CN102352312A - Integrated system of detection and injection of getting gene to cell and technology thereof - Google Patents
Integrated system of detection and injection of getting gene to cell and technology thereof Download PDFInfo
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- CN102352312A CN102352312A CN2011102040245A CN201110204024A CN102352312A CN 102352312 A CN102352312 A CN 102352312A CN 2011102040245 A CN2011102040245 A CN 2011102040245A CN 201110204024 A CN201110204024 A CN 201110204024A CN 102352312 A CN102352312 A CN 102352312A
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Abstract
The invention discloses a new integrated system of the detection and the injection of getting a gene to a cell and a preparation technology thereof. The main content of the preparation technology is characterized in that: a cantilever beam with internal microchannels and a nanotip are used as basic units to construct the system for the detection and the evaluation of getting the gene to the cell, that is, methods of etching and bonding are used to construct the cantilever beam with the internal microchannels, methods of electron beam direct writing, overlaying and the like are used to manufacture the nanotip, the nanotip is communicated with the cantilever beam, the other end of the cantilever beam is connected with a micro-injection control system, and above prepared system can be used for the detection and the injection through the further integration with an atomic force microscopic system.
Description
Technical field
The present invention relates to a kind of gene and get into cell detection and injection integral system and manufacturing process thereof, with regard to its function, this system can realize detecting and injection simultaneously.With regard to its structure, this system is made up of nano-probe and micron pipeline.
Background technology
Some major diseases such as acquired immune deficiency syndrome (AIDS), hepatitis, malignant tumour, genetic diseases, traditional treatment not only spend huge, bring huge economical load for society and family, and result of treatment are undesirable.These diseases can be fundamentally treated in gene therapy; The recurrence of tumour can not appear; The lifelong puzzlement that hepatitis virus, genetic diseases are brought to the patient; Thereby really reach therapeutic purpose; Having caused worldwide extensive concern, is the important directions and the inevitable development trend of disease treatment from now on research.
It is that the matter of utmost importance of gene therapy is again a key problem that gene is sent into cell, and the gene main method of sending into cell has at present:. coprecipitation of calcium phosphate, electroporation, DEAE-dextran and polybrene, mechanical process and lipofectamine reagent.The common issue with that above method exists: provide gene to get into the environment of cell; Can't control and detect the entering process, gene gets into cell and has very big randomness, has influenced result of treatment; Be the difficult problem that present gene therapy research occurs, make gene therapy to further investigate.
Inject gene to present mechanical process, do not handle accurately, be mostly to lean on the experience and the detection in later stage to judge that gene gets into the effect of cell, ignored the process in early stage that gene gets into cell for gene.What has promptly taken place on cytolemma on earth changed the degree of this variation.How to control and utilize this variation, the monitoring of this variation of cytolemma and evaluation.This patent proposes to make and has function of injecting nanometer detection probe concurrently; The accurate importing of realization gene entering cell and the integral system of monitoring are integrated; Completion is controlled injection gene in cell; Utilize the intuitive of AFM and the variation that directly obtains the Performance Detection surface of cell membrane of frictional force; Carry out mark at surface of cell membrane; The zone of action is accurate to nanometer range; Get rid of accurately operability and uncontrollability that conventional method exists; Disclose the mechanism that gene gets into cell; The mechanism problem in the present gene therapy research was clarified in the pairing biology performance of the variation of cell membrane when the refinement gene got into cell.
Summary of the invention
In order to overcome defective or the deficiency that traditional method exists, the object of the present invention is to provide a kind of new gene to get into the detection injection integral system manufacturing process of cell.
Technical scheme of the present invention is achieved in that
A kind of gene gets into cell detection and injection integral system; Comprise laser generator, speculum, photoelectric detector in the atomic force microscope, injection pipeline, micro-Controlling System are connected with the atomic force microscope probe of injection pipeline composition in probe, the probe in probe, the probe; The laser that laser generator sends is beaten on the socle girder of probe; Read by photoelectric detector through speculum; Probe is connected with an end of socle girder; Pipeline is arranged in the socle girder; Pipeline in probe two side hole and the socle girder is connected, and the other end of socle girder connects micro-Controlling System, and the probe tip diameter is 8nm～10nm; Long 20～30 μ m in the square-section of socle girder internal pipeline, wide 1 μ m.
The micro-injection system comprises deceleration device, micro pump, Controlling System and signal detection again.
A kind of gene gets into cell detection and injection integral system preparation technology, and concrete steps are following:
1) spin coating photoresist material thickness 0.4～0.9 μ m on the silicon chip made public 45 seconds, and etching obtains groove structure, formed the socle girder with internal channel structure through bonding techniques then;
2) at socle girder one end spin coating electron beam resist thickness 0.5 μ m, etching obtains the needle point of diameter 8～10 nanometers, makes positioning reference, and needle point both sides cover carves the circular hole of diameter 200～300nm;
3) micro-Controlling System is regulated micro pump through Controlling System, is realized the accurate entering cell of microfluid by deceleration device.
