CN107764994A - The device spread using magnetic bead control DNA in the sequencing experiment of solid nano hole - Google Patents
The device spread using magnetic bead control DNA in the sequencing experiment of solid nano hole Download PDFInfo
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- CN107764994A CN107764994A CN201711161341.7A CN201711161341A CN107764994A CN 107764994 A CN107764994 A CN 107764994A CN 201711161341 A CN201711161341 A CN 201711161341A CN 107764994 A CN107764994 A CN 107764994A
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- 239000011324 bead Substances 0.000 title claims abstract description 55
- 238000002474 experimental method Methods 0.000 title claims abstract description 20
- 238000012163 sequencing technique Methods 0.000 title description 12
- 239000007787 solid Substances 0.000 title description 7
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 230000033001 locomotion Effects 0.000 claims abstract description 19
- 238000007672 fourth generation sequencing Methods 0.000 claims abstract description 12
- 238000009792 diffusion process Methods 0.000 claims abstract 10
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 9
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 9
- 239000000956 alloy Substances 0.000 claims description 3
- 229910045601 alloy Inorganic materials 0.000 claims description 3
- 239000004519 grease Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 3
- 238000004804 winding Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000005672 electromagnetic field Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- UGKDIUIOSMUOAW-UHFFFAOYSA-N iron nickel Chemical compound [Fe].[Ni] UGKDIUIOSMUOAW-UHFFFAOYSA-N 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 230000005415 magnetization Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical group C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Manipulator (AREA)
Abstract
本发明公开了一种利用磁珠控制DNA在固态纳米孔测序实验中扩散的装置，其特征在于，设有磁镊系统，所述磁镊系统包括操作台、显微镜、磁针和控制磁针移动轨迹的微操作器，所述操作台上设有液池，显微镜镜头对准所述液池，磁针在靠近其尖端的位置设有线圈，通电之后会在液池范围内产生磁场，通过移动磁针调节磁场的位置，进而控制液池中磁珠的移动。本发明通过通电产生磁场的磁针控制液池中的磁珠移动，对磁珠的吸引释放自如，通过调节电流大小即可方便地调整磁场大小，操作方便，易于实现，且控制精准，使用效果好，适合推广使用。
The invention discloses a device for controlling the diffusion of DNA in a solid-state nanopore sequencing experiment by using magnetic beads. Micromanipulator, the operating table is provided with a liquid pool, the microscope lens is aimed at the liquid pool, and the magnetic needle is provided with a coil near its tip. After power on, a magnetic field will be generated within the range of the liquid pool, and the magnetic field can be adjusted by moving the magnetic needle position, thereby controlling the movement of the magnetic beads in the liquid pool. The invention controls the movement of the magnetic beads in the liquid pool by means of a magnetic needle that generates a magnetic field through energization, and the attraction to the magnetic beads can be released freely. The size of the magnetic field can be conveniently adjusted by adjusting the magnitude of the current. The operation is convenient, easy to implement, and the control is accurate and the use effect is good , suitable for promotional use.
Description
Technical field
The invention belongs to bio-medical technology field, is specially that one kind is sequenced using magnetic bead control DNA in solid nano hole
The device spread in experiment.
Background technology
Gene sequencing technology is not only that the fundamental biological knowledge researchs such as announcement and the gene expression regulation of hereditary information provide weight
Data are wanted, and are also played an important role in the application study such as gene diagnosis and gene therapy.Since 1977 first
Since being come out for sequencing technologies, by the effort of thirties years, gene sequencing technology had been achieved for major progress, first
On the basis of generation and second generation sequencing technologies, the sequencing technologies of future generation with the characteristics of single-molecule sequencing arise at the historic moment.Nano-pore
Sequencing technologies are considered as that it is not only the possibility to current DNA most possibly as one of technology of gene sequencing of future generation
The cost of sequence analysis substantially reduces, and analyze speed greatly improves, will also promote based on the personalized medicine of genome-based technologies to
Reality is stepped further, so as to cause the once change of medical field.Solid nano hole sequencing technologies are as emerging forth generation DNA
Sequencing technologies, there is the characteristics of low cost, high reading is long, easy of integration, have very big advantage in cost, speed etc..
