CN104293898A - miRNA detection chip, manufacturing method and application thereof - Google Patents
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Abstract
The invention relates to an miRNA detection chip, which comprises a substrate, a chromium film arranged on the substrate, a gold film located on the chromium film, 12- sulfydryl lauric acid self-assembled monolayer introduced by a self-assembly way on the gold film and an RNA probe combined with the self- assembly monolayer. The specific RNA sequence of the miRNA detection chip can hybridize with cDNA formed by miRNA reverse transcriptase to form a hybridization sequence. When the cDNA formed by reverse transcriptase of the miRNA to be tested can hybridize with the specific RNA sequence, a RNase H is used for enzymatic hydrolysis of the specific RNA sequence in the RNA-cDNA hybrid, and the released cDNA can repeatedly combine with specific RNA sequence on the chip surface, and then hydrolysis is carried out until the RAN probe on the chip surface is completely hydrolyzed, thus forming an amplification detection based on non-PCR. The amplification detection has simple operation, and can realize high throughput and rapid detection of miRNA In addition, the invention also relates to a manufacturing method and application of the miRNA detection chip.
Description
Technical field
The present invention relates to biochemistry detection field, especially relate to a kind of miRNA detection chip, its making method and application.
Background technology
MicroRNA (Microrna, also known as miRNA) is the strand microRNA of body endogenous expression, is positioned at Genome noncoding regions, has high conservative type, timing and tissue specificity.MiRNA stops protein expression by being combined with the Incomplete matching of said target mrna.Single miRNA can regulate and control thousands of target gene.Research shows, miRNA can be produced by virus, and such as influenza virus have expressed 8 species specificity miRNA (miRNA-1254 in host cell copies, miRNA-1272, miRNA-17-5p, miRNA-17-3p, miRNA-106B, miRNA-106B, miRNA-124-a and miRNA-124).Research also shows that the most of miRNA of serum is wrapped up by vesica, the opposing of RNA enzyme is prevented to the degraded of nuclease, comparatively stable, room temperature Absorbable organic halogens 4h, multigelation twice is all unaffected, circulation ratio and comparison of coherence good, be applicable to very much making clinical serum mark, for the early diagnosis of disease provides new approaches.
In genome research, biochip technology platform provides effective means support for understanding gene function, Rapid identification, is widely used in the field such as qualification, medical diagnosis of gene expression analysis, virus and bacterium.But traditional biochip technology often needs the mark such as fluorescence, radioactivity, enzyme or needs PCR to realize the amplification of signal, operate too loaded down with trivial details, easily produce pollution and also cost higher, be difficult to the analysis adapting to large-scale gene.
Summary of the invention
Based on this, be necessary to provide a kind of easy and simple to handle and the miRNA detection chip of high throughput testing, its making method and application can be realized.
A kind of miRNA detection chip, comprises substrate, is located at described suprabasil chromium film, the golden film be located on described chromium film, the 12-sulfydryl dodecylic acid self-assembled monolayer introduced in self-assembly mode on described golden film and the rna probe be combined with described self-assembled monolayer; Described self-assembled monolayer is fixed on described golden film on the surface by sulfydryl; Described rna probe contains the specific sequence for detecting miRNA to be measured, and described specific sequence can be hybridized with the cDNA formed by miRNA reverse transcription; One end of described RAN probe is connected by amido linkage with between self-assembled monolayer.
Wherein in an embodiment, the thickness of described chromium film is 1 ~ 2nm.
Wherein in an embodiment, the thickness of described golden film is 50nm.
Wherein in an embodiment, the other end of described rna probe is connected with vitamin H.
Wherein in an embodiment, described rna probe is respectively equipped with 20 thymus pyrimidines be directly connected with described specific sequence at the two ends of described specific sequence.
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, the SEQ ID NO.2 in sequence table and at least one in SEQ ID NO.3.
A kind of miRNA detection kit containing the miRNA detection chip described in above-mentioned any embodiment.
