CN104458682A - Fluorescence detection method of multifunctional oligonucleotide chain for multiple substances - Google Patents
Fluorescence detection method of multifunctional oligonucleotide chain for multiple substances Download PDFInfo
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- CN104458682A CN104458682A CN201410613314.9A CN201410613314A CN104458682A CN 104458682 A CN104458682 A CN 104458682A CN 201410613314 A CN201410613314 A CN 201410613314A CN 104458682 A CN104458682 A CN 104458682A
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Abstract
The invention discloses a fluorescence detection method of a multifunctional oligonucleotide chain for multiple substances. The detected substances comprise a complementary chain of oligonucleotide chains, thrombin, Hg<2+> and L-cysteine. The method comprises the following steps: (1) preparing a solution, a, Tris-HNO3 buffer solution; and b, graphene oxide dispersion; (2) preheating a fluorescence probe and slowly cooling to room temperature; (3) mixing detected standard substances of different concentrations with the fluorescence probe solution, and incubating at room temperature; (4) adding the graphene oxide dispersion and the buffer solution, measuring a fluorescence emission spectrum, and establishing a standard curve of the detected substances according to the detected fluorescence signal intensity; and (5) reacting the sample solution instead of the detected substance, thereby obtaining the content of the detected substance in the sample solution according to the detected fluorescence signal intensity and the corresponding standard curve. The method disclosed by the invention has the advantages that the method is high in sensitivity, small in interference and high in selectivity, and the aim of rapidly detecting four different substances such as the complementary chain, thrombin, Hg<2+> and L-cysteine can be achieved.
Description
Technical field
The present invention relates to the research that multiprobe realizes many kinds of substance detection, specifically a kind of multi-functional oligonucleotide chain is used for complementary strand cTBA, fibrin ferment, Hg
2+with the fluorescence detection method of Cys many kinds of substance.
Background technology
Graphene oxide is a kind of Novel Carbon Nanomaterials, and its thickness is only single carbon atom particle size, but has wide two dimensional surface, and graphene oxide backbone carbon atoms is all with sp
2form hydridization, makes graphene oxide contain abundant pi-electron.Have report to find, graphene oxide has superpower fluorescent quenching ability as fluorescent receptor to the end-labelled fluorescent dye of single stranded DNA.Its principle is that the nucleotide base in single stranded DNA is adsorbed in graphene oxide plane by the hydrophobic forces of pi-pi accumulation, also there is FRET (fluorescence resonance energy transfer) FRET effect, so the fluorescence of dyestuff is by quencher in the fluorescent dye FAM of DNA end and the distance of graphene oxide of effectively having furthered; And after the single stranded DNA of dye marker and the single stranded DNA of its complete complementary form double-stranded DNA, the water wettability acting force of the phosphate backbones that outside is exposed is occupied an leading position, double-stranded DNA is inconjunction with FAM and departs from surface of graphene oxide, distance between fluorogenic donor FAM and fluorescent receptor graphene oxide becomes far away, and FRET effect disappears.Utilize this phenomenon, successfully can realize the detection ssDNA of high selectivity high sensitivity, and the difference of single base can be identified.
But the research reported all can only realize an oligonucleotide chain and detect a kind of target, when needing to detect different targets, can only be realized by the sequence changing oligonucleotide chain.
Summary of the invention
Based on this present situation, this invention exploits a kind of multi-functional oligonucleotide chain that can be used for many kinds of substance fluoroscopic examination, namely an oligonucleotide chain realizes complementary strand, fibrin ferment, Hg
2+with the detection of Cys four kinds of different materials.Its Cleaning Principle and above-mentioned prior art similar, have the single stranded DNA of FAM as fluorescence probe using end mark, this probe called after FAM-TBA.When only containing FAM-TBA and graphene oxide in system, FAM-TBA is freely curling, is adsorbed in surface of graphene oxide, and FRET effect produces, and fluorescence is by quencher.When adding complementary strand, fibrin ferment or Hg
2+after, FAM-TBA forms double-stranded DNA, G-tetra-serobilas/antithrombin complex and " T-Hg respectively
2+-T " structure; FAM-TBA departs from surface of graphene oxide, and the distance between fluorophor FAM and graphene oxide is zoomed out, and hinders the generation of FRET effect; the fluorescence of system is restored, the intensity that fluorescence recovers respectively with the complementary strand cTBA introduced, fibrin ferment and Hg
2+amount be closely related.When introducing Cys Cys in system, the sulfhydryl-group activity of Cys and Hg
2+complexing, by it from " T-Hg
2+-T " remove in structure, FAM-TBA recovers again nature rolled state, and rapid oxidized graphenic surface absorption, FRET effect produces again, fluorescence generation quencher, and the amount of the Cys of now fluorescent quenching degree and introducing is closely related, and can realize the detection of Cys.