The content of the invention
The technical purpose of the present invention is to provide a kind of be sequenced using magnetic bead control DNA in solid nano hole in experiment and spread
Device, its technical scheme is：
A kind of device for controlling DNA to be spread in the sequencing experiment of solid nano hole using magnetic bead, it is characterised in that be provided with
Magnetic tweezers system, the magnetic tweezers system includes operating desk, microscope, needle and the micromanipulator for controlling needle motion track, described
Operating desk is provided with liquid pool, liquid pool described in microscope alignment lenses, and needle is being provided with coil close to its sophisticated position, is being powered
Magnetic field can be produced in the range of liquid pool afterwards, the position in magnetic field is adjusted by mobile needle, and then controls the shifting of magnetic bead in liquid pool
It is dynamic.
On the basis of such scheme, further improve or preferable scheme also includes：
The a diameter of 2mm of needle, the cone height where tip is 7mm, and coil winding is apart from the cone 10mm
Within scope.
In normal experiment, needle normally only controls one or several magnetic beads, thus the diameter at cone tip must and magnetic
Pearl is in the same order of magnitude, described sophisticated a diameter of 10 μm or so.
The coil repeats coiling on needle using diameter 0.04mm fine enamelled wire and formed, and the number of turn of coiling is
500, all-in resistance is 50 Ω, and the enamel-covered wire of the both ends of fine enamelled wire then with a diameter of 0.2mm is connected, the electric current applied to coil
For 60mA.
The needle is made of 1j85 alloy bars, i.e., nickel content is 85% iron nickel soft magnetic materials, manufactured needle tool
There are low-coercivity and high magnetic susceptibility, be easy to magnetization and be also easy to demagnetize.
The liquid pool is attached on slide by the PMMA plates provided with through hole and formed.Preferably, the thickness of PMMA plates is set to
2mm, the diameter of through hole are set to 20mm.
The binding face of the PMMA plates and slide is coated with vacuum grease around through hole.
The magnetic tweezers system is provided with double needles, and the motion of magnetic bead is controlled by double needles.
Beneficial effect:
The needle that the present invention produces magnetic field by being powered controls the magnetic bead in liquid pool to move, and the attraction to magnetic bead is released from
Such as, magnetic field size can be easily adjusted by adjusting size of current, it is easy to operate, it is easy to accomplish, and control accurate, use
Effect is good, is adapted to promote the use of.
Brief description of the drawings
Fig. 1 is the operating diagram of the present invention；
Fig. 2 is liquid pool structural representation；
Fig. 3 is the structural representation of needle and liquid pool；
Fig. 4 is motion of the magnetic bead in single magnetic field；
Fig. 5 is Fig. 4 partial enlarged drawing；
Fig. 6 is motion of the magnetic bead in double magnetic fields；
Fig. 7 is Fig. 6 partial enlarged drawing；
Fig. 8 is needle tip axis magnetic field distribution；
Fig. 9 is needle tip axis magnetic bead stress point.
Embodiment
For the technical scheme and operation principle that the present invention is furture elucidated, below in conjunction with the accompanying drawings with specific embodiment pair
The present invention is described further.
A kind of device for controlling DNA to be spread in the sequencing experiment of solid nano hole using magnetic bead, provided with magnetic tweezers system, institute
Stating magnetic tweezers system includes operating desk, microscope, pump type vibration isolators, screening cage, needle and the microoperation for controlling needle motion track
The parts such as device.
Operating desk is respectively arranged on the left side and the right side a micromanipulator, and the micromanipulator is provided with various dimensions displacement mechanism, left
Right two needles are clamped on the end effector mechanism of corresponding micromanipulator respectively, on microscope alignment lenses operating desk
Liquid pool 2.
