CN103529001A - dsDNA (double-stranded Deoxyribonucleic Acid) high-sensitivity detection method based on monochrome fluorescence off-on switching system - Google Patents
dsDNA (double-stranded Deoxyribonucleic Acid) high-sensitivity detection method based on monochrome fluorescence off-on switching system Download PDFInfo
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Abstract
The invention relates to a dsDNA (double-stranded Deoxyribonucleic Acid) high-sensitivity detection method based on a monochrome fluorescence off-on switching system and belongs to the field of chemistry and biomedicine. The dsDNA high-sensitivity detection method comprises the following steps: synthesizing CdTe quantum dots (QDs) coated by water-soluble glutathione (GSH); controlling the fluorescence-emission wavelength of the quantum dots to be 605mm; mixing the CdTe quantum dots coated by the water-soluble glutathione with a metal ruthenium coordination compound [Ru(phen)2(dppz)]2+(Ru) to enable the fluorescence of the CdTe quantum dots coated by the water-soluble glutathione to be completely quenched, so as to obtain a QDs-Ru assembling group; adding dsDNA into the QDs-Ru assembling group and gradually enhancing the monochrome fluorescence strength at the wavelength of 605mm along with gradual increase of the concentration of the dsDNA so as to rapidly and flexibly defect the dsDNA. The detection system can be used for enabling the detection limit of the dsDNA to reach 10pg/mL. Compared with the prior art in a background technology, the sensitivity is improved by 500 times; the operation is simple and rapid; the specificity is good and the cost is low; the whole process can be finished within 30 minutes.
Description
Technical field
The present invention relates to a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system, belong to chemistry and biomedical sector.
Background technology
Quantum dot (QDs), as good fluorescent nano material, has chemical stability good, and fluorescence efficiency is high, wide excitation spectrum, narrow many unique properties such as emission spectrum, can be combined and prepare biological fluorescent labeling with biomolecule, for detection and the sensing of biomolecule.DNA is as the carrier of hereditary information, and its fast high-sensitive detects more and more important in medical application, and measurement based on fluorescence analysis is the important foundation of DNA research with characterizing.Therefore, utilizing novel fluorescence labelling technique, is particularly the dynamically new of nearest research field development with quantum dot marker DNA.In recent years, the fluorescence probe of modifying based on quantum dot, and the biology sensor that fluorescent energy resonance transfer (FRET) occurs between the object of dye marker causes people's broad research.Yet the biology sensor based on resonance energy transfer, the most important thing is to design a pair of energy donor meeting the demands and acceptor, based on quantum dot fluorescence and simple switch system, for the detection display of biological target molecules, go out larger advantage.Quantum dot first with quencher effect generation fluorescent quenching, and after target analytes and quencher specific binding, will impel quencher to depart from from quantum dot surface, thereby the fluorescence of quantum dot is recovered, finally reach the object that rapid sensitive detects target analytes.
Dan Zhao etc. (Anal.Chem.2009) have reported the detection method of a kind of Two Colour Fluorescence signal that utilizes the design of quantum dot and ruthenium complex to dsDNA, and simple to operate but its detection sensitivity is lower fast, and the detectability of dsDNA only has 5ng/mL.
Summary of the invention
The object of the invention is in order to solve the technology of existing detection dsDNA, the problem that detection sensitivity is lower, provides a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system.
The object of the invention is to be achieved through the following technical solutions.