Described detection injection integral system, this system essentially consist unit is probe and the socle girder with internal passages.
The material that injection pipeline is adopted in probe, the probe is silicon or silicon nitride.
The cell that is adopted is tumour cell or normal cell.
This innovative technology compared with prior art has following advantage:
1. can accurately make probe, be easy to scale operation with internal channel structure; Can inject through the socle girder internal passages, realize detecting integrated with injection through probe.
2. be convenient to detection and monitoring that gene gets into, operation can be controlled and guide, the best site that can accurately find gene to get into.
3. being more suitable for studying gene gets into the mechanism of cell and accurately estimates cytogenetic variation.
Description of drawings
Fig. 1 is a system of the present invention ultimate principle structure iron.
Fig. 2 has the probe system of passage.
The manufacturing process of Fig. 3 probe passage.
The manufacturing process of Fig. 4 probe tip two wing passages.
To combine accompanying drawing that the structure and the technical process of this support are detailed below.
Embodiment
A kind of gene gets into cell detection and injection manufacturing process, it is characterized in that: nano level probe is communicated with socle girder forms a detection injecting systems; Wherein:
There is passage socle girder inside, and an end connects micro-Controlling System; Probe can be accomplished and detect and imaging, finds the position of specificity binding site such as acceptor, and gene can accurately be injected in the cell from this position then.
Described probe, socle girder material adopt silicon, silicon nitride etc.
The manufacturing process of said system is characterized in that: comprise the following steps:
1) at first utilize photoetching, etching, the bonding processing and manufacturing has the socle girder of internal passages, and wherein passage length is between 140～170 μ m, and Entry Interface is a rectangle, and is long at 20～30 μ m, wide 1 μ m;
2) utilize electron-beam direct writing on the socle girder, etching, the circular hole that alignment forms probe and probe both sides links to each other with passage.
3) the socle girder the other end connects micro-Controlling System.
Fig. 1 is that system uses basic block diagram.This system has internal channel structure, is used to accomplish function of injection, can also detect and form images, and can be connected control and the evaluation of accomplishing gene entering cell with the atomic power microscopic system, forms bigger detection system.
Fig. 2 comprises the probe 13 that has passage, silicon chip 14 and 17, passage 15 and 16.
Fig. 3 and Fig. 4 are respectively the manufacturing process of probe passage and probe tip two wing passages.
Concrete technology combines accompanying drawing 3,4 explanations as follows:
At first utilize computer aided design (CAD) (CAD) software (like PROE, UG etc.) design socle girder internal circulation pipeline and probe structure, socle girder internal passages length is between 140～170 μ m, and Entry Interface is a rectangle, and is long at 20～30 μ m, wide 1 μ m; The probe tip diameter is to design the used mask plate of etching between 8nm～10nm then.
Fig. 3 silicon chip applies photoresist material 18, and exposure 19 develops 20, and etching 21 obtains having the silicon chip 22 of groove structure, accomplishes the socle girder with internal passages through the bonding encapsulation.
Fig. 4 probe material adopts the silicon chip of 100 metallographics, applies electron beam resist 23, carries out electron-beam direct writing 24; Etch probe 25, make positioning reference 26, confirm the circular hole 27 of needle point both sides; Cover carves the circular hole 28 of needle point both sides, and etching obtains the socle girder the other end and is connected with micro-Controlling System.
It is even to guarantee to contact strength during etching, during the demoulding, should carefully peel off, and damages internal channel structure and needle point when avoiding the demoulding.
Because the gene size is minimum, be 0.34nm like the length of a pair of base pair, 21～22 base sequences can be sent into the cell change gene at present.And this base sequence length of 21～22 is about 6～7 nanometers, therefore will get into cell to gene and operate and detect cytogenetic variation simultaneously, and existing technique means can't reach.At first make and have function of injecting nanometer detection probe concurrently; The accurate importing of realization gene entering cell and the integral system of monitoring are integrated; Completion is controlled injection gene in cell; Utilize the intuitive of AFM and the variation that directly obtains the Performance Detection surface of cell membrane of frictional force; Carry out mark at surface of cell membrane; The zone of action is accurate to nanometer range; Get rid of accurately operability and uncontrollability that conventional method exists; Disclose the mechanism that gene gets into cell; The mechanism problem in the present gene therapy research was clarified in the pairing biology performance of the variation of cell membrane when the refinement gene got into cell.
Claims (6)
1. a gene gets into cell detection and injection integral system; Comprise laser generator (1), speculum (2), photoelectric detector (3) in the atomic force microscope, injection pipeline (5), micro-Controlling System (6) are connected with the atomic force microscope probe of injection pipeline (5) composition in probe (4), the probe in probe (4), the probe; The laser that laser generator (1) sends is beaten on the socle girder of probe (4); Read by photoelectric detector through speculum (2); It is characterized in that; Probe (4) is connected with an end of socle girder, and pipeline is arranged in the socle girder, and the pipeline in probe (4) two side hole and the socle girder is connected; The other end of socle girder connects micro-Controlling System (6); The probe tip diameter is 8nm～10nm, long 20～30 μ m in the square-section of socle girder internal pipeline, wide 1 μ m.