The needle 1 is being provided with coil close to its sophisticated position, after needle 1 is probeed into liquid pool 2, to coil electricity, magnetic
Pin 1 can produce magnetic field in the range of liquid pool 2, and the position in the regulation of needle 1 magnetic field, and then controllable liquid are moved by micromanipulator
The movement of magnetic bead in pond 2.In the present embodiment, the needle 1 is made of 1j85 alloy bars, and a diameter of a of needle is 2mm, length
B is 40mm, and the cone height c where tip is 7mm, and a diameter of 2.8 μm of magnetic bead, the sophisticated diameter of cone is with magnetic bead same
One order of magnitude, about 10 μm.
The coil winding exempts to weld thin enamel-cover in the scope within cone 10mm, coil using diameter 0.04mm's
Line repeats coiling multilayer and formed, and the number of turn of coiling is 500, and all-in resistance is 50 Ω, and upper diameter is welded at the both ends of the fine enamelled wire
For 0.2mm enamel-covered wire, during work, the electric current applied to coil is 60mA.
In view of the difficulty that liquid pool is processed on slide is high, in the present embodiment, we use PMMA (polymethylacrylic acid
Methyl esters) liquid pool material is used as, the through hole that diameter f is 20mm is processed on the rectangle PMMA plates that thickness is 2mm, in through hole week
Vacuum grease is smeared in the PMMA plate faces enclosed, and ultra-thin slide is sticked into the face, that is, constitutes the liquid pool.
Single needle experiment：We first produce magnetic field with single needle in liquid pool, and solution is the mM of 0.1M KCl in 10
Tris-HCl, 1mM EDTA buffer (pH=8.0) and 0.1%Tween 20, pass to 60mA electric current, and shown with CCD
Micro mirror records experiment video, then extracts video frames to layers with photoshop, obtains the video photography at intervals of 0.3s,
Whole flow process such as Fig. 4, (a) needle are stood in the solution, and (b) needle tip has attracted a magnetic bead, are magnetic bead in circle, (c)
Motion of second magnetic bead in magnetic field, (d) second magnetic bead are attracted on needle.Wherein, after powered up 1 minute or so
It is found that needle starts to attract magnetic bead, and subsequent, as Fig. 4 (c) has found that a magnetic bead close to needle, is by 0.3s
It has been attracted on needle, has illustrated to be significantly stronger than other spaces by subapical magnetic field.Due to the shape and coil winding of needle
During the inevitable reason such as uneven coiling, we can use ANSYS electromagnetic modules to carry out the magnetic field of ideal conditions
Analyze to explain its phenomenon.By figure centre circle part amplification such as Fig. 5 (e).
Magnetic simulation:We simulate to Distribution of Magnetic Field caused by two energization needle surrounding spaces, and simulated conditions are such as
Shown on, two needle magnetic directions different (N-S) and identical (N-N) situation, the N-S on the axis of needle tip are simulated respectively
Situation magnetic direction can be superimposed, and N-N directions magnetic direction can be offset, but from the point of view of analog result, two needle tips compared with
In small scope, magnetic field intensity does not make a big difference, illustrates that magnetic field can't be to another needle caused by a needle
Produce excessive influence, and this influences to can be ignored for the sophisticated magnetic field of itself, and this exactly we control magnetic
The main region of pearl.In addition we are also simulated to magnetic field caused by single needle, in needle tip a small range
As a result the result with the simulation of double needles has no significant difference, it was demonstrated that we can use list in the space in double needle magnetic fields
Individual needle controls the motion for the magnetic bead that this needle attracted, without being influenceed by another needle.