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100, ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Beneficial effect
1, a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, for realizing simple and quick, highly sensitive, the specific detection of dsDNA, we utilize the coated CdTe quantum dot (QDs) of water-soluble glutathione and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) design a kind of monochromatic fluorescence " off-on " switching system.CdTe QDs uses glutathione (GSH) as surface ligand, and QDs is electronegative in aqueous solution, ruthenium complex [Ru (phen)
2(dppz)]
2+from not fluorescing and positively charged in aqueous solution, both can, by electrostatic interaction combination, form QDs-Ru assembling group, the fluorescence generation quencher of QDs.DsDNA adds, specificity and ruthenium complex [Ru (phen)
2(dppz)]
2+in conjunction with forming Ru-dsDNA compound, at 605nm place, fluoresce, ruthenium complex [Ru (phen)
2(dppz)]
2+from the disengaging on QDs surface, can make the fluorescence of QDs be restored, under the exciting of Same Wavelength, the fluorescence of the fluorescence of QDs and Ru-dsDNA compound produces stack at 605nm place, thereby can highly sensitive detection dsDNA.Detection system of the present invention, dsDNA detectability can reach 10pg/mL, compares with prior art in background technology, and sensitivity has improved 500 times.
2, a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, without any need for covalent modification or probe design, object dsDNA is also without any modification, easy and simple to handle, fast, highly sensitive, specificity is good, cost is low, only detects and need in 30 minutes, just can complete.
3, the present invention can be widely used in the dsDNA detection of biomedical sector, comprises all dsDNA virus, and the detection that causes the clinical sample that in body fluid, DNA content changes, and is expected to be designed to DNA Rapid detection test strip.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet that the present invention detects dsDNA;
Fig. 2 is uv absorption (a, c) and the fluorescence emission spectrum (b, d) of CdTe QDs of the present invention and Ru-dsDNA compound;
Fig. 3 is ruthenium complex of the present invention [Ru (phen)
2(dppz)]
2+fluorescence spectrum to the fluorescent quenching of QDs, the concentration of QDs is 1nM, from a to m, ruthenium complex [Ru (phen)
2(dppz)]
2+concentration be respectively 0,1,5,10,20,30,40,50,60,70,80,90,100nM;
Fig. 4 is the fluorescence spectrum after 1nM QDs-Ru assembling group of the present invention and variable concentrations dsDNA effect, and from a to o, dsDNA concentration is respectively 0,0.05,0.5,5,10,20,40,60,80,100,200,400,600,800,1000ng/mL;
Fig. 5 be fluorescence intensity enhancing value of the present invention corresponding to the linear relationship curve of dsDNA concentration change, the dsDNA linearity test concentration range of this system is 0.5ng/mL-100ng/mL.
Embodiment
Following examples and accompanying drawing are further described the present invention.
Embodiment 1
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, as shown in Figure 1, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Synthesis step: by the Te powder of 63.8mg and the NaBH of 100mg
4mix, then add 5mL N
2saturated deionized water, mixed liquor is at N
2the lower reaction 2h of protection is until solution becomes pale pink, and this solution is referred to herein as the precursor liquid of Te.
By the CdCl of 0.2312g (1mmol)
22.5H
2o, the glutathione (GSH) of 0.3685g (1.2mmol), 100mL deionized water mixes, and under magnetic agitation, dropwise dripping NaOH adjusting pH is 8-9, obtains mixed liquor.Logical 30min N
2after, the precursor liquid of Te is injected into rapidly in mixed liquor, at N
2under protection, reaction solution is slowly heated to 100 ℃, and sustained response 5.5h, stops reaction, as shown in Figure 2 until obtain the CdTe quantum dot of required emission wavelength.Isopropyl alcohol sedimentation washing 3 times for CdTe quantum dot, in vacuum drying chamber, the dry pressed powder that obtains, uses in order to subsequent experimental.
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100; Ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
The CdTe QDs (100nM) that adds 1 μ L in the PBS (0.01M, pH7.4) of 89 μ L, obtains QDs solution, then by the [Ru (phen) of 10 μ L variable concentrations
2(dppz)]
2+join in QDs solution, under room temperature, react 10min, the fluorescence generation quencher of QDs, forms QDs-Ru assembling group.By fluorescence spectrophotometer, detect QDs fluorescence, excitation wavelength is 390nm, as shown in Figure 3.
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Described dsDNA is calf thymus DNA, and mass concentration refers to table 1.
