CN104293898B - MiRNA detection chip, its manufacture method and application - Google Patents
MiRNA detection chip, its manufacture method and application Download PDFInfo
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- CN104293898B CN104293898B CN201310302860.6A CN201310302860A CN104293898B CN 104293898 B CN104293898 B CN 104293898B CN 201310302860 A CN201310302860 A CN 201310302860A CN 104293898 B CN104293898 B CN 104293898B
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The present invention relates to a kind of miRNA detection chip, it includes substrate, is located at suprabasil chromium film, the golden film being located on chromium film, 12 sulfydryl dodecylic acid self-assembled monolayers introduced in self assembly mode on gold film and the rna probe being combined with self-assembled monolayer.Specific RNA sequence in this miRNA detection chip can form hybridization sequences with the cDNA hybridization formed by miRNA reverse transcription, when the cDNA that miRNA reverse transcription to be measured is formed can hybridize with this specific RNA sequence, re-use specific RNA sequence in RNase H enzymolysis RNA cDNA crossbred, the cDNA simultaneously discharged can be combined with the specific RNA sequence of chip surface repeatedly, hydrolyze again, until the RAN probe of chip surface is hydrolyzed completely, thus define a kind of amplification detection effect based on non-PCR, easy and simple to handle, the high flux that can realize miRNA quickly detects.Additionally, the present invention also relates to manufacture method and the application of a kind of miRNA detection chip.
Description
Technical field
The present invention relates to biochemistry detection field, especially relate to a kind of miRNA detection chip, its manufacture method and application.
Background technology
MicroRNA (Microrna, also known as miRNA) is the strand microRNA of body endogenous expression, is positioned at genome
Noncoding region, has high conservative type, timing and tissue specificity.MiRNA can be tied by the Incomplete matching with said target mrna
Close and stop protein expression.Single miRNA can regulate and control thousands of target gene.Research shows, miRNA can be produced by virus
Raw, such as influenza virus have expressed in host cell replicates 8 species specificity miRNA (miRNA-1254, miRNA-1272,
MiRNA-17-5p, miRNA-17-3p, miRNA-106B, miRNA-106B, miRNA-124-a and miRNA-124).Research is also
Show that the most of miRNA of serum is wrapped up by vesicle, RNase opposing is prevented the degraded of nuclease, the most stable, room
Temperature can stablize 4h, and multigelation twice is the most unaffected, and repeatability and comparison of coherence are good, are especially suitable for making clinical serum mark
Thing, the early diagnosis for disease provides new approaches.
In genome research, biochip technology platform provides effective hands for deciphering gene function, Rapid identification
Section is supported, is widely used in the fields such as the qualification of gene expression analysis, virus and antibacterial, medical diagnosis.But traditional gene
Chip technology generally requires the labellings such as fluorescence, radioactivity, enzyme or needs PCR to realize the amplification of signal, operates the most loaded down with trivial details, holds
It is easily generated pollution and relatively costly, it is difficult to adapt to the analysis of large-scale gene.
Summary of the invention
Based on this, it is necessary to provide a kind of easy and simple to handle and that high throughput testing can be realized miRNA detection chip, its making
Method and application.
A kind of miRNA detection chip, including substrate, be located at described suprabasil chromium film, the golden film that is located on described chromium film,
Described gold film on the 12-sulfydryl dodecylic acid self-assembled monolayer introduced in self assembly mode and with described self-assembled monolayer
In conjunction with rna probe；Described self-assembled monolayer is fixed on described gold film surface by sulfydryl；Described rna probe contain for
Detecting the specific sequence of miRNA to be measured, described specific sequence can be with the cDNA hybridization formed by miRNA reverse transcription；Described
It is connected by amido link between one end of RAN probe and self-assembled monolayer.
Wherein in an embodiment, the thickness of described chromium film is 1～2nm.
Wherein in an embodiment, the thickness of described gold film is 50nm.
Wherein in an embodiment, the other end of described rna probe connects biotin.
