CN108060075A - Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method - Google Patents
Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method Download PDFInfo
- Publication number
- CN108060075A CN108060075A CN201810091807.9A CN201810091807A CN108060075A CN 108060075 A CN108060075 A CN 108060075A CN 201810091807 A CN201810091807 A CN 201810091807A CN 108060075 A CN108060075 A CN 108060075A
- Authority
- CN
- China
- Prior art keywords
- micro
- layer
- detection chip
- gene expression
- biochemical analysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of micro-fluidic unicellular gene expression detection chip and its method of work and preparation method, which includes basal layer, passivation layer, magnet layer, fluidized bed and pneumatic key-course；The passivation layer is set on the base layer；The magnet layer is set over the passivation layer；The fluidized bed is arranged on magnet layer；The pneumatic control layer is arranged on fluidized bed；Electrode is provided on basal layer；The pneumatic control layer is divided into biochemical analysis cell array；The biochemical analysis cell array includes several biochemical analysis units；Biochemical analysis dividing elements are cell capture zones and biochemical reaction zone.Precision, sensitivity and efficiency of Molecular Detection etc. can be effectively improved, and reduces sample cross contamination, Operating Complexity and reagent consumption.
Description
Technical field
The invention belongs to chip fields, and in particular to a kind of micro-fluidic unicellular gene expression detection chip and its work side
Method and preparation method.
Background technology
It is micro-/receive the main research characteristic size of manufacture micron, the functional structure of nanometer range, device and system design,
Problem in science in manufacture.Cell is the base unit of life 26S Proteasome Structure and Function, and Living single cell gene expression detection is cell
The limiting condition of life analytical technology for the generation of announcement major disease and Evolvement Mechanism, promotes newtype drug research and development etc. to have
Significance.Although microflow control technique supports fundamental biological knowledge and clinical medical great discovery to a certain extent, accelerate
The process of many basic scientific research hot subjects, but existing chip there is it is complicated, integration is poor, detection flux it is low,
The deficiencies of expensive problem.
The content of the invention
The object of the present invention is to provide a kind of micro-fluidic unicellular gene expression detection chip and its method of work and preparations
Method.
The present invention uses following technical scheme：A kind of micro-fluidic unicellular gene expression detection chip, including basal layer,
Passivation layer, magnet layer, fluidized bed and pneumatic key-course；The passivation layer is set on the base layer；The magnet layer is arranged on blunt
Change on layer；The fluidized bed is arranged on magnet layer；The pneumatic control layer is arranged on fluidized bed；Electricity is provided on basal layer
Pole；The pneumatic control layer is divided into biochemical analysis cell array；The biochemical analysis cell array includes several biochemistry point
Analyse unit；Biochemical analysis dividing elements are cell capture zones and biochemical reaction zone.
In an embodiment of the present invention, the first multiplexer, the second multiplexing are provided on the pneumatic control layer
Device.
In an embodiment of the present invention, micro- magnetic bead of surface coating specific primer is provided on the cell capture zones.
In an embodiment of the present invention, micro-heater and temperature sensor are provided on the basal layer；Micro- heating
Device, temperature sensor are connected respectively with electrode.
Further, the micro-heater and resistance temperature sensor ratio are 1:2.7.
In an embodiment of the present invention, evaporation inhibition layer is set in the fluidized bed.
In an embodiment of the present invention, the biochemical analysis cell array includes 400 biochemical analysis units.
The present invention also provides a kind of method of work of micro-fluidic unicellular gene expression detection chip as described above, bags
Include following steps：Step S1：When cell is on carrier flow to detection chip；It is unicellular to stop and split by chemical cracking liquid
Solution, the mRNA released are captured by micro- magnetic bead of surface coating specific primer；Step S2：If if in biochemical analysis cell array
A dry biochemical analysis unit is all completely completed intracellular mRNA and is captured, then starts the micro-heater on basal layer, complete reverse transcription
It is even reacted with real time fluorescent quantitative poly；Step S3：Target gene fragment amplification signal is captured by a fluorescence microscope, the letter
It number is uploaded to computer and its gray value is analyzed by computer；Real time fluorescent quantitative poly connects reaction signal by a fluorescence microscopy
Mirror and CCD acquisitions, the signal are uploaded to computer and are analyzed by computer.