The CdTe QDs (100nM) of 1 μ L, [the Ru (phen) of 10 μ L
2(dppz)]
2+(1 μ M) joins in 89 μ L PBS (0.01M, pH7.4) successively, and mixed at room temperature 10min forms QDs-Ru assembling group, obtains QDs-Ru assembling group solution.Then to the dsDNA that adds respectively 1 μ L variable concentrations in QDs-Ru assembling group solution, under room temperature, hatch 15min, by fluorescence spectrophotometer, carry out fluoroscopic examination, excitation wavelength is 390nm, as shown in Figure 4, dsDNA linearity test concentration range as shown in Figure 5 for fluorescence spectrum figure.All fluorescence intensities detect (n=3) all in triplicate, calculate the inventive method batch in coefficient of variation Intra-assay CV, CV is that the coefficient of variation also can be described as relative standard deviation (Relative Standard Deviation, RSD), CV%=standard deviation (SD)/mean value (mean).Repeatability is the results detailed in Table 1.
The monochromatic fluorescence of table 1 " off-on " switching system detect calf thymus DNA batch in otherness
The result of this test: monochromatic fluorescence of the present invention " off-on " switching system detects dsDNA, and along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually.Detection sensitivity is high, and detectability can reach 10pg/mL.Linearity test concentration range is 0.5ng/mL-100ng/mL, as shown in Figure 5.And batch in coefficient of variation be only 0.91%, illustrate that detection system of the present invention is reproducible.
Embodiment 2
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, as shown in Figure 1, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Synthesis step: by the Te powder of 63.8mg and the NaBH of 100mg
4mix, then add 5mL N
2saturated deionized water, mixed liquor is at N
2the lower reaction 2h of protection is until solution becomes pale pink, and this solution is referred to herein as the precursor liquid of Te.
By the CdCl of 0.2312g (1mmol)
22.5H
2o, the glutathione (GSH) of 0.3685g (1.2mmol), 100mL deionized water mixes, and under magnetic agitation, dropwise dripping NaOH adjusting pH is 8-9, obtains mixed liquor.Logical 30min N
2after, the precursor liquid of Te is injected into rapidly in mixed liquor, at N
2under protection, reaction solution is slowly heated to 100 ℃, and sustained response 5.5h, stops reaction, as shown in Figure 2 until obtain the CdTe QDs of required emission wavelength.Isopropyl alcohol sedimentation washing 3 times for CdTe QDs, in vacuum drying chamber, the dry pressed powder that obtains, uses in order to subsequent experimental.
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100, ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
The CdTe QDs (100nM) that adds 1 μ L in the PBS (0.01M, pH7.4) of 89 μ L, obtains QDs solution, then by the [Ru (phen) of 10 μ L variable concentrations
2(dppz)]
2+join in QDs solution, under room temperature, react 10min, the fluorescence generation quencher of QDs, forms QDs-Ru assembling group.By fluorescence spectrophotometer, detect QDs fluorescence, excitation wavelength is 390nm, as shown in Figure 3.
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Described dsDNA is three kinds of different lengths of any mass concentration ratio mixing, the dsDNA (12bp dsDNA+36bp dsDNA+54bp dsDNA) of different base compositions, and mass concentration refers to table 2.
The CdTe QDs (100nM) of 1 μ L, [the Ru (phen) of 10 μ L
2(dppz)]
2+(1 μ M) joins in 89 μ L PBS (0.01M, pH7.4) successively, and mixed at room temperature 10min forms QDs-Ru assembling group, obtains QDs-Ru assembling group solution.Then in QDs-Ru assembling group solution, add respectively three kinds of different lengths of above-mentioned any mass concentration ratio mixing of 1 μ L variable concentrations, the dsDNA of different base compositions, under room temperature, hatch 15min, by fluorescence spectrophotometer, carry out fluoroscopic examination, excitation wavelength is 390nm.Fluorescence intensity testing result refers to table 2.
The result of this test: along with mixing the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Embodiment 3
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, as shown in Figure 1, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Synthesis step: by the Te powder of 63.8mg and the NaBH of 100mg
4mix, then add 5mL N
2saturated deionized water, mixed liquor is at N
2the lower reaction 2h of protection is until solution becomes pale pink, and this solution is referred to herein as the precursor liquid of Te.