A making method for miRNA detection chip, comprises the steps:
Build the specific sequence for detecting miRNA to be measured, and carry out amido modified in one end of described specific sequence, described specific sequence can be hybridized with the cDNA formed by miRNA reverse transcription;
At substrate surface evaporation one deck chromium film of cleaning, again at described chromium film surface evaporation one deck gold film, substrate containing described chromium film and described golden film being cleaned up and being placed on concentration is in the 12-sulfydryl dodecylic acid ethanolic soln of 10mmol/L, ambient temperature overnight is hatched, 12-sulfydryl dodecylic acid is made to be self-assembled to the surface of described golden film by sulfydryl, take out afterwash, and dry up with nitrogen, obtain the chip being modified with carboxyl;
The described chip being modified with carboxyl being placed in 1-ethyl-3(3-dimethylamino-propyl) carboxyl of mixing solutions to described golden film surface of carbodiimide and N-hydroxysuccinimide carry out activation treatment, by described specific sequence point sample to described chip surface, amino and the described carboxyl reaction of described specific sequence one end form amido linkage and described specific sequence are fixed on described chip surface, namely obtain described miRNA detection chip.
A detection method of miRNA, comprises the steps:
Extract miRNA to be measured, and with the described miRNA extracted for template, under the effect of reversed transcriptive enzyme, the cDNA sample that synthesis is complementary;
MiRNA detection chip described in above-mentioned any embodiment or the miRNA detection chip that makes according to the method described above are placed in the reaction tank of surface plasma resonance imaging instrument, take concentration as 10mM, pH be that the phosphoric acid buffer of 7.4 is as moving phase, flow rate set is 2 μ L/s, after the baseline stability of instrument to be imaged, passing into concentration is that the solution of streptavidin of 10 μ g/mL makes the vitamin H of the free terminal of rna probe connect upper Streptavidin, thus the degraded of follow-up rna probe has signal amplification effect;
RNase H is added in cDNA sample, and the cDNA sample containing RNase H is placed in described reaction tank, described imager is used to detect the rna probe degraded situation on miRNA detection chip surface in real time, obtain the degradation amount of the rna probe on miRNA detection chip surface by comparing the baseline changing value passed into before and after cDNA sample, thus obtain the content of miRNA to be measured.
Specific RNA sequence in above-mentioned miRNA detection chip can be hybridized with the cDNA formed by miRNA reverse transcription and formed hybridization sequences, when the cDNA that miRNA reverse transcription to be measured is formed can be hybridized 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, be hydrolyzed 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-throughput rapid detection of miRNA can be realized.
Accompanying drawing explanation
Fig. 1 is the structural representation of the miRNA detection chip of an embodiment;
Fig. 2 be surface plasma resonance imaging instrument monitoring pass into H1N1cDNA analyte (concentration containing RNase H is 1.0 units/μ L) afterwards miRNA detection chip surface rna probe degradation process schematic diagram.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments miRNA detection chip, its preparation method and application are described in further detail below.
As shown in Figure 1, the miRNA detection chip 100 of an embodiment comprises substrate 110, chromium film 120, golden film 130, self-assembled monolayer 140 and rna probe 150.
Substrate 110 is flat glass substrate.Chromium film 120 is located at the side of substrate 110 on the surface, and thickness is 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 golden film 120 on the surface by sulfydryl, and the other end is carboxyl terminal.
In the present embodiment, rna probe 150 comprises one according to the specific sequence of the conserved regions sequences Design of Respirovirus, 20 thymus pyrimidines (T) laying respectively at these specific sequence two ends, the vitamin H be connected with the thymus pyrimidine end of one end and the amino (-NH be connected with the thymus pyrimidine end of the other end
2).Concrete in the present embodiment, the sequence of rna probe 150 comprises SEQ ID NO.1, SEQ ID NO.2 in sequence table and SEQ ID NO.3.Be appreciated that in other embodiments, the structure of rna probe 150 is not limited thereto, as can be do not had thymus pyrimidine tumor-necrosis factor glycoproteins or only arranging the thymus pyrimidine sequence etc. of repetition in one end of specific sequence; The quantity of thymus pyrimidine is also not limited to 20; The sequence of rna probe 150 also can be other diseases conserved viral region sequence etc.The end of thymus pyrimidine at one end connects vitamin H, can form the amplification that vitamin H-Streptavidin system realizes detection signal in follow-up testing process.