In an embodiment of the present invention, micro-heater control source is provided by a digital power, resistance temperature sensor by
One digital multimeter is read；Cell current-carrying sample introduction is controlled by a micro syringe pump, and pneumatic control layer is controlled by a nitrogen pressure.
The present invention provides a kind of preparation method of micro-fluidic unicellular gene expression detection chip as described above, including
Following steps：
Step 1：Vacuum evaporation thickness 20nm chromium and 110nm gold, then micro-heater and temperature sensor are obtained by wet etching,
Micro-heater is 1 with resistance temperature sensor ratio:2.7；Step 2：Spin coating thickness is 80 μm of negative photoresist SU-8, by purple
Outer exposure method processes pneumatic control layer glued membrane in silicon chip substrate, and casting polydimethylsiloxane and cures and obtain final gas
Dynamic layer；Step 3：Spin coating thickness is 30 μm of negative photoresist SU-8, processes pneumatic control in silicon chip substrate by uv-exposure method
Preparative layer glued membrane, the PDMS that casts twice obtain the fluidized bed of built-in evaporation inhibition layer；Step 4：By PDMS card punch, core is processed
Piece fluidized bed, the entrance of pneumatic control layer, outlet；Step 5：It is obtained by reactive ion etching RIE encapsulation steps one to four
Multilayered structure obtains detection chip.
Compared with prior art, the present invention has the following advantages：Micro-/manufacturing science of receiving is ground with technology and basic biomedical
Study carefully and be combined, can realize the accurate manipulation and detection in real time of low abundance biological sample, micro-fluidic chip can effectively improve point
Precision, sensitivity and efficiency of son detection etc., and reduce sample cross contamination, Operating Complexity and reagent consumption.
Description of the drawings
Fig. 1 is the detection chip structure broken away view of one embodiment of the invention.
Fig. 2 is the detection chip enlarged diagram of one embodiment of the invention.
Fig. 3 is a micro-fluidic unicellular gene expression detection schematic diagram of the invention.
Fig. 4 is the detection chip work connection diagram of one embodiment of the invention.
Fig. 5 is the detection chip preparation flow schematic diagram of a specific embodiment of the invention.
Specific embodiment
Explanation is further explained to the present invention in the following with reference to the drawings and specific embodiments.
A kind of micro-fluidic unicellular gene expression detection chip of high throughput, including basal layer 1, passivation layer 2, magnet layer 3,
Fluidized bed 4 and pneumatic key-course 5；The passivation layer 2 is arranged on basal layer 1；The magnet layer 3 is arranged on passivation layer 2；Institute
Fluidized bed 4 is stated to be arranged on magnet layer 3；The pneumatic control layer 5 is arranged on fluidized bed 4；Electrode 6 is provided on basal layer 1；
The pneumatic control layer 5 is divided into biochemical analysis cell array；The biochemical analysis cell array includes several biochemical analysises
Unit 5-1；Biochemical analysis unit draws 5-1 points as cell capture zones 5-1-1 and biochemical reaction zone 5-1-2.Primary structure schematic diagram
Referring to Fig. 1-2.
In an embodiment of the present invention, the multiplexing of the first multiplexer 7, second is provided on the pneumatic control layer
Device 8.
In an embodiment of the present invention, micro- magnetic bead of surface coating specific primer is provided on the cell capture zones.
In an embodiment of the present invention, micro-heater and temperature sensor are provided on the basal layer；Micro- heating
Device, temperature sensor are connected respectively with electrode.Referring to Fig. 1, the present invention has two pairs of electrodes；Micro-heater and one pair of which electrode
Connection；Temperature sensor is connected with one pair of which electrode.
Further, the micro-heater and resistance temperature sensor ratio are 1:2.7.
In an embodiment of the present invention, evaporation inhibition layer is set in the fluidized bed.
In an embodiment of the present invention, the biochemical analysis cell array includes 400 biochemical analysis units.