By the CdCl of 0.2312g (1mmol)
22.5H
2o, the glutathione (GSH) of 0.3685g (1.2mmol), 100mL deionized water mixes, and under magnetic agitation, dropwise dripping NaOH adjusting pH is 8-9, obtains mixed liquor.Logical 30min N
2after, the precursor liquid of Te is injected into rapidly in mixed liquor, at N
2under protection, reaction solution is slowly heated to 100 ℃, and sustained response 5.5h, stops reaction, as shown in Figure 2 until obtain the CdTe quantum dot of required emission wavelength.Isopropyl alcohol sedimentation washing 3 times for CdTe quantum dot, in vacuum drying chamber, the dry pressed powder that obtains, uses in order to subsequent experimental.
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100; Ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
The CdTe QDs (100nM) that adds 1 μ L in the PBS (0.01M, pH7.4) of 89 μ L, obtains QDs solution, then by the [Ru (phen) of 10 μ L variable concentrations
2(dppz)]
2+join in QDs solution, under room temperature, react 10min, the fluorescence generation quencher of QDs, forms QDs-Ru assembling group.By fluorescence spectrophotometer, detect QDs fluorescence, excitation wavelength is 390nm, as shown in Figure 3.
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Described dsDNA is two kinds of different lengths of any mass concentration ratio mixing, the dsDNA (12bp dsDNA+36bp dsDNA) of different base compositions, and mass concentration refers to table 2.
The CdTe QDs (100nM) of 1 μ L, [the Ru (phen) of 10 μ L
2(dppz)]
2+(1 μ M) joins in 89 μ L PBS (0.01M, pH7.4) successively, and mixed at room temperature 10min forms QDs-Ru assembling group, obtains QDs-Ru assembling group solution.Then in QDs-Ru assembling group solution, add respectively two kinds of different lengths of above-mentioned any mass concentration ratio mixing of 1 μ L variable concentrations, the dsDNA of different base compositions, under room temperature, hatch 15min, by fluorescence spectrophotometer, carry out fluoroscopic examination, excitation wavelength is 390nm.Fluorescence intensity testing result refers to table 2
The result of this test: along with mixing the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Embodiment 4
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, as shown in Figure 1, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Synthesis step: by the Te powder of 63.8mg and the NaBH of 100mg
4mix, then add 5mL N
2saturated deionized water, mixed liquor is at N
2the lower reaction 2h of protection is until solution becomes pale pink, and this solution is referred to herein as the precursor liquid of Te.
By the CdCl of 0.2312g (1mmol)
22.5H
2o, the glutathione (GSH) of 0.3685g (1.2mmol), 100mL deionized water mixes, and under magnetic agitation, dropwise dripping NaOH adjusting pH is 8-9, obtains mixed liquor.Logical 30min N
2after, the precursor liquid of Te is injected into rapidly in mixed liquor, at N
2under protection, reaction solution is slowly heated to 100 ℃, and sustained response 5.5h, stops reaction, as shown in Figure 2 until obtain the CdTe quantum dot of required emission wavelength.Isopropyl alcohol sedimentation washing 3 times for CdTe quantum dot, in vacuum drying chamber, the dry pressed powder that obtains, uses in order to subsequent experimental.
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100; Ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
The CdTe QDs (100nM) that adds 1 μ L in the PBS (0.01M, pH7.4) of 89 μ L, obtains QDs solution, then by the [Ru (phen) of 10 μ L variable concentrations
2(dppz)]
2+join in QDs solution, under room temperature, react 10min, the fluorescence generation quencher of QDs, forms QDs-Ru assembling group.By fluorescence spectrophotometer, detect QDs fluorescence, excitation wavelength is 390nm, as shown in Figure 3.
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Described dsDNA is two kinds of different lengths of any mass concentration ratio mixing, the dsDNA (36bp dsDNA+54bp dsDNA) of different base compositions, and mass concentration refers to table 2.