Amino on rna probe 150 can generate amido linkage with the carboxyl reaction of self-assembled monolayer 140, thus the amido linkage that RAN probe 140 generates by reaction is fixed on golden film 130.
Present embodiment additionally provides a kind of miRNA detection kit containing the miRNA detection chip described in above-mentioned any embodiment.This miRNA detection kit further comprises solution of streptavidin and ribonuclease H (RNase H) etc.
In addition, present embodiment additionally provides a kind of making method of miRNA detection chip, and it comprises the steps:
Step one: build the specific sequence for detecting miRNA to be measured, and carry out amido modified in one end of specific sequence, specific sequence can be hybridized with the cDNA formed by miRNA reverse transcription.
In the present embodiment, in step one, the specific sequence two ends being also included in structure connect the step of 20 thymus pyrimidines respectively, then the end of thymus pyrimidine in specific sequence one end is carried out amido modified.Further, the step of biotin modification is carried out in the end being also included in the thymus pyrimidine of the specific sequence the other end, forms vitamin H-Streptavidin system to realize the amplification of detection signal to facilitate in subsequent detection process.
Step 2: at substrate surface evaporation one deck chromium film of cleaning, again at chromium film surface evaporation one deck gold film, substrate containing chromium film and golden film being cleaned up and being placed on concentration is in the 12-sulfydryl dodecylic acid ethanolic soln of 10mmol/L, ambient temperature overnight is hatched, 12-sulfydryl dodecylic acid is made to be self-assembled to the surface of golden film by sulfydryl, take out afterwash, and dry up with nitrogen, obtain the chip being modified with carboxyl.
In this enforcement side 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) carboxyl of mixing solutions to golden film surface of carbodiimide and N-hydroxysuccinimide carry out activation treatment, by specific sequence point sample to chip surface, the amino of specific sequence one end and carboxyl reaction form amido linkage and specific sequence are fixed on chip surface, namely obtain miRNA detection chip.
Specific RNA sequence in above-mentioned miRNA detection chip can be hybridized with the cDNA formed by miRNA reverse transcription and formed hybridization sequences, when the cDNA that miRNA reverse transcription to be measured is formed can be hybridized 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, be hydrolyzed 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-throughput rapid detection of miRNA can be realized.
Further, present embodiment additionally provides the detection method of a kind of miRNA, and it comprises the steps:
Extract miRNA to be measured, and with this miRNA extracted for template, under the effect of reversed transcriptive enzyme, the cDNA sample that synthesis is complementary;
The miRNA detection chip of above-mentioned making is placed in surface plasma resonance (Surface Plasmon Resonance Imaging, in the reaction tank of SPR) imager, take concentration as 10mM, pH be that the phosphoric acid buffer of 7.4 is as moving phase, flow rate set is 2 μ L/s, after the baseline stability of instrument to be imaged, passing into concentration is that the solution of streptavidin of 10 μ g/mL makes the vitamin H of the free terminal of rna probe connect upper Streptavidin, thus the degraded of follow-up rna probe has signal amplification effect;
RNase H is added in cDNA sample, and the cDNA sample containing RNase H is placed in the reaction tank of SPR imager, SPR imager detects the rna probe degraded situation on miRNA detection chip surface in real time, obtain the degradation amount of the rna probe on miRNA detection chip surface by comparing the baseline changing value passed into before and after cDNA sample, thus obtain the content of miRNA to be measured.
Be below specific embodiment part:
Reagent: RNA extracts test kit (Beijing Tian Ze gene prod), diethanolamine hydrochloride (2-Aminoethanol Hydrochloride) (Japanese TCI Products); 1-ethyl-3(3-dimethylamino-propyl) carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydr-ochloride, EDC) (Canadian Bio Basic Inc Products); 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 analytical pure.
