CN102492772A - Molecule detection signal amplification technique - Google Patents
Molecule detection signal amplification technique Download PDFInfo
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- CN102492772A CN102492772A CN2011103947086A CN201110394708A CN102492772A CN 102492772 A CN102492772 A CN 102492772A CN 2011103947086 A CN2011103947086 A CN 2011103947086A CN 201110394708 A CN201110394708 A CN 201110394708A CN 102492772 A CN102492772 A CN 102492772A
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Abstract
The invention relates to novel molecule detection signal amplification technique, which is characterized in that the molecule detection signal amplification technique is a biomolecule detection method with high sensitivity which is formed by streptavidin, an antigen, an antibody, polypeptide and the like which are marked by quantum dots through tyramine signal amplification and silver strengthening dyeing signal amplification. The detection method comprises the following steps: the biomolecule to be detected is combined with nucleic acid, the antibody, the antigen, the polypeptide and the like which are fixed on a solid phase material, the quantum dots are introduced to molecule to be detected through biomolecule specificity combination, silver strengthening dyeing is carried out on the quantum dots by using silver dyeing reagents, and signals are amplified. Obtained signals can be scanned through common optical scanners, analyzed through common imaging instruments or observed through naked eyes. The detection method achieves high sensitivity detection of the biomolecule on one hand, and reduces application cost of biomolecule detection on the other hand.
Description
Technical field
The present invention relates to a kind of molecular detection technology.
Background technology
Biomolecules comprises protein, nucleic acid, lipid, carbohydrate.Molecular detecting method mainly comprises analyses immunity percolation, immunochromatography, biochip technology, round pcr etc.Wherein biochip technology comprises gene chip, protein chip, cell chip, organization chip etc. again; It is a biological new and high technology of gathering numerous subjects such as life science, microtronics, physics, chemistry and Materials science; Be the important means of efficiently obtaining bioinformations such as nucleic acid, protein on a large scale, have broad application prospects in various fields such as gene expression profile research, disease Molecular Detection and large-scale medicine screenings.At present biochip test is generally used fluorescence detection, the material of use such as Cy3, Cy5, FITC etc., directly or indirect labelling on DNA or RNA, antibody, albumen equimolecular, the fluorescent signal of generation adopts laser confocal scanning to detect.The shortcoming of this method is that detection sensitivity is low, detecting instrument costs an arm and a leg, and fluorescent signal is prone to saturated, easy cancellation and poor stability in addition.Thereby limited the use of biochip, especially in the promotion and application of middle-size and small-size medical institutions.
(quantum dots QDs) claims semiconductor-quantum-point again to quantum dot.The semiconductor nanocrystal of the size＜100nm that forms by II-VI family element (like cadmium selenide, Cadmium Sulfide etc.) or III-V family element (like indium phosphide, indium arsenide).Quantum dot has unique physics and chemical property such as surface-area effects, small-size effect, microcosmic quantum tunneling effect, makes it have unique optics and electrical properties.Quantum dot is little because of its grain diameter, and specific surface area is big, and the avtive spot that the surface has is many, and quantum dot exists such as nanometer gold, nanometer silver similarly as the electron transport carrier, but in the presence of reductive agent catalysis Ag
+Be reduced into simple substance Ag and be deposited on surperficial catalytic capability.Small-size effect makes the quantum dot particle have very strong catalytic reduction effect, can silver ion reduction on every side be become silver-colored particle; And silver-colored particle can catalytic reduction silver ions on every side.It is many more that the katalysis of this cascade waterfall makes that silver-colored particle gathers more, tightly wraps up the quantum dot particle and be accumulated into bulk silver shell, forms almost macroscopic black particle, is may observe with common light microscopic even naked eyes.Utilize the catalysis characteristics of quantum dot during the distribution situation of quantum dot in tissue visualization such as Chou, strengthen the direct qualitative CdSe/ZnS quantum dot of having observed of staining in each in-house distribution through tissue slice silver.Compare with fluorescence imaging method simultaneously, silver staining method more can accurately be located the distribution situation of quantum dot, does not receive quenching of fluorescence factor affecting in the organism, and method is quick, directly perceived, easy.Xu Guofeng etc. are detected object with the human IgG, and the quantum dot visible detection method is studied.
TSA (tyramine signal amplification, the tyrasamine signal amplifies) is proposed in 1989 by people such as Bobrow.The principle of TSA is a large amount of tyrasamine molecule depositions under the catalysis of horseradish peroxidase.1992, Adams at first introduced immunohistochemical methods with the principle of TSA, is applied to antigen or detection of antibodies.Compare with the vitamin H-Streptavidin method of routine, this technology can improve 100 to 1000 times with detection sensitivity.The TSA technology has obtained widespread use in fields such as immunoblotting, enzyme linked immunologicals.