The CdTe QDs (100nM) of 1 μ L, [the Ru (phen) of 10 μ L
2(dppz)]
2+(1 μ M) joins in 89 μ L PBS (0.01M, pH7.4) successively, and mixed at room temperature 10min forms QDs-Ru assembling group, obtains QDs-Ru assembling group solution.Then in QDs-Ru assembling group solution, add respectively two kinds of different lengths of above-mentioned any mass concentration ratio mixing of 1 μ L variable concentrations, the dsDNA of different base compositions, under room temperature, hatch 15min, by fluorescence spectrophotometer, carry out fluoroscopic examination, excitation wavelength is 390nm.Fluorescence intensity testing result refers to table 2
The result of this test: along with mixing the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Fluorescence intensity under table 2 QDs-Ru assembling group and variety classes dsDNA variable concentrations
DNA1:ctDNA in table 2 (calf thymus DNA), DNA2:12bp dsDNA+36bp dsDNA+54bp dsDNA, DNA3:12bp dsDNA+36bp dsDNA, DNA4:36bp dsDNA+54bpdsDNA.
The result of table 2 shows, monochromatic fluorescence of the present invention " off-on " switching system, increase gradually along with dsDNA concentration, monochromatic fluorescence intensity at 605nm place strengthens gradually, and the gross mass that the sensitivity responding for dsDNA and detectability only depend on dsDNA, and irrelevant with species composition and the length of dsDNA.
Embodiment 5
A kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system of the present invention, as shown in Figure 1, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Synthesis step: by the Te powder of 63.8mg and the NaBH of 100mg
4mix, then add 5mL N
2saturated deionized water, mixed liquor is at N
2the lower reaction 2h of protection is until solution becomes pale pink, and this solution is referred to herein as the precursor liquid of Te.
By the CdCl of 0.2312g (1mmol)
22.5H
2o, the glutathione (GSH) of 0.3685g (1.2mmol), 100mL deionized water mixes, and under magnetic agitation, dropwise dripping NaOH adjusting pH is 8-9, obtains mixed liquor.Logical 30min N
2after, the precursor liquid of Te is injected into rapidly in mixed liquor, at N
2under protection, reaction solution is slowly heated to 100 ℃, and sustained response 5.5h, stops reaction, as shown in Figure 2 until obtain the CdTe quantum dot of required emission wavelength.Isopropyl alcohol sedimentation washing 3 times for CdTe quantum dot, in vacuum drying chamber, the dry pressed powder that obtains, uses in order to subsequent experimental.
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix, mixing mol ratio is 1:100; Ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
The CdTe QDs (100nM) that adds 1 μ L in the PBS (0.01M, pH7.4) of 89 μ L, obtains QDs solution, then by the [Ru (phen) of 10 μ L variable concentrations
2(dppz)]
2+join in QDs solution, under room temperature, react 10min, the fluorescence generation quencher of QDs, forms QDs-Ru assembling group.By fluorescence spectrophotometer, detect QDs fluorescence, excitation wavelength is 390nm, as shown in Figure 3.
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
Described dsDNA is clinical sample DNA, can comprise all DNA virus, and the clinical sample that in body fluid, DNA content changes.
First need clinical sample to carry out the extraction of DNA, thereby obtain clinical sample DNA.Subsequently, the CdTe QDs (100nM) of 1 μ the L, [Ru (phen) of 10 μ L
2(dppz)]
2+(1 μ M) joins in 89 μ L PBS (0.01M, pH7.4) successively, and mixed at room temperature 10min forms QDs-Ru assembling group, obtains QDs-Ru assembling group solution.Then in QDs-Ru assembling group solution, add respectively the above-mentioned clinical sample DNA obtaining that extracts through DNA of 1 μ L, under room temperature, hatch 20min, by fluorescence spectrophotometer, carry out fluoroscopic examination, excitation wavelength is 390nm.