Object of the present invention can only realize the problem of the detection of a target just for oligonucleotide chain in prior art, and provides a kind of multi-functional oligonucleotide chain for the fluorescence detection method of many kinds of substance.The detected object chosen is respectively complementary strand, fibrin ferment, Hg
2+with Cys Cys.As everyone knows, mankind's numerous disease is relevant to the single base mutation of gene, and thus the detection of the DNA of single base mismatch seems particularly important.And fibrin ferment, be a kind of serine protease, generation that can be a large amount of in wound, is combined with blood platelet and produces hemoglutination, but also can cause thrombosis under pathological conditions, and therefore its detection has great importance to biomedical diagnostic.Further, Hg
2+to intestines, kidney and brain, there is serious corrosive attack, Hg
2+there is very strong affinity with the ligand containing sulphur atom in human body, the sulfydryl agllutination of protein, enzyme and film can be caused.It can cause severe inflammation symptom in human body alimentary canal, usually within a few hours, namely abdominal cramps occurs and accompanies by Nausea and vomiting and bloody diarrhea etc.Therefore, identify and detect Hg
2+tool is of great significance.Finally, Cys is a kind of amino acid with physiological function, is the only amino acid with reductibility group sulfydryl in 20 several amino acids of constitutive protein matter, and therefore, identification and detection Cys tool are of great significance.The present invention, by a kind of fluorescence probe, analyzes the detection of thing in different detection system and application realize target.
The object of the invention is that technical scheme in the following manner realizes:
Multi-functional oligonucleotide chain is used for a fluorescence detection method for many kinds of substance, is to complementary strand, fibrin ferment, Hg
2+fluoroscopic examination with Cys tetra-kinds of materials, specifically comprises the following steps:
1) obtain solution: a, Tris-HNO
3buffer solution; B, graphene oxide dispersion;
2) by the fluorescence probe solution thermal treatment of 100 μMs, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature;
3) by the complementary strand of variable concentrations, fibrin ferment, Hg
2+mix with fluorescence probe solution respectively with Cys standard solution, incubated at room temperature half an hour;
4) in above-mentioned solution, add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, reacts half an hour under room temperature; Measure the fluorescence emission spectrum of solution, set up fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution
2+and the typical curve between Cys;
5) sample solution is replaced standard solution, carry out above-mentioned reaction, bring the fluorescence signal intensity of the solution detected into typical curve, obtain complementary strand in sample solution, fibrin ferment, Hg
2+with the content of Cys.
Described complementary strand cTBA, fibrin ferment, Hg
2+be respectively with the concentration of Cys Cys standard solution: the cTBA of 0.005,0.01,0.04,0.07,0.1,0.3,0.5,0.7 and 1 μM; 0.01,0.05,0.1,0.5,1,2,3,4,5,6,7,8,9,10,15 and 20 μ g mL
-1fibrin ferment; The Hg of 0.5,1,3,5,8,10,20,30 and 50 μM
2+; The Cys of 10,9,8,7,6,5,4,3,2,1 and 0.5 μMs.
Described Tris-HNO
3buffer solution composition is: 20 mM Tris, 100 mM NaNO
3, pH=8.0.
Described fluorescence probe sequence is 5'FAM-GG TTG GTG TGG TTG G-3'.
Instrument for fluorescence measurement is Hitachi F-4600 fluorospectrophotometer.
Described fluorescence spectral measuring condition: excite and launch slit width and be respectively 5.0 nm and 10.0nm, voltage is 700 V, and excitation wavelength is 480 nm, emission wavelength sweep limit 500-580 nm, and sample cell is the quartz colorimetric utensil of 0.60 mL.
In the present invention, complementary strand, fibrin ferment, Hg
2+as follows with the method that Cys four kinds of materials detect respectively:
(1) detection of complementary strand, as shown in Figure 1.By fluorescence probe solution thermal pretreatment, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature.The cTBA solution of variable concentrations is mixed with fluorescence probe solution, incubated at room temperature half an hour.Add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, reacts half an hour under room temperature, measures fluorescence emission spectrum.In this sensor of investigation is optionally tested cTBA, basic step is consistent with the step detecting cTBA, unlike, replace cTBA, by passing judgment on the specificity of this sensor in the detection of 520 nm place fluorescent emission intensity to sample with other oligonucleotide chain of same concentrations.