Instrument: C1000Thermal cycler PCR amplification instrument (Bio Rad Laboratories), whizzer, eddy blending machine (German IKAMS company), Hybex Microsamp Incubator, ND-1000 trace ultra-violet and visible spectrophotometer (Nanodrop company of the U.S.) (purposes: be applied to the concentration measuring protein and DNA), SPR imager (
analyzer s-generation product).
The synthesis of rna probe:
First stream H1N1:Biotin-T
20-GGACUACCACGAUUCAAAUGUG-T
20-NH
2(SEQ ID NO.1);
Respiratory syncytial virus (RSV): Biotin-T
20-CCAUGUGAAUUCCCUGCAUCAAU-T
20-NH
2(SEQ ID NO.2);
Adenovirus (ADV): Biotin-T
20-CACCGAGACGUACUUCAGCCUG-T
20-NH
2(SEQ ID NO.3);
Wherein, Biotin is vitamin H.
Experimental procedure:
The synthesis of 1.RAN probe: from the GenBank file needed for NCBI download, utilize the specific sequence of software premier5 designated rna probe in the conserved regions sequence of Respirovirus (H1N1, RSV and ADV).Biotin modification has been carried out again, with the effect making biotin labeled probe can be realized signal amplification by Streptavidin at 5 ' end of this specific sequence.Carry out at the other end of rna probe amido modified, fix 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 ' end of specific sequence).Poly(T) rna probe can be made fully to stretch at solid phase surface as spacerarm and be beneficial to the fixing of probe and hybridization.The sequence of rna probe of synthesis is SEQ ID NO.1, SEQ ID NO.2 in sequence table and SEQ ID NO.3.
2.miRNA detection chip makes: at the glass substrate surface evaporation of cleaning or the chromium film of magnetron sputtering 1 ~ 2nm thickness, then at the surperficial evaporation of chromium film or the golden film of magnetron sputtering 50nm.The chip obtained is soaked in 10mmol/L12-sulfydryl dodecylic acid ethanolic soln immediately after cleaning, ambient temperature overnight is hatched, make sulfydryl orderly be self-assembled to golden film surface, the carboxyl of 12-sulfydryl dodecylic acid dissociates, after taking-up, repeatedly rinse 5 times with dehydrated alcohol, then clean with deionized water, nitrogen dries up; The chip that above-mentioned 12-sulfydryl dodecylic acid the is modified carboxylic group of the EDC/NHS solution activating surface of new configuration, the rna probe high precision point sample instrument Genetix Q-Array Mini be correlated with by Respirovirus or manual point sample, in the specific region of chip, obtain described miRNA detection chip.
3. the process of sample: the RNA of Respirovirus extracts and adopts the RNA of Beijing Tian Ze Reagent Company to extract test kit, and specific experiment operating process is according to the explanation step-by-step operation of test kit.With the miRNA extracted in sample for template, under the effect of reversed transcriptive enzyme, synthesize complementary cDNA sample.
4.SPR rapid detection: the SPR imager of employing is
analyzer s-generation product, is arranged on the miRNA detection chip of making in SPR instrument according to the operating process of instrument.And with phosphate buffer solution (PBS buffer, concentration is 10mM, pH is 7.4), as moving phase, flow rate set is 2L/s.After the baseline stability of instrument to be imaged, pass into Streptavidin (Streptavidin, concentration is 10g/mL) 500s, make the end of rna probe in conjunction with Streptavidin, make the degraded of follow-up RNA have signal amplification effect.In cDNA sample, add RNase H, make the concentration of final RNase H be 1.0 units/L.Pass in the reaction tank of SPR imager by the cDNA sample containing RNase H, SPR detects surperficial RNA degraded situation in real time.The degradation amount of surperficial rna probe is obtained by the baseline changing value comparing the front and back passing into sample.
Experimental result
The present embodiment is to detect influenza virus H1N1 for experimental group, RSV and ADV is control group, utilizes throat swab to extract viral miRNA, with the miRNA extracted for template, under the effect of reversed transcriptive enzyme, synthesizes complementary cDNA as detection sample.The acquisition target of throat swab is through Shenzhen port immigration, infrared detecting group has heating sign, trial inspection has flu symptoms case.