The result of this test: the difference of clinical sample DNA concentration, the monochromatic fluorescence intensity detecting at 605nm place is also different, linear relationship curve according to fluorescence intensity enhancing value corresponding to dsDNA concentration change, as shown in Figure 5, can calculate the concentration of clinical sample DNA, thereby realize, the fast high-sensitive of clinical sample DNA be detected.
In sum, coated CdTe QDs and the Ru-dsDNA compound of water-soluble glutathione that the present invention synthesizes all has absorption at 350-500nm place, and there is fluorescent emission at the 605nm place that coexists, as shown in Figure 2, build a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence " off-on " switching system.Ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) be a kind of good quencher, himself does not fluoresce in aqueous solution, as QDs and ruthenium complex [Ru (phen)
2(dppz)]
2+mix, along with ruthenium complex [Ru (phen)
2(dppz)]
2+the increase of concentration, the fluorescence of QDs declines gradually, and when both molar ratios reach QDs:Ru=1:100, the fluorescence of QDs is by quencher completely, as shown in Figure 3.While adding dsDNA in the QDs-Ru assembling group to the complete quencher of QDs fluorescence quilt, along with the increase gradually of dsDNA concentration, the monochromatic fluorescent emission at 605nm place strengthens gradually, as shown in Figure 4.This shows ruthenium complex [Ru (phen)
2(dppz)]
2+be again a kind of dsDNA molecular light switch, dsDNA adds, specificity and ruthenium complex [Ru (phen) simultaneously
2(dppz)]
2+in conjunction with forming Ru-dsDNA compound, at 605nm place, fluoresce, ruthenium complex [Ru (phen)
2(dppz)]
2+from the disengaging on QDs surface, can make the fluorescence of QDs be restored, under the exciting of Same Wavelength, the fluorescence of the fluorescence of QDs and Ru-dsDNA compound produces stack at 605nm place, thereby can highly sensitive detection dsDNA.DsDNA linearity test concentration range is 0.5ng/mL-100ng/mL, as shown in Figure 5.
By above various detections, prove that the inventive method can quick, highly sensitive, specific detection dsDNA, simple to operate, reproducible, reliable results, cost is low, for the detectability of dsDNA, can reach 10pg/mL.The inventive method provides a kind of new method to the detection of other clinical sample DNA, can be applied to the detection of all dsDNA viruses.And this method only depends on dsDNA gross mass for dsDNA detectability, and it is irrelevant with species composition and the length of dsDNA, therefore can be applied to detect cause the detection of the clinical sample that in body fluid, DNA content changes by DNA damage, and be expected to be designed to DNA Rapid detection test strip.
Claims (2)
1. the dsDNA high-sensitivity detecting method based on monochromatic fluorescence off-on switching system, is characterized in that, concrete steps are as follows:
Step 1, the coated CdTe quantum dot (QDs) of synthesizing water-solubility glutathione (GSH), control the fluorescent emission wavelength of this quantum dot at 605nm;
Step 2, by the coated CdTe quantum dot (QDs) of the water-soluble glutathione of step 1 gained and ruthenium complex [Ru (phen)
2(dppz)]
2+(Ru) mix ruthenium complex [Ru (phen)
2(dppz)]
2+by the CdTe quantum dot that electrostatic interaction is coated with water-soluble glutathione, be combined, cause the coated complete quencher of CdTe quantum dot fluorescence of water-soluble glutathione, obtain QDs-Ru assembling group;
Step 3, in the QDs-Ru assembling group of step 2 gained, add after dsDNA, along with the increase gradually of dsDNA concentration, the monochromatic fluorescence intensity at 605nm place strengthens gradually, thereby realize, dsDNA fast high-sensitive is detected.
2. a kind of dsDNA high-sensitivity detecting method based on monochromatic fluorescence off-on switching system as claimed in claim 1, is characterized in that: coated CdTe quantum dot and the ruthenium complex [Ru (phen) of water-soluble glutathione described in step 2
2(dppz)]
2+mix, mixing mol ratio is 1:100.
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