The present invention also provides a kind of method of work of micro-fluidic unicellular gene expression detection chip as described above, bags
Include following steps：Step S1：When cell is on carrier flow to detection chip；It is unicellular to stop and split by chemical cracking liquid
Solution, the mRNA released are captured by micro- magnetic bead of surface coating specific primer；Step S2：If if in biochemical analysis cell array
Dry biochemical analysis unit is all completely completed mRNA and is captured, then starts the micro-heater on basal layer, complete reverse transcription in real time
Fluorescent quantitative poly even reacts（PCR）；Step S3：Target gene fragment amplification signal is captured by a fluorescence microscope, the letter
It number is uploaded to computer and its gray value is analyzed by computer；Real time fluorescent quantitative poly connects reaction signal by a fluorescence microscopy
Mirror and CCD acquisitions, the signal are uploaded to computer and are analyzed by computer.
Main flow schematic diagram is referring to Fig. 3.Detection chip using the present invention, experiment whole process avoid the behaviour such as sample transfer
Make step.It solves the problems such as traditional experiment integrated level is poor, complicated for operation, can reduce to researcher's biomedicine ability of experiment
Horizontal requirement.Correspondingly, microfluidic platform, temperature control platform and bio signal detection platform are developed, is respectively used to live body
Manipulation of single cells and a variety of reaction reagents it is accurate, stablize sample introduction, the real-time detection and accurate control of chip reaction temperature and raw
Object fluorescence signal acquisition and analysis in real time.
In an embodiment of the present invention, micro-heater control source is provided by a digital power, resistance temperature sensor by
One digital multimeter is read；Cell current-carrying sample introduction is controlled by a micro syringe pump, and pneumatic control layer is controlled by a nitrogen pressure.
Fig. 4 is the corresponding detection platform schematic diagram of one embodiment of the invention chip.Wherein, micro-heater control source is by counting
Word power supply provides, and microsensor resistance is read by digital multimeter；Cell current-carrying sample introduction is controlled by micro syringe pump, and pneumatic layer is by nitrogen
Gas pressure control；Signal in PCR reactions is gathered by fluorescence microscope and CCD, and final result can be by the phase on computer
Software is answered to calculate analysis.
The present invention provides a kind of preparation method of micro-fluidic unicellular gene expression detection chip as described above, including
Following steps：
Step 1：Vacuum evaporation thickness 20nm chromium and 110nm gold, then micro-heater and temperature sensor are obtained by wet etching,
Micro-heater is 1 with resistance temperature sensor ratio:2.7；Step 2：Spin coating thickness is 80 μm of negative photoresist SU-8, by purple
Outer exposure method processes pneumatic control layer glued membrane in silicon chip substrate, and casting polydimethylsiloxane and cures and obtain final gas
Dynamic layer；Step 3：Spin coating thickness is 30 μm of negative photoresist SU-8, processes pneumatic control in silicon chip substrate by uv-exposure method
Preparative layer glued membrane, the PDMS that casts twice obtain the fluidized bed of built-in evaporation inhibition layer；Step 4：By PDMS card punch, core is processed
Piece fluidized bed, the entrance of pneumatic control layer, outlet；Step 5：It is obtained by reactive ion etching RIE encapsulation steps one to four
Multilayered structure obtains detection chip.Preparation flow schematic diagram is referring to Fig. 5.
The present invention integrates multiplexing pneumatic control microflow control technique, fully integrated single cell analysis and quantitative fluorescent PCR skill
Art can realize that the parallelization of 400 human body cells manipulates and the high-throughput inspection of intracellular extremely low abundance DNA sample in nanoliter magnitude
It surveys.Integrated level, accuracy and sensitivity of the present invention etc. will be better than similar research both at home and abroad at present.
The above are preferred embodiments of the present invention, all any changes made according to the technical solution of the present invention, and generated function is made
During with scope without departing from technical solution of the present invention, all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of micro-fluidic unicellular gene expression detection chip, it is characterised in that：Including basal layer, passivation layer, magnet layer, stream
Dynamic layer and pneumatic key-course；The passivation layer is set on the base layer；The magnet layer is set over the passivation layer；The fluidized bed
It is arranged on magnet layer；The pneumatic control layer is arranged on fluidized bed；Electrode is provided on basal layer；The pneumatic control layer
It is divided into biochemical analysis cell array；The biochemical analysis cell array includes several biochemical analysis units；Biochemical analysis list
Member is divided into cell capture zones and biochemical reaction zone.
