CN102352312B - 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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- CN102352312B CN102352312B CN 201110204024 CN201110204024A CN102352312B CN 102352312 B CN102352312 B CN 102352312B CN 201110204024 CN201110204024 CN 201110204024 CN 201110204024 A CN201110204024 A CN 201110204024A CN 102352312 B CN102352312 B CN 102352312B
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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 enter 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 comprised of nano-probe and micron pipeline.
Background technology
Some major diseases such as acquired immune deficiency syndrome (AIDS), hepatitis, malignant tumour, genetic diseases, traditional methods for the treatment of not only spends huge, bring huge economical load for society and family, and result for the treatment of is 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 important directions and the inevitable development trend of from now on disease treatment research.
It is that the matter of utmost importance of gene therapy is again 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 enter the environment of cell, can't control and detect the process of entering, gene enters cell and has very large randomness, has affected result for the treatment of, the difficult problem that present gene therapy research occurs, so that gene therapy can't be furtherd investigate.
Inject gene for present mechanical process, do not handle accurately for gene, be mostly to judge that by experience and the detection in later stage gene enters the effect of cell, ignored the process in early stage that gene enters cell.Namely at cytolemma what having occured has changed the degree of this variation on earth.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, it is integrated to realize that gene enters the integral system of the accurate importing of cell and monitoring, finish controlled injection gene in the cell, utilize the intuitive of atomic force microscope and the directly variation of the Performance Detection surface of cell membrane of acquisition 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 traditional method exists, disclose the mechanism that gene enters cell, the mechanism problem in the present gene therapy research was clarified in the corresponding biology performance of the variation of cytolemma when the refinement gene entered cell.
Summary of the invention
The defective or the deficiency that exist in order to overcome traditional method the object of the present invention is to provide a kind of new gene to enter the detection injection integral system manufacturing process of cell.
Technical scheme of the present invention is achieved in that
A kind of gene enters 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 by 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 again deceleration device, micro pump, Controlling System and signal detection.
A kind of gene enters cell detection and injection integral system preparation technology, and concrete steps are as follows:
1) spin coating photoresist material thickness 0.4～0.9 μ m on the silicon chip exposed 45 seconds, and etching obtains groove structure, then formed the socle girder with internal channel structure by bonding techniques;
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 datum, and needle point both sides cover carves the circular hole of diameter 200～300nm;
3) micro-Controlling System is regulated micro pump by Controlling System, accurately enters cell by what deceleration device was realized microfluid.
Described detection injection integral system, this system essentially consist unit is probe and the socle girder with internal passages.
The material that injection pipeline adopts in probe, the probe is silicon or silicon nitride.
The cell that adopts is tumour cell or normal cell.
The innovation technology compared with prior art has following advantage:
1. can accurately make the probe with internal channel structure, be easy to scale operation; Can inject by the socle girder internal passages, realize detecting with injection integrated by probe.
2. be convenient to the detecting ﹠ monitoring that gene enters, operation can be carried out control and guidance, the best site that can accurately find gene to enter.
3. be more suitable for studying mechanism and the cytogenetic variation of accurate evaluation that gene enters cell.
Description of drawings
Fig. 1 is system of the present invention ultimate principle structure iron.
Fig. 2 is with the probe system of passage.
The manufacturing process of Fig. 3 probe passage.
The manufacturing process of Fig. 4 probe tip two wing passages.
Described in detail below in conjunction with the structure ﹠processes flow process of accompanying drawing to this support.
Embodiment
A kind of gene enters cell detection and injection manufacturing process, it is characterized in that: nano level probe is communicated with socle girder and forms a detection injecting systems; Wherein:
There is passage socle girder inside, and an end connects micro-Controlling System; Probe can be finished and detect and imaging, finds the position of specific binding position such as acceptor, and then gene can accurately be injected in the cell from this position.