As shown in figure 8, the numerical value for some the specific magnetic field intensity B being also obtained in the present embodiment on needle axis, in view of
The accuracy of ANSYS network analogs, the numerical value at interval of 2.5 μm is can only obtain, it is apart 50 μm to calculate respectively with tip, 60 μ
M, 70 μm, the Distribution of Magnetic Field of 80 μm of four kinds of spacing, as shown in fig. 7, coordinate x=0 represents the center of axis, both ends are respectively pin
Sharp position, it can be seen that there is great magnetic field in the place close to needle point, and with the increase of distance, magnetic field intensity is drastically
Decline.Do not considering the influence of the coefficient of viscosity of water and magnetic bead in gravitational field in itself, bead diameter is 2.8 μm, and inside is
Fe2O3, outside is PDMS, is to be uniformly distributed, therefore motion of the magnetic bead in magnetic field can be seen as particle by we, only be examined
Consider the motion of barycenter, stress of the magnetic bead in magnetic field is：
Wherein, m is the magnetic moment of little magnetic bead, and read up the literature to obtain m=1.42 × 10-13Am2,For magnetic field gradient, i.e. Fig. 8 curves
Slope, thus calculate to obtain the stress of magnetic bead in the solution such as Fig. 9, it can be seen that close to needle region in magnetic bead stress phase
When big, and it is far longer than the power 50pN required for DNA stretchings, the diameter of the scope in this region also than magnetic bead in itself is big, and
In intermediate region, the bigger magnetic field gradient of needle distance is smaller, almost levels off to 0, therefore from Fig. 9 it can also be seen that this region
The stress of magnetic bead is substantially equal to 0, therefore we can control the motion of magnetic bead with double needles, so as to further to its institute
The DNA of binding carries out the control of via motion, and double needle experimentations are as shown in Figure 6, Figure 7.
The present embodiment device can realize the high-precision control of micro-meter scale and various dimensions using narishige clamping needle
Needle, so as to further control movement locus and its position of magnetic bead.Only narishige and the operation being arranged in screening cage
Platform etc. does not interfere with the device of electromagnetic field, and power supply is placed in outside screening cage, and attraction release of the needle to magnetic bead is freely, no excessive
External factor interference, can easily adjust magnetic field size by adjusting size of current, pass through CCD microscopie unit energy
It is visually known experimental result.The present embodiment can in the motion of the scope control magnetic bead of 50 μm (λ DNA length be 46 μm),
And then the DNA molecular that biotin molecules are connected with magnetic bead is stretched through, suppress its warm-up movement in the solution.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, this hair
Bright claimed scope is by appended claims, specification and its equivalent thereof.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711161341.7A CN107764994A (en) | 2017-11-21 | 2017-11-21 | The device spread using magnetic bead control DNA in the sequencing experiment of solid nano hole |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711161341.7A CN107764994A (en) | 2017-11-21 | 2017-11-21 | The device spread using magnetic bead control DNA in the sequencing experiment of solid nano hole |
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| CN107764994A true CN107764994A (en) | 2018-03-06 |
Family
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| CN201711161341.7A Pending CN107764994A (en) | 2017-11-21 | 2017-11-21 | The device spread using magnetic bead control DNA in the sequencing experiment of solid nano hole |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111455034A (en) * | 2020-04-09 | 2020-07-28 | 南京罗岛纳米科技有限公司 | Single molecule detection method and system based on solid-state nanopore mechanism |
| CN114522649A (en) * | 2022-02-15 | 2022-05-24 | 浙江大学 | Acoustic particle capturing and track control method based on magnetofluid reconstruction |
| US11892445B2 (en) | 2021-12-08 | 2024-02-06 | Western Digital Technologies, Inc. | Devices, systems, and methods of using smart fluids to control translocation speed through a nanopore |
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| US11892445B2 (en) | 2021-12-08 | 2024-02-06 | Western Digital Technologies, Inc. | Devices, systems, and methods of using smart fluids to control translocation speed through a nanopore |
| CN114522649A (en) * | 2022-02-15 | 2022-05-24 | 浙江大学 | Acoustic particle capturing and track control method based on magnetofluid reconstruction |
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