When passing through the cDNA sample of H1N1 virus in experiment, the spr signal of the rna probe point of H1N1 significantly declines, and in 1 hour, spr signal decline reaches 1000RU, shows the Mass lost on miRNA detection chip surface.Decline that the rna probe point that other two kinds of probe RSV and ADV are corresponding is slightly small (only decline in 5 hours 260 and 382RU), specifically 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.
Above experimental result shows that adopting RNase H specific for hydrolysis to detect Respirovirus has good specificity and sensitivity.
Be appreciated that, in other embodiments, Respirovirus is not limited to for miRNA to be measured, the detection method of this miRNA can be widely used in the detection of various miRNA, the result obtained can, as the middle diagnostic data of some diseases, can be also both that the function studying miRNA is further offered help as scientific data.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a miRNA detection chip, it is characterized in that, comprise substrate, be located at described suprabasil chromium film, the golden film be located on described chromium film, the 12-sulfydryl dodecylic acid self-assembled monolayer introduced in self-assembly mode on described golden film and the rna probe be combined with described self-assembled monolayer; Described self-assembled monolayer is fixed on described golden film on the surface by sulfydryl; Described rna probe contains the specific sequence for detecting miRNA to be measured, and described specific sequence can be hybridized with the cDNA formed by miRNA reverse transcription; One end of described RAN probe is connected by amido linkage with between self-assembled monolayer.
2. miRNA detection chip as claimed in claim 1, it is characterized in that, the thickness of described chromium film is 1 ~ 2nm.
3. miRNA detection chip as claimed in claim 1, it is characterized in that, the thickness of described golden film is 50nm.
4. miRNA detection chip as claimed in claim 1, it is characterized in that, the other end of described rna probe is connected with vitamin H.
5. miRNA detection chip as claimed in claim 4, it is characterized in that, described rna probe is respectively equipped with 20 thymus pyrimidines be directly connected with described specific sequence at the two ends of described specific sequence.
6. miRNA detection chip as claimed in claim 5, it is characterized in that, described specific sequence is the conserved sequence of Respirovirus.
7. miRNA detection chip as claimed in claim 6, it is characterized in that, the sequence of described rna probe is SEQ ID NO.1, the SEQ ID NO.2 in sequence table and at least one in SEQ ID NO.3.
8. the miRNA detection kit containing, for example the miRNA detection chip according to any one of claim 1-7.
9. a making method for miRNA detection chip, is characterized in that, comprises the steps:
Build the specific sequence for detecting miRNA to be measured, and carry out amido modified in one end of described specific sequence, described specific sequence can be hybridized with the cDNA formed by miRNA reverse transcription;
At substrate surface evaporation one deck chromium film of cleaning, again at described chromium film surface evaporation one deck gold film, substrate containing described chromium film and described golden film being cleaned up and being placed on concentration is in the 12-sulfydryl dodecylic acid ethanolic soln of 10mmol/L, ambient temperature overnight is hatched, 12-sulfydryl dodecylic acid is made to be self-assembled to the surface of described golden film by sulfydryl, take out afterwash, and dry up with nitrogen, obtain the chip being modified with carboxyl;
The described chip being modified with carboxyl being placed in 1-ethyl-3(3-dimethylamino-propyl) carboxyl of mixing solutions to described golden film surface of carbodiimide and N-hydroxysuccinimide carry out activation treatment, by described specific sequence point sample to described chip surface, amino and the described carboxyl reaction of described specific sequence one end form amido linkage and described specific sequence are fixed on described chip surface, namely obtain described miRNA detection chip.