Wherein in an embodiment, described rna probe is respectively arranged at two ends with 20 and institute at described specific sequence
State the thymus pyrimidine that specific sequence is directly connected to.
Wherein in an embodiment, described specific sequence is the conserved sequence of Respirovirus.
Wherein in an embodiment, the sequence of described rna probe is SEQ ID NO.1, SEQ ID in sequence table
At least one in NO.2 and SEQ ID NO.3.
A kind of miRNA detection kit containing the miRNA detection chip described in any of the above-described embodiment.
The manufacture method of a kind of miRNA detection chip, comprises the steps:
Build for detecting the specific sequence of miRNA to be measured, and carry out amino in one end of described specific sequence and repair
Decorations, described specific sequence can be with the cDNA hybridization formed by miRNA reverse transcription；
At clean substrate surface one layer of chromium film of evaporation, then it is deposited with one layer of golden film on described chromium film surface, will be containing described
The substrate of chromium film and described gold film cleans up and is placed in the 12-sulfydryl dodecylic acid ethanol solution that concentration is 10mmol/L,
Ambient temperature overnight is hatched, and makes 12-sulfydryl dodecylic acid be self-assembled to the surface of described gold film by sulfydryl, takes out afterwash, and use
Nitrogen dries up, and obtains being modified with the chip of carboxyl；
The described chip being modified with carboxyl is placed in 1-ethyl-3(3-dimethylamino-propyl) carbodiimide and N-hydroxyl fourth
In the mixed solution of imidodicarbonic diamide, the carboxyl to described gold film surface carries out activation processing, by described specific sequence point sample to institute
State chip surface, the amino of described specific sequence one end and described carboxyl reaction form amido link and by described specific sequence
It is fixed on described chip surface, i.e. obtains described miRNA detection chip.
The detection method of a kind of miRNA, comprises the steps:
Extracting miRNA to be measured, and with the described miRNA that extracts as template, under the effect of reverse transcriptase, synthesis is complementary
CDNA sample；
MiRNA detection chip described in any of the above-described embodiment or the miRNA that makes according to the method described above are detected core
Sheet is placed in the reaction tank of surface plasma resonance imaging instrument, using concentration as 10mM, pH be that the phosphate buffer of 7.4 is as flowing
Phase, flow rate set is 2 μ L/s, after the baseline stability of instrument to be imaged, is passed through the solution of streptavidin that concentration is 10 μ g/mL and makes
The biotin of the free terminal of rna probe connects upper Streptavidin, thus the degraded of follow-up rna probe has signal and amplifies effect
Really；
In cDNA sample, add RNase H, and the cDNA sample containing RNase H is placed in described reaction tank, make
The rna probe degraded situation on miRNA detection chip surface is detected in real time, before comparing and being passed through cDNA sample with described imager
After baseline changing value obtain the degradation amount of rna probe on miRNA detection chip surface, thus obtain the content of miRNA to be measured.
Specific RNA sequence in above-mentioned miRNA detection chip can hybridize shape with the cDNA formed by miRNA reverse transcription
Become hybridization sequences, when the cDNA that miRNA reverse transcription to be measured is formed can hybridize with this specific RNA sequence, re-use RNase H
Specific RNA sequence in enzymolysis RNA-cDNA crossbred, the cDNA simultaneously discharged can repeatedly with the specific RNA of chip surface
Sequence combines, then hydrolyzes, until the RAN probe of chip surface is hydrolyzed completely, thus defines a kind of amplification based on non-PCR
Detection effect, easy and simple to handle, it is possible to achieve the high flux of miRNA quickly detects.
Accompanying drawing explanation
Fig. 1 is the structural representation of the miRNA detection chip of an embodiment；
Fig. 2 is that the monitoring of surface plasma resonance imaging instrument is passed through H1N1cDNA analyte (concentration containing RNase H is
1.0 units/μ L) the degradation process schematic diagram of miRNA detection chip surface rna probe afterwards.