Summary of the invention
The technical problem that the present invention will solve be to detection signal in the present molecular detecting method be prone to saturated, signal stabilization is poor, detection sensitivity is low, the expensive problem of detecting instrument, provides that a kind of detection signal is stable, detection sensitivity is high and do not need expensive detecting instrument equipment testing method.
Technical solution provided by the invention is the detection method that just quantum dot-labeled, tyrasamine signal amplification technique and silver-colored enhancing signal amplifying technique combine, and biomolecules to be detected is combined with probe, antibody, polypeptide, antigen on being fixed on solid phase; Nucleic acid, antigen, antibody, polypeptide, albumen combine with the molecules detected specificity; Combine quantum dot is incorporated on the testing molecule through the biomolecules specificity; Use silver-colored transfection reagent to carry out silver and strengthen dyeing, signal is amplified; The gained signal can also can detect by an unaided eye with common optical scanning equipment scanning analysis.Present method has realized the high-sensitivity detection of biomolecules on the one hand, has realized the visual detection of biomolecules on the other hand.
A kind of Molecular Detection signal amplification technique detection method, its characteristics may further comprise the steps:
One, the application in the gene chip
1. the purifying of testing gene, amplification and mark: purification process is referring to molecular cloning experiment guide (Science Press, author J. Sa nurse Brooker); Gene amplification method adopts polymerase chain reaction (PCR) or post transcription cloning polymerase chain reaction (RT-PCR); Nucleic acid labeling methods is that the downstream primer employing end-labelling of PCR is introduced vitamin H, carries out PCR or RT-PCR reaction then.
2. the preparation of chip and hybridization: some system institute design probe microarray on aldehyde group modified sheet glass; PCR product and gene chip hybridization with gene to be detected.
3. tyrasamine signal amplification process: the respective regions that will hybridize the gene chip of completion adds the horseradish peroxidase (Streptavidin-HRP) of marked by streptavidin.Attend the vitamin H and the specific introducing HRP that combines of streptavidin of institute's mark through target; Add biotin labeled tyrasamine (Biotin-Tyramine) then, make the tyrasamine deposition of a large amount of mark vitamin Hs through enzyme catalysis.
4. quantum dot silver dyes process: the respective regions at gene chip adds marked by streptavidin quantum dot (Streptavidin-QDs).Utilize specific combination of vitamin H and Streptavidin that quantum dot is introduced once more; Add silver-colored staining reagent and utilize quantum dot small size, catalytic reduction effect, can silver ion reduction on every side be become silver-colored particle; And silver-colored particle can catalytic reduction silver ions on every side.Form macroscopic detection signal.
5. signal processing: hybridization signal can visual inspection also can be used common optical scanning equipment scanning analysis.
Two, the application in the protein chip
1. the preparation of chip: some system antigen or antibody on aldehyde group modified sheet glass, sealing.
2. testing sample combines with the protein chip specificity: the respective regions at protein chip adds testing sample.
3. biotin labeled antigen or antibody combine with the protein chip specificity: the respective regions at protein chip adds biotin labeled antigen or antibody.
4. silver strengthens dyeing signal amplification process: the respective regions at protein chip adds marked by streptavidin quantum dot (Streptavidin-QDs).Utilize specific combination of vitamin H and Streptavidin that quantum dot is introduced once more; Add silver-colored staining reagent and utilize quantum dot small size, catalytic reduction effect, can silver ion reduction on every side be become silver-colored particle; And silver-colored particle can catalytic reduction silver ions on every side.Form macroscopic detection signal.
5. signal processing; Hybridization signal can visual inspection also can be used common optical scanning equipment scanning analysis.
The present invention has the following advantages and effect:
1. present method provides a kind of Molecular Detection signal amplification technique.This detection method has higher detection sensitivity and specificity.
2. present method detection signal can be used common optical scanning equipment scanning analysis or visual inspection, has reduced the Molecular Detection cost, helps the molecular detection technology promotion and application.
3. present method detection signal is stable, helps result's preservation and comparison.
Description of drawings
Fig. 1 dyes visual detection method synoptic diagram for the quantum dot-labeled silver of gene chip.
Fig. 2 is the detected result signal scanning figure of Ev71 virus, and relatively the outer transcribe rna template of Ev71 virosome is (10
6Copies/ μ L to 10
2Copies/ μ L) the intensity of hybridization signal time.