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 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 for finishing function of injection, can also detect and imaging, and can be connected with the atomic force microscopy system and finish control and the evaluation that gene enters cell, forms larger detection system.
Fig. 2 comprises the probe 13 with 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.
At first utilize computer aided design (CAD) (CAD) software (such as 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 rectangle, and is long at 20～30 μ m, wide 1 μ m; The probe tip diameter is then to design the used mask plate of etching between 8nm～10nm.
Fig. 3 silicon chip applies photoresist material 18, and exposure 19 develops 20, and etching 21 obtains the silicon chip 22 with groove structure, finishes the socle girder with internal passages by bonding packaging.
Fig. 4 probe material adopts the silicon chip of 100 metallographics, and coating electron beam resist 23 carries out electron-beam direct writing 24, etch probe 25, make positioning datum 26, determine 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 such as 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 enter cell to gene and operate and detect simultaneously cytogenetic variation, and existing technique means can't reach.At first make and have function of injecting nanometer detection probe concurrently, it is integrated to realize that gene enters the integral system of the accurate importing of cell and monitoring, finish controlled injection gene in the cell, utilize the intuitive of atomic force microscope and the directly variation of the Performance Detection surface of cell membrane of acquisition 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 traditional method exists, disclose the mechanism that gene enters cell, the mechanism problem in the present gene therapy research was clarified in the corresponding biology performance of the variation of cytolemma when the refinement gene entered cell.
Claims (5)
1. a gene enters 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 by 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;
Described micro-Controlling System comprises again deceleration device (7), micro pump (8), Controlling System (9) and signal detection (10); The trace Controlling System is regulated micro pump by Controlling System, accurately enters cell by what deceleration device was realized microfluid;
Described 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 datum, and needle point both sides cover carves the circular hole of diameter 200～300nm.
2. gene as claimed in claim 1 enters the preparation technology of cell detection and injection integral system, it is characterized in that concrete steps are as follows:
1) spin coating photoresist material thickness 0.4～0.9 μ m on the silicon chip exposed 45 seconds, and etching obtains groove structure, then formed the socle girder with internal channel structure by bonding techniques;
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 datum, and needle point both sides cover carves the circular hole of diameter 200～300nm;
3) micro-Controlling System (6) is regulated micro pump by Controlling System, accurately enters cell by what deceleration device was realized microfluid.
3. gene according to claim 2 enters 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.
4. gene according to claim 2 enters cell detection and injection integral system preparation technology, it is characterized in that the material that injection pipeline (5) adopts in probe (4), the probe is silicon or silicon nitride.
5. gene according to claim 2 enters cell detection and injection integral system preparation technology, it is characterized in that the cell that adopts 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 CN102352312A (en) | 2012-02-15 |
| CN102352312B true CN102352312B (en) | 2013-04-17 |
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| CN104758058B (en) * | 2015-03-11 | 2017-06-13 | 苏州大学 | A kind of haemocyte mechanical stress deformation pulse laser synchronization micro-imaging observation device |
| CN105738254B (en) * | 2016-02-03 | 2019-07-12 | 苏州大学 | A kind of Mechanobiology coupling testing system and method |
| EP3339424B1 (en) * | 2016-12-23 | 2019-05-22 | Eppendorf AG | Manual injector 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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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 |
|---|
| "Development of Global Vision System for Biological Automatic Micro-Manipulation System";xudong Li,et al;《Proceedings of the 2001 IEEE international Conference on Robotics and Automation,Seoul,Korea》;20011231;第1卷;127-132 * |
| "光纤纳米生物传感器的现状及发展";周李承 等;《传感器技术》;20021231;第21卷(第12期);56-59 * |
| xudongLi et al."Development of Global Vision System for Biological Automatic Micro-Manipulation System".《Proceedings of the 2001 IEEE international Conference on Robotics and Automation |
| 周李承 等."光纤纳米生物传感器的现状及发展".《传感器技术》.2002,第21卷(第12期), |
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