10. a detection method of miRNA, is characterized in that, comprises the steps:
Extract miRNA to be measured, and with the described miRNA extracted for template, under the effect of reversed transcriptive enzyme, the cDNA sample that synthesis is complementary;
MiRNA detection chip such as according to any one of claim 1-7 is placed in the reaction tank of surface plasma resonance imaging instrument, take concentration as 10mM, pH be that the phosphoric acid buffer of 7.4 is as moving phase, flow rate set is 2 μ L/s, after the baseline stability of instrument to be imaged, passing into concentration is that the solution of streptavidin of 10 μ g/mL makes the vitamin H of the free terminal of rna probe connect upper Streptavidin, thus the degraded of follow-up rna probe has signal amplification effect;
RNase H is added in cDNA sample, and the cDNA sample containing RNase H is placed in described reaction tank, described imager is used to detect the rna probe degraded situation on miRNA detection chip surface in real time, obtain the degradation amount of the rna probe on miRNA detection chip surface by comparing the baseline changing value passed into before and after cDNA sample, thus obtain the content of miRNA to be measured.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039564A (en) * | 2015-08-18 | 2015-11-11 | 深圳国际旅行卫生保健中心 | miRNA (microribonucleic acid) detection chip, and manufacturing method and application thereof |
| 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 |
| WO2019090886A1 (en) * | 2017-11-09 | 2019-05-16 | 清华大学 | Method for preparing pattern visible under polarized light |
| CN110554300A (en) * | 2019-09-05 | 2019-12-10 | 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 |
| CN111595736A (en) * | 2020-05-13 | 2020-08-28 | 南京大学 | Plasmon phase imaging system of single nano particle and coupling effect measuring method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181579A (en) * | 2011-04-11 | 2011-09-14 | 深圳国际旅行卫生保健中心 | Respiratory virus detection kit and detection method |
| WO2012009373A2 (en) * | 2010-07-12 | 2012-01-19 | Gen-Probe Incorporated | Compositions and assays to detect swine h1n1 influenza a virus, seasonal h1 influenza a virus and seasonal h3 influenza a virus nucleic acids |
| US20120208722A1 (en) * | 2010-10-19 | 2012-08-16 | Richard Dluhy | Surface enhanced raman spectroscopy platforms and methods |
| CN103898194A (en) * | 2012-12-24 | 2014-07-02 | 深圳国际旅行卫生保健中心 | Photo-crosslinking functionalized gene chip, preparation method thereof and detection kit |
-
2013
- 2013-07-17 CN CN201310302860.6A patent/CN104293898B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012009373A2 (en) * | 2010-07-12 | 2012-01-19 | Gen-Probe Incorporated | Compositions and assays to detect swine h1n1 influenza a virus, seasonal h1 influenza a virus and seasonal h3 influenza a virus nucleic acids |
| US20120208722A1 (en) * | 2010-10-19 | 2012-08-16 | Richard Dluhy | Surface enhanced raman spectroscopy platforms and methods |
| CN102181579A (en) * | 2011-04-11 | 2011-09-14 | 深圳国际旅行卫生保健中心 | Respiratory virus detection kit and detection method |
| CN103898194A (en) * | 2012-12-24 | 2014-07-02 | 深圳国际旅行卫生保健中心 | Photo-crosslinking functionalized gene chip, preparation method thereof and detection kit |
Non-Patent Citations (3)
| Title |
|---|
| ROBERTA D’AGATA,GIUSEPPE SPOTO: "Surface plasmon resonance imaging for nucleic acid detection", 《ANAL BIOANAL CHEM》 * |
| TERRY T. GOODRICH, ET AL: "Enzymatically Amplified Surface Plasmon Resonance Imaging Method Using RNase H and RNA Microarrays for the Ultrasensitive Detection of Nucleic Acids", 《ANAL. CHEM.》 * |
| 徐华，顾大勇: "基于等离子共振原理的兽药联检芯片的建立", 《广东省生物医学工程学会成立32周年纪念大会暨2012广州（国际）生物医学工程学术大会论文集》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039564A (en) * | 2015-08-18 | 2015-11-11 | 深圳国际旅行卫生保健中心 | miRNA (microribonucleic acid) detection chip, and manufacturing method and application thereof |
| 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 |
| WO2019090886A1 (en) * | 2017-11-09 | 2019-05-16 | 清华大学 | Method for preparing pattern visible under polarized light |
| CN110554300A (en) * | 2019-09-05 | 2019-12-10 | 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 |
| 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 |
| CN111595736A (en) * | 2020-05-13 | 2020-08-28 | 南京大学 | Plasmon phase imaging system of single nano particle and coupling effect measuring method |
| 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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