Fig. 3 is the detected result signal scanning figure of rotavirus, and relatively rotavirus in-vitro transcription RNA template is (10
6Copies/ μ L to 10
2Copies/ μ L) the intensity of hybridization signal time.
Fig. 4 is the detected result signal scanning figure of the Sa Qi B3 of section virus, the Sa Qi B4 of section virus, the Sa Qi B5 of section virus, poliomyelitis 1 virus, poliomyelitis 2 viruses, poliomyelitis 3 viruses, Ai Ke 30 viruses, the Sa Qi A16 of section virus, enterovirns type 71 and rotavirus virus.
Fig. 5 is for detecting the hepatitis B surface antigen HbsAg in the different hepatitis B patient serum.The consequential signal scintigram.
Embodiment
Embodiment 1
Detect Ev71 virus and rotavirus
1. self-control digestive tube virus detects gene chip.
2. with Ev71 virus and rotavirus cell culture fluid, press the operation of QIAamp Viral RNA Mini Kit nucleic acid extraction kit (QIAGEN) specification sheets, obtain purified RNA.Use reverse transcription test kit PrimeScript One Step RT-PCR Kit Ver.2 (precious biotechnology ltd) to carry out reverse transcription; Reaction system is 20 μ L:2 * One Step Buffer10 μ L, PrimeScript 1 Step Enzyme Mix 0.8 μ L, 0.2 μ L (upstream primer; 10 μ M), 1 μ L (downstream primer; 10 μ M), RNA template 5 μ L, nuclease free water is supplied volume to 20 μ L system.Reaction conditions: rt: 50 ℃, 30min; Sex change in advance: 94 ℃, 2min; Pcr amplification: 94 ℃, 30s; 50 ℃, 30s; 72 ℃, 30s; Extend: 72 ℃, 10min.Totally 45 are circulated in pcr amplification.
3. the gene chip that will prepare completion cleans 20s with 0.2%SDS, then with washed with de-ionized water 20s, dries subsequent use.
4. be template RT-PCR amplification with Ev71 virus and the outer transcribe rna of rotavirus respectively, delivery plate concentration is 10
6Copies/ μ L, 10
5Copies/ μ L, 10
4Copies/ μ L, 10
3Copies/ μ L and 10
2The RT-PCR amplified production of copies/ μ L respectively get 5 μ L respectively with 5 μ L hybridization solution (5 * SSC, 2.5% methane amide, 0.2%SDS) uniform mixing; Mixed solution is joined chip zone accordingly, and chip places hybridizing box, 45 ℃ of incubation 60min; After hybridization is accomplished chip successively with washing lotion A (1 * SSC, 0.2%SDS), washing lotion B (0.2 * SSC); (0.1 * SSC) respectively washs 20s to washing lotion C, dries.
5. get the gene chip of having hybridized completion and add Streptavidin-HRP (dilution in 1: 500 in corresponding zone; 1 * PBS-0.5%BSA) 10 μ L; 37 ℃ of incubation 30min, (three each 20s of 1 * PBS+0.05%Tween20) repeated washing dry with PBST to take out chip; (37 ℃ of incubation 30min take out chip with three each 20s of PBST repeated washing, dry for dilution in 1: 1000,1 * PBS-0.5%BSA) 10 μ L to add Biotin-Tyramine at respective regions.
6. add Streptavidin-QDs (dilution in 1: 50,2XPBS-0.1%BSA-0.01% Thiomersalate) at respective regions, with three each 20s of PBST washing, dry behind 37 ℃ of incubation 30min; Silver strengthens reagent A liquid and B liquid equal-volume mixes, and each zone is with the about 30 μ L of mixed solution, and lucifuge colour developing 5-6min develops the color, and signal is visual visible, and colour developing finishes with deionized water rinsing.
7. the hybridization signal naked eyes promptly can be observed, with common optical scanning equipment sweep record and analysis.
The Sa Qi B3 of detection section virus, the Sa Qi B4 of section virus, the Sa Qi B5 of section virus, poliomyelitis 1 virus, poliomyelitis 2 viruses, poliomyelitis 3 viruses, Ai Ke 30 viruses, the Sa Qi A16 of section virus, enterovirns type 71, rotavirus.
Self-control digestive tube virus detects gene chip.