Detailed description of the invention
And should be used as further miRNA detection chip, its preparation method mainly in combination with drawings and the specific embodiments below
Detailed description.
As it is shown in figure 1, the miRNA detection chip 100 of an embodiment include substrate 110, chromium film 120, gold film 130, from
Assemble monolayer 140 and rna probe 150.
Substrate 110 is flat glass substrate.Chromium film 120 is located on a side surface of substrate 110, thickness be 1～
2nm.Gold film 130 is located on chromium film 120, and thickness is 50nm.
Self-assembled monolayer 140 one end that 12-sulfydryl dodecylic acid is formed is fixed on gold film 120 surface by sulfydryl, separately
One end is c-terminus.
In the present embodiment, rna probe 150 includes one according to the specificity of the conserved region sequential design of Respirovirus
Sequence, lay respectively at the biology that 20 thymus pyrimidines (T) at these specific sequence two ends are connected with the thymus pyrimidine end of one end
Element and the amino (-NH being connected with the thymus pyrimidine end of the other end2).The most in the present embodiment, rna probe 150
Sequence includes the SEQ ID NO.1 in sequence table, SEQ ID NO.2 and SEQ ID NO.3.It is appreciated that other embodiment party
In formula, the structure of rna probe 150 is not limited to this, as can be do not had thymus pyrimidine repetitive sequence or only at specific sequence
One end arranges the thymus pyrimidine sequence etc. of repetition；The quantity of thymus pyrimidine is also not necessarily limited to 20；The sequence of rna probe 150 also may be used
Think other diseases conserved viral region sequence etc..The end of thymus pyrimidine at one end connects biotin, can be in follow-up inspection
Constitute biotin-Streptavidin system during survey and realize the amplification of detection signal.
Amino on rna probe 150 can generate amido link with the carboxyl reaction of self-assembled monolayer 140, thus RAN probe
140 can be fixed on gold film 130 by the amido link that reaction generates.
Present embodiment additionally provides a kind of miRNA containing the miRNA detection chip described in any of the above-described embodiment inspection
Test agent box.This miRNA detection kit further comprises solution of streptavidin and ribonuclease H (RNase H) etc..
Additionally, present embodiment additionally provides the manufacture method of a kind of miRNA detection chip, it comprises the steps:
Step one: build the specific sequence for detecting miRNA to be measured, and carry out amino in one end of specific sequence
Modifying, specific sequence can be with the cDNA hybridization formed by miRNA reverse transcription.
In the present embodiment, in step one, the specific sequence two ends being additionally included in structure connect 20 breasts respectively
The step of gland pyrimidine, then the end of the thymus pyrimidine in specific sequence one end carries out amido modified.Further, it is additionally included in spy
The end of the thymus pyrimidine of the opposite sex sequence other end carries out the step of biotin modification, is formed during subsequent detection to facilitate
Biotin-Streptavidin system realizes detecting the amplification of signal.
Step 2: at clean substrate surface one layer of chromium film of evaporation, then be deposited with one layer of golden film on chromium film surface, will be containing chromium
The substrate of film and gold film cleans up and is placed in the 12-sulfydryl dodecylic acid ethanol solution that concentration is 10mmol/L, room temperature mistake
Night hatches, and makes 12-sulfydryl dodecylic acid be self-assembled to the surface of gold film by sulfydryl, takes out afterwash, and dry up with nitrogen,
To the chip being modified with carboxyl.
In this embodiment party is, the thickness of the chromium film of making is 1～2nm；The thickness of the golden film made is 50nm.
Step 3: the chip being modified with carboxyl is placed in 1-ethyl-3(3-dimethylamino-propyl) carbodiimide and N-hydroxyl
In the mixed solution of succimide, the carboxyl to gold film surface carries out activation processing, by specific sequence point sample to chip list
Face, the amino of specific sequence one end forms amido link with carboxyl reaction and specific sequence is fixed on chip surface, to obtain final product
To miRNA detection chip.