2. a kind of gene according to claim 1 gets into cell detection and injection integral system, it is characterized in that the micro-injection system comprises deceleration device (7), micro pump (8), Controlling System (9) and signal detection (10) again.
3. a gene gets into cell detection and injection integral system preparation technology, it is characterized in that concrete steps are following:
1) spin coating photoresist material thickness 0.4～0.9 μ m on the silicon chip made public 45 seconds, and etching obtains groove structure, formed the socle girder with internal channel structure through bonding techniques then;
2) at socle girder one end spin coating electron beam resist thickness 0.5 μ m, etching obtains the needle point of diameter 8～10 nanometers, makes positioning reference, and needle point both sides cover carves the circular hole of diameter 200～300nm;
3) micro-Controlling System (6) is regulated micro pump through Controlling System, is realized the accurate entering cell of microfluid by deceleration device.
4. a kind of gene according to claim 3 gets into cell detection and injection integral system preparation technology, it is characterized in that, described detection injection integral system, and this system essentially consist unit is probe and the socle girder with internal passages.
5. system according to claim 3 is characterized in that, the material that injection pipeline (5) is adopted in probe (4), the probe is silicon or silicon nitride.
6. system according to claim 3 is characterized in that the cell that is adopted is tumour cell or normal cell.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110204024 CN102352312B (en) | 2011-07-21 | 2011-07-21 | Integrated system of detection and injection of getting gene to cell and technology thereof |
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| CN 201110204024 CN102352312B (en) | 2011-07-21 | 2011-07-21 | Integrated system of detection and injection of getting gene to cell and technology thereof |
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| CN102352312A true CN102352312A (en) | 2012-02-15 |
| CN102352312B CN102352312B (en) | 2013-04-17 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758058A (en) * | 2015-03-11 | 2015-07-08 | 苏州大学 | Synchronous pulse laser microimaging observation device for mechanical stress deformation of blood cells |
| CN105738254A (en) * | 2016-02-03 | 2016-07-06 | 苏州大学 | Mechanobiological coupling testing system and method |
| CN108779428A (en) * | 2016-12-23 | 2018-11-09 | 埃佩多夫股份公司 | Manually syringe for cell manipulation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763170A (en) * | 2004-10-19 | 2006-04-26 | 章维一 | Cell microinjection instrument |
| CN101435818A (en) * | 2008-12-12 | 2009-05-20 | 清华大学深圳研究生院 | Portable Mediterranean anemia disease screening apparatus based on micro-flow control chip image technology |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763170A (en) * | 2004-10-19 | 2006-04-26 | 章维一 | Cell microinjection instrument |
| CN101435818A (en) * | 2008-12-12 | 2009-05-20 | 清华大学深圳研究生院 | Portable Mediterranean anemia disease screening apparatus based on micro-flow control chip image technology |
Non-Patent Citations (4)
| Title |
|---|
| 《Proceedings of the 2001 IEEE international Conference on Robotics and Automation,Seoul,Korea》 20011231 xudong Li,et al "Development of Global Vision System for Biological Automatic Micro-Manipulation System" 127-132 第1卷, * |
| 《传感器技术》 20021231 周李承 等 "光纤纳米生物传感器的现状及发展" 第21卷, 第12期 * |
| XUDONG LI,ET AL: ""Development of Global Vision System for Biological Automatic Micro-Manipulation System"", 《PROCEEDINGS OF THE 2001 IEEE INTERNATIONAL CONFERENCE ON ROBOTICS AND AUTOMATION,SEOUL,KOREA》, vol. 1, 31 December 2001 (2001-12-31), pages 127 - 132, XP010550140, DOI: doi:10.1109/ROBOT.2001.932541 * |
| 周李承 等: ""光纤纳米生物传感器的现状及发展"", 《传感器技术》, vol. 21, no. 12, 31 December 2002 (2002-12-31) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758058A (en) * | 2015-03-11 | 2015-07-08 | 苏州大学 | Synchronous pulse laser microimaging observation device for mechanical stress deformation of blood cells |
| CN105738254A (en) * | 2016-02-03 | 2016-07-06 | 苏州大学 | Mechanobiological coupling testing system and method |
| CN105738254B (en) * | 2016-02-03 | 2019-07-12 | 苏州大学 | A kind of Mechanobiology coupling testing system and method |
| CN108779428A (en) * | 2016-12-23 | 2018-11-09 | 埃佩多夫股份公司 | Manually syringe for cell manipulation |
| CN108779428B (en) * | 2016-12-23 | 2022-03-15 | 埃佩多夫股份公司 | Manual syringe for cell manipulation |
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| CN102352312B (en) | 2013-04-17 |
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