1. with the Sa Qi B3 of section virus, the Sa Qi B4 of section virus, the Sa Qi B5 of section virus, poliomyelitis 1 virus, poliomyelitis 2 viruses, poliomyelitis 3 viruses, Ai Ke 30 viruses, the Sa Qi A16 of section virus, Enterovirus 71, rotavirus cell culture fluid; Press the operation of QIAamp Viral RNA Mini Kit nucleic acid extraction kit (QIAGEN) specification sheets, obtain purified RNA solution.Use reverse transcription test kit PrimeScript One Step RT-PCR Kit Ver.2 (precious biotechnology ltd) to carry out reverse transcription; Reaction system is 20 μ L:10 μ L (2 * One Step Buffer), 0.8 μ L (PrimeScript 1Step Enzyme Mix), 0.2 μ L (upstream primer; 10 μ M), 1 μ L (downstream primer; 10 μ M), 5 μ L (LRNA template), nuclease free water is supplied volume to 20 μ L system.Reaction conditions: rt: 50 ℃, 30min; Sex change in advance: 94 ℃, 2min; Pcr amplification: 94 ℃, 30s; 50 ℃, 30s; 72 ℃, 30s; Extend: 72 ℃, 10min.Totally 45 are circulated in the pcr amplification stage.
2. the gene chip that will prepare completion cleans 20s with 0.2%SDS, then with washed with de-ionized water 20s, dries subsequent use.
3. be template RT-PCR amplification with the Sa Qi B3 of section virus, the Sa Qi B4 of section virus, the Sa Qi B5 of section virus, poliomyelitis 1 virus, poliomyelitis 2 viruses, poliomyelitis 3 viruses, Ai Ke 30 viruses, the Sa Qi A16 of section virus, Enterovirus 71 and rotavirus in-vitro transcription RNA respectively, delivery plate concentration is 10
4The RT-PCR amplified production of copies/ μ L respectively get 5 μ L respectively with 5 μ L hybridization solution (5 * SSC, 2.5% methane amide, 0.2%SDS) uniform mixing; Mixed solution is joined chip zone accordingly, and chip places hybridizing box, 45 ℃ of incubation 60min; After hybridization is accomplished chip successively with washing lotion A (1 * SSC, 0.2%SDS), washing lotion B (0.2 * SSC); (0.1 * SSC) respectively washs 20s to washing lotion C, dries.
4. get the gene chip of having hybridized completion and add Streptavidin-HRP (dilution in 1: 500 in corresponding zone; 1 * PBS-0.5%BSA) 10 μ L; 37 ℃ of incubation 30min, (three each 20s of 1 * PBS+0.05%Tween20) repeated washing dry with PBST to take out chip; (37 ℃ of incubation 30min take out chip with three each 20s of PBST repeated washing, dry for dilution in 1: 1000,1 * PBS-0.5%BSA) 10 μ L to add Biotin-Tyramine at respective regions.
5. add Streptavidin-QDs (dilution in 1: 50,2XPBS-0.1%BSA-0.01% Thiomersalate) at respective regions, with three each 20s of PBST washing, dry behind 37 ℃ of incubation 30min; Silver strengthens reagent A liquid and B liquid equal-volume mixes, and each zone is with the about 30 μ L of mixed solution, and lucifuge colour developing 5-6min develops the color, and signal is visual visible, and colour developing finishes with deionized water rinsing.
6. the hybridization signal naked eyes promptly can be observed, with common optical scanning equipment sweep record and analysis.
Embodiment 3
Detect the hepatitis B surface antigen HBsAg among the patients serum
Chip preparation: some system HBsAb (0.5mg/mL) on aldehyde group modified sheet glass; BSA (1mg/mL) negative control.Behind 4 ℃ of held 12h,, use PBST (1 * PBS+0.5% tween) to give a baby a bath on the third day after its birth then time to dry 4 ℃ of preservations subsequent use with confining liquid (1 * PBS+25% calf serum) sealing 2h.
1. get the chip that has prepared and add sample serum (dilution in 1: 5) successively, 37 ℃ of incubation 30min, (three each 20s of 1 * PBS+0.05%Tween20) repeated washing dry with PBST to take out chip.
Respective regions add HbsAb-Biotin (1: 1000,1 * PBS-0.5%BSA, 1mg/mL) 37 ℃ of incubation 30min, (three each 20s of 1 * PBS+0.05%Tween20) repeated washing dry with PBST to take out chip.
3. add Streptavidin-QDs (dilution in 1: 50,2XPBS-0.1%BSA-0.01% Thiomersalate) at respective regions, with three each 20s of PBST washing, dry behind 37 ℃ of incubation 30min; Silver strengthens reagent A liquid and B liquid equal-volume mixes, and each zone is with the about 30 μ L of mixed solution, and lucifuge colour developing 5-6min develops the color, and signal is visual visible, and colour developing finishes with deionized water rinsing.