Specific RNA sequence in above-mentioned miRNA detection chip can hybridize shape with the cDNA formed by miRNA reverse transcription
Become hybridization sequences, when the cDNA that miRNA reverse transcription to be measured is formed can hybridize with this specific RNA sequence, re-use RNase H
Specific RNA sequence in enzymolysis RNA-cDNA crossbred, the cDNA simultaneously discharged can repeatedly with the specific RNA of chip surface
Sequence combines, then hydrolyzes, until the RAN probe of chip surface is hydrolyzed completely, thus defines a kind of amplification based on non-PCR
Detection effect, easy and simple to handle, it is possible to achieve the high flux of miRNA quickly detects.
Further, present embodiment additionally provides the detection method of a kind of miRNA, and it comprises the steps:
Extracting miRNA to be measured, and with this miRNA of extracting as template, under the effect of reverse transcriptase, synthesis is complementary
CDNA sample；
The miRNA detection chip of above-mentioned making is placed in surface plasma resonance (Surface Plasmon Resonance
Imaging, SPR) imager reaction tank in, using concentration as 10mM, pH be 7.4 phosphate buffer as flowing phase, flow velocity
It is set as 2 μ L/s, after the baseline stability of instrument to be imaged, is passed through the solution of streptavidin that concentration is 10 μ g/mL and makes rna probe
The biotin of free terminal connects upper Streptavidin, thus the degraded of follow-up rna probe has signal amplification effect；
In cDNA sample, add RNase H, and the cDNA sample containing RNase H is placed in the reaction of SPR imager
Chi Zhong, SPR imager detects the rna probe degraded situation on miRNA detection chip surface in real time, is passed through cDNA sample by comparing
Baseline changing value front and back obtains the degradation amount of the rna probe on miRNA detection chip surface, thus obtains containing of miRNA to be measured
Amount.
It is below specific embodiment part:
Reagent: RNA extracts test kit (Beijing Tian Ze gene prod), diethanolamine hydrochloride (2-Aminoethanol
Hydrochloride) (Japan's TCI Products)；1-ethyl-3(3-dimethylamino-propyl) carbodiimide (1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydr-ochloride, EDC) (Canada Bio Basic Inc is public
Department's product)；N-hydroxysuccinimide (N-hydroxysuccini-mide, NHS) (Shanghai covalent chemical Science and Technology Ltd.
Product)；Dehydrated alcohol (purity: analytical pure, 1Guanghua Chemical Plant Co., Ltd., Guangdong's product)；Every other reagent is analysis
Pure.
Instrument: C1000Thermal cycler PCR amplification instrument (Bio Rad Laboratories), centrifuge, eddy blending machine (moral
IKAMS company of state), Hybex Microsamp Incubator, the ND-1000 trace ultra-violet and visible spectrophotometer (U.S.
Nanodrop company) (purposes: be applied to measure protein and the concentration of DNA), SPR imager (Analyzer
Second filial generation product).
The synthesis of rna probe:
Swine flu H1N1:Biotin-T20-GGACUACCACGAUUCAAAUGUG-T20-NH2(SEQ ID NO.1)；
Respiratory syncytial virus (RSV): Biotin-T20-CCAUGUGAAUUCCCUGCAUCAAU-T20-NH2(SEQ ID
NO.2)；
Adenovirus (ADV): Biotin-T20-CACCGAGACGUACUUCAGCCUG-T20-NH2(SEQ ID NO.3)；
Wherein, Biotin is biotin.