2. micro-fluidic unicellular gene expression detection chip according to claim 1, it is characterised in that：The pneumatic control
The first multiplexer, the second multiplexer are provided on layer.
3. the micro-fluidic unicellular gene expression detection chip of high throughput according to claim 1, it is characterised in that：It is described thin
Micro- magnetic bead of surface coating specific primer is provided on born of the same parents capture area.
4. micro-fluidic unicellular gene expression detection chip according to claim 1, it is characterised in that：On the basal layer
It is provided with micro-heater and temperature sensor；The micro-heater, temperature sensor are connected respectively with electrode.
5. the micro-fluidic unicellular gene expression detection chip of high throughput according to claim 4, it is characterised in that：It is described micro-
Heater is 1 with resistance temperature sensor ratio:2.7.
6. micro-fluidic unicellular gene expression detection chip according to claim 1, it is characterised in that：In the fluidized bed
Evaporation inhibition layer is set.
7. micro-fluidic unicellular gene expression detection chip according to claim 1, it is characterised in that：The biochemical analysis
Cell array includes 400 biochemical analysis units.
8. a kind of method of work based on micro-fluidic unicellular gene expression detection chip described in claim 1, feature exist
In：Comprise the following steps：
Step S1：When cell is on carrier flow to detection chip；It is unicellular to stop and cracked by chemical cracking liquid, it releases
MRNA by surface coating specific primer micro- magnetic bead capture；
Step S2：It captures, starts if several biochemical analysis units in biochemical analysis cell array all completely complete mRNA
Micro-heater on basal layer completes reverse transcription, real time fluorescent quantitative poly even reaction and target gene fragment amplification；
Step S3：Target gene fragment amplification signal is captured by a fluorescence microscope, which is uploaded to computer and by calculating
Machine analyzes its gray value；Real time fluorescent quantitative poly connects reaction signal and is gathered by a fluorescence microscope and a CCD, on the signal
It reaches computer and is analyzed by computer.
9. the method for work of micro-fluidic unicellular gene expression detection chip according to claim 8, it is characterised in that：It is micro-
Heater voltage input is provided by a digital power, and resistance temperature sensor is read by a digital multimeter；Cell current-carrying sample introduction
It is controlled by a micro syringe pump, pneumatic control layer is controlled by a nitrogen pressure.
10. a kind of preparation method of micro-fluidic unicellular gene expression detection chip as described in claim 1, feature exist
In：Comprise the following steps：
Step 1：Vacuum evaporation thickness 20nm chromium and 110nm gold, then micro-heater and temperature sensor are obtained by wet etching,
Micro-heater is 1 with resistance temperature sensor ratio:2.7；
Step 2：Spin coating thickness is 80 μm of negative photoresist SU-8, processes pneumatic control in silicon chip substrate by uv-exposure method
Preparative layer glued membrane, casting polydimethylsiloxane and curing obtain final pneumatic layer；
Step 3：Spin coating thickness is 30 μm of negative photoresist SU-8, processes pneumatic control in silicon chip substrate by uv-exposure method
Preparative layer glued membrane, the PDMS that casts twice obtain the fluidized bed of built-in evaporation inhibition layer；
Step 4：By PDMS card punch, processing chip fluidized bed, the entrance of pneumatic control layer, outlet；
Step without：By the multilayered structure that reactive ion etching RIE encapsulation steps one to four obtain, detection chip is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810091807.9A CN108060075A (en) | 2018-01-30 | 2018-01-30 | Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810091807.9A CN108060075A (en) | 2018-01-30 | 2018-01-30 | Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108060075A true CN108060075A (en) | 2018-05-22 |
Family
ID=62134275
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810091807.9A Pending CN108060075A (en) | 2018-01-30 | 2018-01-30 | Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060075A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022165706A1 (en) * | 2021-02-04 | 2022-08-11 | Singleron (Nanjing) Biotechnologies, Ltd. | Method of reverse transcription |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI20085299A0 (en) * | 2008-04-10 | 2008-04-10 | Valtion Teknillinen | Microfluidic chip devices and their use |