4. the detection signal naked eyes promptly can be observed, with common optical scanning equipment sweep record and analysis.
Claims (12)
1. biomolecule detection signal amplification technique is characterized in that quantum dot-labeled Streptavidin, antigen, antibody, polypeptide and the biomolecules specificity cohesive process, tyrasamine signal amplification process and the silver that are fixed on the solid phase material are strengthened dyeing signal amplification process.
2. quantum dot according to claim 1 is characterized in that:
Quantum dot is that diameter is less than the 100nm semiconductor nano material.
3. like the said quantum dot of claim 2, it is characterized in that:
Quantum dot is by two or more is elementary composition and by II-VI family or III-V family element and other elementary composition nano particles in II-VI family or the III-V family element.
4. like the said quantum dot of claim 2, it is characterized in that:
Quantum dot be diameter between 1-100nm, the semiconductor nanocrystal particle with nucleocapsid structure that can emitting fluorescence is like ZnS, CdS, HgS, CdSe, CdTe, MgSe; MgS, MgTe, ZnO, ZnTe, CdSe/HgSe, CdS/ZnS, CdS/Ag2S; CdS/PbS, CdS/Cd (OH) 2, CdS/HgS, CdSe/CdS, MgS, MgSe, MgTe; CaS, CaSe, CaTe, SrS, SrSe, BaS, BaSe.Form and be the nano-complex particle that karyomorphism becomes by above any two kinds and above nanoparticle by a kind of and more than one nanoparticles.
5. solid phase material according to claim 1 is characterized in that:
Solid phase material comprises: glass, plastics, metal, macromolecular material and PS, NC film, cellulose membrane, 96 orifice plates.
6. detection method according to claim 1 is characterized in that may further comprise the steps:
(1) molecules detected combines with the probe molecule specificity;
(2) antigen or antibody combine with the molecules detected specificity;
(3) tyrasamine signal amplification process;
(4) silver strengthens dyeing signal amplification process;
(5) signal processing;
7. detection method according to claim 1 is characterized in that may further comprise the steps:
(1) molecules detected combines with the probe molecule specificity;
(2) biotin labeled antigen, antibody or albumen combine with the molecules detected specificity;
(3) quantum dot-labeled Streptavidin specificity combines;
(4) silver strengthens dyeing signal amplification process;
(5) signal processing;
8. detection method according to claim 1 is characterized in that may further comprise the steps:
(1) purifying of testing gene, amplification are introduced vitamin H to the downstream primer end-labelling of amplification and are carried out nucleic acid marking;
(2) hybridization;
(3) quantum dot-labeled Streptavidin specificity combines;
(4) silver strengthens dyeing signal amplification process;
(5) signal processing;
9. detection method according to claim 1 is characterized in that may further comprise the steps:
(1) purifying of testing gene, amplification are introduced vitamin H to the downstream primer end-labelling of amplification and are carried out nucleic acid marking;
(2) hybridization;
(3) tyrasamine signal amplification process;
(4) silver strengthens dyeing signal amplification process;
(5) signal processing;
10. according to claim 6,9 described detection methods, it is characterized in that step (3) tyrasamine signal amplification process:
Accomplish the horseradish peroxidase that specificity bonded respective regions adds the streptavidin mark.Through the specific introducing HRP that combines of vitamin H and streptavidin; Add biotin labeled tyrasamine then, make the tyrasamine molecule deposition of a large amount of mark vitamin Hs through enzyme catalysis.
11., it is characterized in that step (4) silver strengthens dyeing signal amplification process: add streptavidin mark quantum dot at respective regions according to claim 6,7,8,9 described detection methods.Vitamin H combines with Streptavidin is specific, and quantum dot is incorporated on the molecules detected; Add silver-colored staining reagent and utilize the effect of quantum dot small size catalytic reduction, can silver ion reduction on every side be become silver-colored particle; And the silver ions of silver-colored particle around can catalytic reduction forms macroscopic detection signal.
12., it is characterized in that step (5) signal processing according to claim 6,7,8,9 described detection methods:
Signal can visual inspection also can be used common optical scanning equipment scanning analysis or common Image-forming instrument record.
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| CN102978295A (en) * | 2012-08-30 | 2013-03-20 | 重庆西南医院 | Pathogenic microorganism nucleic acid amplification-free detection and typing method |
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| CN103333967A (en) * | 2013-07-12 | 2013-10-02 | 湖南工程学院 | Nucleic acid detection method based on microfluidic microbead array chip |
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