Experimental procedure:
The synthesis of 1.RAN probe: the GenBank file needed for downloading from NCBI, utilizes software premier5 breathing
The specific sequence of designated rna probe in the conserved region sequence in road virus (H1N1, RSV and ADV).Again at this specific sequence
5 ' ends have carried out biotin modification, so that biotin labeled probe can realize, by Streptavidin, the work that signal amplifies
With.The other end at rna probe has carried out amido modified, fixes and ensure probe hybridization efficiency for the ease of probe, further,
The present embodiment with the addition of 20 poly(T at 3 ' and 5 ' ends of specific sequence).Poly(T) rna probe can be made as spacerarm
It is fully extended at solid phase surface and is beneficial to the fixing of probe and hybridization.The sequence of the rna probe of synthesis is in sequence table
SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
2.miRNA detection chip makes: at clean glass substrate surface evaporation or the chromium of magnetron sputtering 1～2nm thickness
Film, then at the surface of chromium film evaporation or the golden film of magnetron sputtering 50nm.The chip obtained is soaked into after over cleaning immediately
In 10mmol/L12-sulfydryl dodecylic acid ethanol solution, ambient temperature overnight is hatched, make sulfydryl orderly be self-assembled to gold film surface,
The carboxyl of 12-sulfydryl dodecylic acid dissociates, and after taking-up, repeatedly rinses with dehydrated alcohol 5 times, then is washed with deionized water clean, nitrogen
Dry up；The chip carboxylic group of newly configured EDC/NHS solution activating surface that above-mentioned 12-sulfydryl dodecylic acid is modified,
By rna probe relevant for Respirovirus with high accuracy point sample instrument Genetix Q-Array Mini or manual point sample in chip
Specific region, obtains described miRNA detection chip.
3. the process of sample: the RNA of Respirovirus extracts and uses the RNA of Beijing Tian Ze Reagent Company to extract test kit,
Specific experiment operating process is according to the explanation step-by-step operation of test kit.The miRNA extracted in sample is as template, at reverse transcriptase
Effect under, synthesize complementation cDNA sample.
4.SPR quickly detection: the SPR imager of employing isAnalyzer second filial generation product, will make
MiRNA detection chip be arranged in SPR instrument according to the operating process of instrument.And with phosphate buffer solution (PBS buffer,
Concentration be 10mM, pH be 7.4) as flowing phase, flow rate set is 2L/s.After the baseline stability of instrument to be imaged, it is passed through strepto-parent
With element (Streptavidin, concentration is 10g/mL) 500s, the end of rna probe is made to combine Streptavidin so that follow-up RNA
Degraded there is signal amplification effect.RNase H is added so that the concentration of final RNase H is 1.0 lists in cDNA sample
Position/L.Being passed through in the reaction tank of SPR imager by cDNA sample containing RNase H, SPR detects surface RNA degraded feelings in real time
Condition.It is passed through the baseline changing value before and after sample obtains the degradation amount of surface rna probe by comparing.
Experimental result
The present embodiment is with detection influenza virus H1N1 as experimental group, RSV and ADV is matched group, utilizes throat swab to extract disease
Poison miRNA, with extract miRNA as template, under the effect of reverse transcriptase, synthesize complementation cDNA as detection sample.
The acquisition target of throat swab is for having heating sign, trial inspection to have influenza through Shenzhen port immigration, infrared detecting set
The case of symptom.
When passing through the cDNA sample of H1N1 virus in experiment, the spr signal of the rna probe point of H1N1 is remarkably decreased, little 1
Time interior spr signal decline reach 1000RU, show the Mass lost on miRNA detection chip surface.Other two kinds of probe RSV and
The decline (only declining 260 and 382RU in 5 hours) that rna probe point corresponding for ADV is the most small, is specifically shown in Table 1 and Fig. 2.
In table 1.RNA micro-array chip, rna probe is degraded situation over time
| Time (s) | 0 | 2800 | 3600 | 5400 | 7200 | 9000 | 10800 | 12600 | 14400 | 16200 | 18000 |
| RSV | 0 | 0 | -8 | -30 | -80 | -100 | -150 | -180 | -200 | -221 | -260 |
| ADV | 0 | 0 | -12 | -42 | -110 | -200 | -230 | -300 | -320 | -360 | -382 |
| H1N1 | 0 | 0 | -112 | -625 | -1100 | -1335 | -1421 | -1534 | -1620 | -1690 | -1723 |
Note: in table 1, unit is: resonance angle unit/Ru.