| CN101429560A (en) * | 2008-12-19 | 2009-05-13 | 清华大学 | Quantitative polyase chain reaction detecting device and method of producing the same |
| RU2385940C1 (en) * | 2008-10-23 | 2010-04-10 | Общество с ограниченной ответственностью "ВИНТЕЛ" | Method for real-time detection of nucleic acids by polymerase chain reaction and device for implementation thereof |
| CN103695307A (en) * | 2007-07-13 | 2014-04-02 | 汉迪实验室公司 | Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
| CN104593256A (en) * | 2015-01-06 | 2015-05-06 | 上海交通大学 | PCR chip with repeatedly used electrode |
| CN106065391A (en) * | 2016-07-20 | 2016-11-02 | 国家纳米科学中心 | For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification |
-
2018
- 2018-01-30 CN CN201810091807.9A patent/CN108060075A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695307A (en) * | 2007-07-13 | 2014-04-02 | 汉迪实验室公司 | Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
| FI20085299A0 (en) * | 2008-04-10 | 2008-04-10 | Valtion Teknillinen | Microfluidic chip devices and their use |
| RU2385940C1 (en) * | 2008-10-23 | 2010-04-10 | Общество с ограниченной ответственностью "ВИНТЕЛ" | Method for real-time detection of nucleic acids by polymerase chain reaction and device for implementation thereof |
| CN101429560A (en) * | 2008-12-19 | 2009-05-13 | 清华大学 | Quantitative polyase chain reaction detecting device and method of producing the same |
| CN104593256A (en) * | 2015-01-06 | 2015-05-06 | 上海交通大学 | PCR chip with repeatedly used electrode |
| CN106065391A (en) * | 2016-07-20 | 2016-11-02 | 国家纳米科学中心 | For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification |
Non-Patent Citations (1)
| Title |
|---|
| 曹际娟 辽宁师范大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022165706A1 (en) * | 2021-02-04 | 2022-08-11 | Singleron (Nanjing) Biotechnologies, Ltd. | Method of reverse transcription |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gao et al. | Recent advances in single cell manipulation and biochemical analysis on microfluidics | |
| Zarei | Advances in point-of-care technologies for molecular diagnostics | |
| Piraino et al. | A digital–analog microfluidic platform for patient-centric multiplexed biomarker diagnostics of ultralow volume samples | |
| Gupta et al. | Lab-on-chip technology: A review on design trends and future scope in biomedical applications | |
| Zhou et al. | Recent innovations in cost-effective polymer and paper hybrid microfluidic devices | |
| Liu et al. | Advancing single-cell proteomics and metabolomics with microfluidic technologies | |
| Derkus | Applying the miniaturization technologies for biosensor design | |
| Chin et al. | Lab-on-a-chip devices for global health: Past studies and future opportunities | |
| US8614056B2 (en) | Microfluidic method for measurement or detection involving cells or biomolecules | |
| TW200928364A (en) | Microfluidic platforms for multi-target detection | |
| CN101587124A (en) | Micro-fluidic chip special for diagnosing syphilis by aid of organic conductor material technology | |
| WO2008082712A2 (en) | Systems and methods for biodosimetry with biochip using gene expression signatures | |
| CN105296349A (en) | Microfluidic chip, detection system and device used for rapid DNA detection | |
| Zhu et al. | A lab-on-a-chip device integrated DNA extraction and solid phase PCR array for the genotyping of high-risk HPV in clinical samples | |
| CN202279815U (en) | Circulating tumor cell capturing device | |
| Emaminejad et al. | Portable cytometry using microscale electronic sensing | |
| Wang et al. | Recent developments in microfluidic‐based point‐of‐care testing (Poct) diagnoses | |
| Zhang et al. | Microfluidic flow cytometry for blood-based biomarker analysis | |
| CN108060075A (en) | Micro-fluidic unicellular gene expression detection chip and its method of work and preparation method | |
| Team | Introduction to lab-on-a-chip 2023: review, history and future | |
| CN201517993U (en) | Micro-fluidic chip detecting device based on photoacoustic technique | |
| Watkins et al. | On a chip | |
| EP3610037B1 (en) | Paper-based analytical microfluidic chip and device, having embossed&debossed processing channel, for nucleic acid diagnosis | |
| Ghubade et al. | Dielectrophoresis assisted concentration of micro-particles and their rapid quantitation based on optical means | |
| CN208684970U (en) | A kind of micro-fluidic unicellular gene expression detection chip of high throughput |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |