal generation)
Electrod-array area is symmetrically respectively in its both sides centered on (1113).
The electromagnetic module (3) includes controllable magnetic generating means, is manipulated for digital microcurrent-controlled chip in drop
The movement of magnetic bead, mix and gather, and ferromagnetic material used is one kind in permanent magnet, electromagnet；Magnet used control material is
One kind in PMMA, 3D printing material.
The chemiluminescence acquisition module (4) includes aperture, optical filter, photomultiplier, photomultiplier transit tube module, operation
Camera bellows；
When the drop comprising magnetic bead is displaced into correspondence position and produces chemiluminescence signal, optical signal passes sequentially through light
It is detected after circle, optical filter into photomultiplier, produces electric signal and export and show after the collection amplification of photomultiplier transit tube module
Show on computer.
For guard electrode surface, prevent the volatilization of drop, fluorocarbon oil (particularly FC-40) is added in clearance layer, at the same time
Play the role of promoting drop movement.
According to electrode power-up sequence difference, the liquid storage tank electrode unit can generate the big droplet of single electrode, 1.5 times
The various sizes of drop such as electrode size drop and 2 times of electrode size drops, under the gap coating systems of different height, is generated
Droplet size can realize the change from 1uL to 10uL.
In order to ensure the movement of pyrophosphoric acid system drop, Pluronic is added in drop based on digital microfluidic technology
For palm pyrosequencing system F68 to prevent bio-adhesive, addition concentration is 0.01%-0.2%.
The plurality of reagents of pyrosequencing system corresponds respectively to the liquid storage tank electrode unit on digital microcurrent-controlled chip, allusion quotation
Liquid storage tank electrode unit on the digital microcurrent-controlled chip of single channel of type is specially that A, D, E, H- are respectively tetra- kinds of nucleosides containing A, C, G, T
The drop of acid and APS substrates；F- carries the drop of magnetic bead-sequence to be detected-sequencing primer compound；B- includes ATP sulphations
The enzyme system drop of enzyme, luciferase and archaeal dna polymerase；C- pyrosequencing washing lotions；G- scrub raffinates.
A kind of palm pyrosequencing system based on digital microfluidic technology, the typical digital microcurrent-controlled pyrophosphoric acid of single channel
Sequencing steps are as follows：
Liquid with magnetic bead, be placed in the liquid storage tank electrode unit of digital microcurrent-controlled chip by step 1, is transferred to step 2；
The control circuit coordination electrode drive circuit of step 2, integrated circuit (2), carries out the break-make of electrode in a certain order
Electric control, so as to produce a drop from liquid storage tank electrode unit, is moved drop by connected channel electrode array
To droplets mixing (signal generation) electrod-array, step 3 is transferred to；
Step 3, generate respectively according to the operation of step 2 drop containing magnetic bead-sequence to be detected-sequencing primer compound,
Enzyme system drop comprising ATP sulfurylases, luciferase and archaeal dna polymerase and containing a kind of in tetra- kinds of nucleotide of A, C, G, T and
The drop of APS substrates, is transferred to step 4；
Step 4, by generated in step 3 three drops droplets mixing (signal generation) electrod-array position mix, make
Drop is mixed thoroughly；
Step 5, mixing one minute after, magnetic bead is gathered using electromagnetic module；
Step 6, detect optical signal using photomultiplier in real time, and is shown from computer；
Step 7, using electromagnetic module make magnetic bead be separated from drop, and is washed using pyrosequencing washing lotion
Wash, until the unglazed signal output of the drop, waste liquid are drained into scrub raffinate liquid storage tank；
Step 8, return to step 2, so circulation, until detecting that chemiluminescence signal just enters next round and is sequenced.
Compared with prior art, the present invention it is advantageous that：
(1) isolation of interior reactant can prevent the dilution of reactor product, reduce reagent consumption, realize quick and smooth friendship
Stream；
(2) multiple sequencing reaction conditions can be performed with independent parallel (such as the nucleotide sequence each reacted)；
(3) enzyme reaction including DNA polymerisations, ATP reactions of formation and luminescence-producing reaction etc. can be isolated into individually
Drop, be allowed in the time or be spatially separated, reaction condition and kinetics etc. is separately optimized；
(4) digital microcurrent-controlled can realize more directly controls the discrete microlayer model of generation with a greater degree of space-time,
There is advantage first in the sky in full automatic treatment and fully-integratedization.
Digital microcurrent-controlled palm pyrosequencing system be one kind can be controlled by external circuit realize on chip it is complete from
The reaction platform of dynamic microlayer model pyrosequencing.Compared with traditional pyrosequencing techniques, its reaction cost can be ignored not
Meter, reaction time greatly shorten, and reaction rate greatly improves, while again without complicated operation skill.Therefore, can be adapted for
In, low side sequencing need scientific research institutions it is even personal, its application prospect is boundless.
Brief description of the drawings
Fig. 1 is the overall structure diagram of the palm pyrosequencing system of the invention based on digital microfluidic technology.
Fig. 2 is the structure diagram of the pyrosequencing chip of the invention based on digital microfluidic technology；Wherein (a) is it
Cross-sectional view, (b) are the digital microcurrent-controlled chip plane structure diagram of single channel.
Fig. 3 is the schematic diagram that the present invention realizes pyrosequencing on the digital microcurrent-controlled chip of Fig. 2, with typical single channel
Exemplified by digital microcurrent-controlled chip, wherein A, D, E, H- are respectively the drop containing tetra- kinds of nucleotide of A, C, G, T and APS substrates；F- bands
There is the drop of magnetic bead-sequence to be detected-sequencing primer compound；B- includes ATP sulfurylases, luciferase and archaeal dna polymerase
Enzyme system drop；C- pyrosequencing washing lotions；G- scrub raffinates；I, J, K are respectively that the corresponding big drop of liquid storage tank is generated
Droplet；L is by the reaction drop that is formed after tri- droplets mixing of I, J, K.
Embodiment
Below in conjunction with the accompanying drawings by taking single channel DNA pyrosequencings on digital microcurrent-controlled chip as an example, to the present invention into advance one
Walk careful description.
As shown in Figure 1, a kind of palm pyrosequencing system based on digital microfluidic technology, including digital microcurrent-controlled core
Piece (1), integrated circuit (2), electromagnetic module (3) and chemiluminescence acquisition module (4)；The digital microcurrent-controlled chip (1) and electricity
Magnetic module (3) is connected with integrated circuit (2), and chemiluminescence acquisition module (4) and electromagnetic module (3) are respectively positioned at digital microcurrent-controlled
Up and down on the same straight line of both sides, integrated circuit (2) and chemiluminescence acquisition module (4) are respectively connected on computer chip.
As shown in Fig. 2 (a, b), the digital microcurrent-controlled chip (1) is bipolar plate structure, including a bottom crown (11),
One top crown (12) and clearance layer (13), upper and lower pole plate are placed in parallel, and top crown is placed in above bottom crown and by with fixation
The clearance layer (13) of height is separated, and forms the sandwich structure that gap includes drop；
The bottom crown includes printed circuit board (111), dielectric layer (112) and hydrophobic layer (113), printed circuit board
(111) directly generated by circuit board printing technology, as the bottom crown substrate and electrode layer of digital microcurrent-controlled chip, dielectric layer
(112) printed circuit board upper surface is evenly applied to, hydrophobic layer (113) is in the upper surface of bottom crown；The top crown from down toward
On include hydrophobic layer (121), ground connection conductive layer (122) and top crown substrate (123) successively；
There is the one layer of effigurate copper of tool (gold-plated) electrode formed by printing speed on printed circuit board (111)
Array includes eight liquid storage tank electrode units (1111), eight groups of drop formation channel electrode arrays as electrode layer, the electrode layer
(1112), droplets mixing (signal generation) electrod-array area (1113), eight groups of drop formation channel electrode arrays (1112) are respectively
It is connected with eight liquid storage tank electrode units (1111), and it is right centered on droplets mixing (signal generation) electrod-array area (1113)
Title is respectively in its both sides.
The digital microcurrent-controlled manufacture craft of the present invention is as follows：
The making of bottom crown：
(a) bottom crown substrate is made of electronics industry PCB processing technologys, and it is heavy with specific structure to be formed in substrate
Gold electrode layer；
(b) dielectric layer material is with high-k, such as insulating materials that thickness is uniform, breakdown characteristics are strong, photoetching
Glue, PDMS, Paralyene-C, Si3N4Deng material, generally formed by way of spin coating or deposition；
(c) hydrophobic layer material is generally Teflon, is formed by spin coating annealing process.
The making of top crown：
(a) top crown substrate is any insulation transparent material, such as glass；
(b) material that conductive selection light transmittance is high, observability is good is grounded, such as ITO, forms by depositing；
(c) top crown hydrophobic material is generally consistent with bottom crown hydrophobic material, generally Teflon, passes through spin coating lehr attendant
Skill is formed.
Separate between upper bottom crown using with certain thickness clearance layer, with droplet formation sandwich knot therein
Structure, drop are introduced by the top crown position perforate above corresponding bottom crown liquid storage tank electrode by auto injection.Sample introduction is completed
Afterwards, digital microcurrent-controlled chip is subjected to oil sealing, plays the role of isolation pollution and promote drop movement.
Then by the control circuit coordination electrode drive circuit of integrated circuit (2), the logical of electrode is carried out in a certain order
Power-off control, so as to produce a drop from liquid storage tank electrode unit, is moved drop by connected channel electrode array
Move to droplets mixing (signal generation) electrod-array.According to operation, generation is compound containing magnetic bead-sequence-sequencing primer to be detected respectively
The drop of thing, the enzyme system drop comprising ATP sulfurylases, luciferase and archaeal dna polymerase and containing tetra- kinds of nucleotide of A, C, G, T
The middle a kind of and drop of APS substrates.By generated in step 3 three drops in droplets mixing (signal generation) electrod-array position
Mixing, is mixed thoroughly drop；After mixing one minute, magnetic bead is gathered using electromagnetic module；Use photomultiplier
Detection optical signal in real time, and shown from computer；Magnetic bead is separated from drop using electromagnetic module, and use
Pyrosequencing washing lotion is washed, until the unglazed signal output of the drop, waste liquid are drained into scrub raffinate liquid storage tank；Return to step
Rapid 2, so circulation, until detecting that chemiluminescence signal just enters next round and is sequenced.
Experiments verify that the sequencing procedure detersive efficiency is high, the reaction time is short, and often wheel sequencing only needs 3 minutes, and length is sequenced
40-60 base can be reached, accuracy rate is up to 100%, and a kind of immediately quick palm sequenator can be used as to use.
Applicant states that the present invention illustrates the detailed features and method detailed of the present invention by above-described embodiment, but
The invention is not limited in above-mentioned detailed features and method detailed, that is, does not mean that the present invention has to rely on above-mentioned detailed features
And method detailed could be implemented.Person of ordinary skill in the field should be understood that any improvement in the present invention, to this hair
Bright selection of equivalence replacement, concrete mode for selecting component etc., belongs within protection scope of the present invention and the open scope.
Claims (8)
- A kind of 1. palm pyrosequencing system based on digital microfluidic technology, it is characterised in that：Including digital microcurrent-controlled core Piece (1), integrated circuit (2), electromagnetic module (3) and chemiluminescence acquisition module (4)；The digital microcurrent-controlled chip (1) and electricity Magnetic module (3) is connected with integrated circuit (2), and chemiluminescence acquisition module (4) and electromagnetic module (3) are respectively positioned at digital microcurrent-controlled Up and down on the same straight line of both sides, integrated circuit (2) and chemiluminescence acquisition module (4) are respectively connected on computer chip；The digital microcurrent-controlled chip (1) is bipolar plate structure, including a bottom crown (11), a top crown (12) and gap Layer (13), upper and lower pole plate are placed in parallel, and top crown is placed in above bottom crown and is separated by the clearance layer (13) with level altitude Come, form the sandwich structure that gap includes drop；The bottom crown includes printed circuit board (111), dielectric layer (112) and hydrophobic layer (113), and printed circuit board (111) is made For the bottom crown substrate and electrode layer of digital microcurrent-controlled chip, dielectric layer (112) is evenly applied to printed circuit board upper surface, dredges Water layer (113) is in the upper surface of bottom crown；The top crown includes hydrophobic layer (121), ground connection conductive layer successively from bottom to up (122) and top crown substrate (123)；On printed circuit board (111) there is electrod-array to include eight liquid storage tank electrode units as electrode layer, the electrode layer (1111), eight groups of drop formation channel electrode arrays (1112), droplets mixing electrod-array area (1113), eight groups of drop formations lead to Road electrod-array (1112) is connected with eight liquid storage tank electrode units (1111) respectively, and with droplets mixing electrod-array area (1113) its both sides is symmetrically respectively in centered on.
- 2. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that：Institute Stating electromagnetic module (3) includes controllable magnetic generating means, for digital microcurrent-controlled chip manipulate the movement of magnetic bead in drop, Mix and gather, ferromagnetic material used is one kind in permanent magnet, electromagnet；Magnet control material used is PMMA, 3D printing One kind in material.
- 3. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that：Institute Stating chemiluminescence acquisition module (4) includes aperture, and optical filter, photomultiplier, photomultiplier transit tube module, operates camera bellows；When the drop comprising magnetic bead is displaced into correspondence position and produces chemiluminescence signal, optical signal passes sequentially through aperture, filter It is detected after mating plate into photomultiplier, produces electric signal and exported after the collection amplification of photomultiplier transit tube module and be shown to meter On calculation machine.
- 4. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that：For Guard electrode surface, the volatilization for preventing drop, add oil in clearance layer, while play the role of promoting drop movement.
- 5. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that：According to According to the difference of electrode power-up sequence, the liquid storage tank electrode unit can generate the big droplet of single electrode, 1.5 times of electrode size liquid The various sizes of drop such as drop and 2 times of electrode size drops, under the gap coating systems of different height, generating droplet size can Realize the change from 1uL to 10uL.
- 6. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that： Palm pyrosequencing system F68s of the Pluronic based on digital microfluidic technology is added in drop to prevent bio-adhesive, Addition concentration is 0.01%-0.2%.
- 7. the palm pyrosequencing system according to claim 1 based on digital microfluidic technology, it is characterised in that：It is burnt The plurality of reagents of phosphoric acid sequencing system corresponds respectively to eight liquid storage tank electrode units on digital microcurrent-controlled chip, be specially A, D, E, H- are respectively the drop containing tetra- kinds of nucleotide of A, C, G, T and APS substrates；F- carries magnetic bead-sequence-sequencing primer to be detected The drop of compound；B- includes the enzyme system drop of ATP sulfurylases, luciferase and archaeal dna polymerase；C- pyrosequencings are washed Liquid；G- scrub raffinates.
- 8. a kind of pyrosequencing method, it uses the palm pyrophosphoric acid based on digital microfluidic technology described in claim 1 Sequencing system, includes the following steps：Liquid with magnetic bead, be placed in the liquid storage tank electrode unit of digital microcurrent-controlled chip by step 1, is transferred to step 2；The control circuit coordination electrode drive circuit of step 2, integrated circuit (2), the break-make for carrying out electrode in a certain order are automatically controlled System, so as to produce a drop from liquid storage tank electrode unit, liquid is moved to by connected channel electrode array by drop Mixed electrode array is dripped, is transferred to step 3；Step 3, according to the operation of step 2 generate the drop containing magnetic bead-sequence to be detected-sequencing primer compound, include respectively The enzyme system drop of ATP sulfurylases, luciferase and archaeal dna polymerase and containing a kind of in tetra- kinds of nucleotide of A, C, G, T and APS bottoms The drop of thing, is transferred to step 4；Step 4, by generated in step 3 three drops droplets mixing electrod-array position mix, drop is thoroughly mixed It is even；Step 5, mixing one minute after, magnetic bead is gathered using electromagnetic module；Step 6, detect optical signal using photomultiplier in real time, and is shown from computer；Step 7, using electromagnetic module make magnetic bead be separated from drop, and is washed using pyrosequencing washing lotion, directly To the unglazed signal output of the drop, waste liquid is drained into scrub raffinate liquid storage tank；Step 8, return to step 2, so circulation, until detecting that chemiluminescence signal just enters next round and is sequenced.
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| CN110170341A (en) * | 2018-11-23 | 2019-08-27 | 复旦大学 | The digital microcurrent-controlled device of particle high throughput sorting is realized using surface acoustic wave techniques |
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| CN109868215A (en) * | 2019-02-02 | 2019-06-11 | 苏州奥素液芯电子科技有限公司 | A kind of detection system, detection method and device, computer readable storage medium |
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| CN113634174A (en) * | 2021-08-24 | 2021-11-12 | 聚光科技(杭州)股份有限公司 | Microchannel-based liquid mixing chip and method |
| CN114113010A (en) * | 2021-10-28 | 2022-03-01 | 山东师范大学 | Automatic bacteria detection system and method based on digital micro-fluidic |
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