More than test result indicate that employing RNase H specific for hydrolysis detection Respirovirus have preferable specificity and
Sensitivity.
It is appreciated that in other embodiments, is not limited to Respirovirus for miRNA to be measured, the detection side of this miRNA
Method can be widely applied to the detection of various miRNA, and the result obtained both can be as diagnosis data in the middle of some diseases, also
Help can be provided as the function that scientific data is research miRNA further.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (3)
1. a miRNA detection chip, it is characterised in that include substrate, be located at described suprabasil chromium film, be located at described chromium film
On golden film, described gold film on the 12-sulfydryl dodecylic acid self-assembled monolayer introduced in self assembly mode and with described
The rna probe that self-assembled monolayer combines；Described self-assembled monolayer is fixed on described gold film surface by sulfydryl；Described RNA visits
Pin contains the specific sequence for detecting miRNA to be measured, and described specific sequence can be with the cDNA formed by miRNA reverse transcription
Hybridization；It is connected by amido link between one end of described rna probe and self-assembled monolayer；The sequence of described rna probe is SEQ
At least one in ID NO.1, SEQ ID NO.2 and SEQ ID NO.3；The thickness of described chromium film is 1～2nm；Described gold film
Thickness be 50nm；The other end of described rna probe connects biotin.
2. the miRNA detection kit containing miRNA detection chip as claimed in claim 1.
3. the manufacture method of a miRNA detection chip, it is characterised in that comprise the steps:
Build the rna probe for detecting miRNA to be measured, and carry out amido modified in one end of described rna probe, described RNA
The other end of probe connects has biotin, described rna probe to contain the specific sequence for detecting miRNA to be measured, described spy
Opposite sex sequence can be with the cDNA hybridization formed by miRNA reverse transcription, and the sequence of described rna probe is SEQ ID NO.1, SEQ ID
At least one in NO.2 and SEQ ID NO.3；
At clean substrate surface one layer of chromium film of evaporation, then it is deposited with one layer of golden film, the thickness of described chromium film on described chromium film surface
Be 1～2nm, described gold film thickness be 50nm, by containing described chromium film and described gold film substrate clean up be placed on dense
In the degree 12-sulfydryl dodecylic acid ethanol solution for 10mmol/L, ambient temperature overnight is hatched, and makes 12-sulfydryl dodecylic acid pass through mercapto
Base is self-assembled to the surface of described gold film, and after taking-up, dehydrated alcohol is cleaned, and dries up with nitrogen, obtains being modified with the core of carboxyl
Sheet；
The described chip being modified with carboxyl is placed in 1-ethyl-3 (3-dimethylamino-propyl) carbodiimide and N-maloyl
In the mixed solution of imines, the carboxyl to described gold film surface carries out activation processing, by described rna probe point sample to described chip
Surface, the amino of described rna probe one end forms amido link with described carboxyl reaction and described rna probe is fixed on described core
Sheet surface, i.e. obtains described miRNA detection chip.
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| CN105039564B (en) * | 2015-08-18 | 2018-09-04 | 深圳国际旅行卫生保健中心 | MiRNA detection chips and preparation method thereof and application |
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
| CN108020258A (en) * | 2017-11-09 | 2018-05-11 | 清华大学 | The method for preparing pattern visual under polarisation |
| CN110554300B (en) * | 2019-09-05 | 2024-03-29 | 佛山市国星半导体技术有限公司 | Detection device and detection method for detecting hydrolysis resistance of LED chip |
| CN110907643A (en) * | 2019-12-02 | 2020-03-24 | 中国科学院重庆绿色智能技术研究院 | Preparation method of escherichia coli detection chip and detection chip |
| CN111595736B (en) * | 2020-05-13 | 2021-04-27 | 南京大学 | Plasmon phase imaging system of single nano particle and coupling effect measuring method |
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