(2) detection of fibrin ferment, as shown in Figure 2.By fluorescence probe solution thermal pretreatment, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature.The thrombin solution of variable concentrations is mixed with fluorescence probe solution, incubated at room temperature half an hour.Add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, reacts half an hour under room temperature, measures fluorescence emission spectrum.In this sensor of investigation is optionally tested fibrin ferment, basic step is consistent with the step detecting fibrin ferment, unlike, replace fibrin ferment, by passing judgment on the specificity of this sensor in the detection of 520 nm place fluorescent emission intensity to sample with the other oroteins of same concentrations.
(3) Hg
2+detection, as shown in Figure 3.By fluorescence probe solution thermal pretreatment, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature.By the Hg of variable concentrations
2+solution mixes with fluorescence probe solution, incubated at room temperature half an hour.Add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, reacts half an hour under room temperature, measures fluorescence emission spectrum.At this sensor of investigation to Hg
2+optionally in experiment, basic step and detection Hg
2+step consistent, unlike, replace Hg with other metallic ion of same concentrations
2+, by passing judgment on the specificity of this sensor in the detection of 520 nm place fluorescent emission intensity to sample.
(4) detection of Cys, as shown in Figure 4.To fluorescence probe solution thermal pretreatment, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature.By Hg
2+solution mixes with fluorescence probe solution, adds the Cys solution of variable concentrations afterwards, and incubated at room temperature 0.5 hour, adds graphene oxide dispersion and buffer solution, makes the final volume treating test sample be 200 μ L, and graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, Hg
2+be 10 μMs, under room temperature, react half an hour, measure fluorescence emission spectrum.In this sensor of investigation is optionally tested Cys, basic step is consistent with the step detecting Cys, unlike, replace Cys, by passing judgment on the specificity of this sensor in the detection of 520 nm place fluorescent emission intensity to sample with other amino acid of same concentrations.
The invention has the advantages that: highly sensitive, the interference of other control sample to measured matter is little, can realize the selective enumeration method to measured matter, thus reaches an oligonucleotide chain and realize complementary strand, fibrin ferment, Hg
2+with the detection of Cys four kinds of different materials.
Accompanying drawing explanation
Fig. 1 is the concentration of complementary strand and fluorescence intensity graph of a relation and interference test.The fluorescence emission spectrogram of Zuo Tu: cTBA (a-i:0.005,0.01,0.04,0.07,0.1,0.3,0.5,0.7 and 1 μM); Right figure: the control test of single base and double alkali yl mispairing.
Fig. 2 is the concentration of fibrin ferment and fluorescence intensity graph of a relation and interference test.Zuo Tu: fibrin ferment (a-p:0.01,0.05,0.1,0.5,1,2,3,4,5,6,7,8,9,10,15 and 20 μ g mL
-1) fluorescence emission spectrogram; Right figure: the control test of other oroteins.
Fig. 3 is Hg
2+concentration and fluorescence intensity graph of a relation and interference test.Zuo Tu: Hg
2+the fluorescence emission spectrogram of (a-i:0.5,1,3,5,8,10,20,30 and 50 μM); Right figure: the control test of other metallic ion.
Fig. 4 is the concentration of Cys and fluorescence intensity graph of a relation and interference test.The fluorescence emission spectrogram of Zuo Tu: Cys (a-k:10,9,8,7,6,5,4,3,2,1 and 0.5 μMs); Right figure: other amino acid whose control test.
Embodiment
The present invention is described further by following instantiation:
Multi-functional oligonucleotide chain is used for a fluorescence detection method for many kinds of substance, specifically comprises the following steps:
1) obtain solution: a, Tris-HNO
3buffer solution; B, graphene oxide dispersion;
2) by the fluorescence probe solution thermal treatment of 100 μMs, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature;
3) by the complementary strand of variable concentrations, fibrin ferment, Hg
2+mix with fluorescence probe solution respectively with Cys standard solution, incubated at room temperature half an hour;
4) in above-mentioned solution, add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe solution is 0.2 μM, reacts half an hour under room temperature;
5) measure the fluorescence emission spectrum of solution, set up fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution
2+and the typical curve between Cys;
6) four of unknown concentration kinds of mark-on solution are replaced standard solution, carry out above-mentioned reaction, bring the fluorescence signal intensity of the solution detected into typical curve, obtain complementary strand cTBA, fibrin ferment, Hg in mark-on solution
2+0.083 μM is respectively, 3.12 μ g mL with the content of Cys Cys
-1, 5.27 μMs and 4.12 μMs.
Claims (6)
1. multi-functional oligonucleotide chain is used for a fluorescence detection method for many kinds of substance, it is characterized in that: be to complementary strand, fibrin ferment, Hg
2+fluoroscopic examination with Cys four kinds of materials, specifically comprises the following steps:
1) obtain solution: a, Tris-HNO
3buffer solution; B, graphene oxide dispersion;
2) by the fluorescence probe solution thermal treatment of 100 μMs, heat 5-10 minute at 90-100 DEG C, progressively cool to room temperature;
3) by the complementary strand of variable concentrations, fibrin ferment, Hg
2+mix with fluorescence probe solution respectively with Cys standard solution, incubated at room temperature half an hour;
4) in above-mentioned solution, add graphene oxide dispersion and buffer solution, make the final volume treating test sample be 200 μ L, graphene oxide concentration is 130 μ g mL
-1, fluorescence probe concentration is 0.2 μM, reacts half an hour under room temperature; Measure the fluorescence emission spectrum of solution, set up fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution
2+and the typical curve between Cys;
5) sample solution is replaced standard solution, carry out above-mentioned reaction, bring the fluorescence signal intensity of the solution detected into typical curve, obtain complementary strand in sample solution, fibrin ferment, Hg
2+with the content of Cys.
2. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, it is characterized in that: described complementary strand, fibrin ferment, Hg
2+be respectively with the concentration of Cys standard solution: the complementary strand of 0.005,0.01,0.04,0.07,0.1,0.3,0.5,0.7 and 1 μM; 0.01,0.05,0.1,0.5,1,2,3,4,5,6,7,8,9,10,15 and 20 μ g mL
-1fibrin ferment; The Hg of 0.5,1,3,5,8,10,20,30 and 50 μM
2+; The Cys of 10,9,8,7,6,5,4,3,2,1 and 0.5 μMs.
3. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, it is characterized in that: described Tris-HNO
3buffer solution composition is: 20 mM Tris, 100 mM NaNO
3, pH=8.0.
4. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, it is characterized in that: described fluorescence probe sequence is 5'FAM-GG TTG GTG TGG TTG G-3'.
5. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, it is characterized in that: the instrument for fluorescence measurement is Hitachi F-4600 fluorospectrophotometer.
6. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, it is characterized in that: described fluorescence spectral measuring condition: excite and launch slit width and be respectively 5.0 nm and 10.0 nm, voltage is 700 V, excitation wavelength is 480 nm, emission wavelength sweep limit 500-580 nm, sample cell is the quartz colorimetric utensil of 0.60 mL.
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| CN107677651A (en) * | 2017-08-03 | 2018-02-09 | 商丘师范学院 | A kind of Single wavelength excites the Hg that dual signal strengthens2+Ratio fluorescent method |
| CN110243794A (en) * | 2019-06-19 | 2019-09-17 | 济南大学 | A kind of fluorescence probe for detecting sulfur dioxide and its application based on graphene quantum dot |
| CN112326613A (en) * | 2020-10-30 | 2021-02-05 | 北京林业大学 | Method for detecting thrombin content |
| CN112934132A (en) * | 2021-03-10 | 2021-06-11 | 瓮福（集团）有限责任公司 | Boron-nitrogen co-doped reduced graphene oxide aerogel and preparation method and application thereof |
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2014
- 2014-11-05 CN CN201410613314.9A patent/CN104458682B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107677651A (en) * | 2017-08-03 | 2018-02-09 | 商丘师范学院 | A kind of Single wavelength excites the Hg that dual signal strengthens2+Ratio fluorescent method |
| CN107677651B (en) * | 2017-08-03 | 2019-10-08 | 商丘师范学院 | A kind of Hg of Single wavelength excitation dual signal enhancing2+Ratio fluorescent method |
| CN110243794A (en) * | 2019-06-19 | 2019-09-17 | 济南大学 | A kind of fluorescence probe for detecting sulfur dioxide and its application based on graphene quantum dot |
| CN110243794B (en) * | 2019-06-19 | 2022-08-02 | 济南大学 | Graphene quantum dot-based fluorescent probe for detecting sulfur dioxide and application thereof |
| CN112326613A (en) * | 2020-10-30 | 2021-02-05 | 北京林业大学 | Method for detecting thrombin content |
| CN112934132A (en) * | 2021-03-10 | 2021-06-11 | 瓮福（集团）有限责任公司 | Boron-nitrogen co-doped reduced graphene oxide aerogel and preparation method and application thereof |
| CN112934132B (en) * | 2021-03-10 | 2023-03-03 | 瓮福（集团）有限责任公司 | Boron-nitrogen co-doped reduced graphene oxide aerogel and preparation method and application thereof |
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