CN103611929B - A kind of method of fluorescence labeling gold nano grain - Google Patents
A kind of method of fluorescence labeling gold nano grain Download PDFInfo
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- 0 CC1(C)c2ccccc2N(CCCCP(O)(O[*+])=O)C1CC=CC(C1(C)C)=[N+](CCCO)C2C1=CC=CC2 Chemical compound CC1(C)c2ccccc2N(CCCCP(O)(O[*+])=O)C1CC=CC(C1(C)C)=[N+](CCCO)C2C1=CC=CC2 0.000 description 1
Abstract
The invention provides a kind of method of fluorescence labeling gold nano grain. The invention provides the method for fluorescence labeling gold nano grain, for G-tetra-serobila DNA moleculars and cyanine dyes, gold nano grain being carried out to fluorescence labeling, obtain fluorescence labeling gold nano grain; Described G-tetra-serobila DNA moleculars are the strand G-tetra-serobila DNA moleculars of strand G-tetra-serobila DNA moleculars or 5 ' end or 3 ' end sulfydryl modification or the strand G-tetra-serobila DNA moleculars of 5 ' end or 3 ' end mark cyanine dyes, its nucleotides sequence is classified GGYGGZGGMGG as, wherein: Y, Z and M represent one or more any bases independently. Of the present invention experimental results show that, the G-of utilization tetra-serobilas provided by the invention connect fluorescence probe mark gold nano grain method tool and have the following advantages: G-tetra-serobilas can effectively be isolated the fluorescence quenching of gold nano grain to fluorescence probe, and can significantly strengthen the fluorescence intensity of fluorescence probe, the detector probe of high fluorescent is provided.
Description
Technical field
The invention belongs to biomedicine field, relate in particular to a kind of method of fluorescence labeling gold nano grain.
Background technology
Nm of gold refers to golden molecule, and its diameter is at 1～100nm, conventionally in the aqueous solution with the form of collaurumExist, there is high electron density, dielectric property and catalytic action, can be combined with multiple large biological molecule, and not affectIts biologically active. The most classical method of preparing collaurum is natrium citricum reducing process at present. According to the kind of reducing agentWith the difference of concentration, can under laboratory condition, prepare the collaurum of different-grain diameter, and method is simple, raw material is inexpensive.
Nm of gold has an absworption peak in 510-550nm limit of visible spectrum, and absorbing wavelength is with the increase of gold grain diameterIncrease. When particle diameter is from small to large time, apparent colour presents greenish orange yellow, grape wine redness, peony and royal purple successivelyLook changes. Except these unique change color, golden nanometer particle also has good biocompatibility and nontoxic pairEffect. Thus, nm of gold has been widely used in biomolecular labeling and detection, nano-biosensing in recent yearsThe technology such as device and nano biological chip.
In biomolecular labeling technology, the macromolecules such as protein and nanogold particle be firm knot because Electrostatic Absorption formsClose, because gold grain has the characteristic of high electron density, at gold mark protein combination place, visible pitchy under the microscopeParticle, in the time that these labels are assembled in a large number at corresponding part place, naked eyes red color visible or pink spot, therebyFor qualitative or semiquantitative tachysynthesis detection method. Because spherical nano Au particle has very strong to proteinAdsorption function, can with staphylococcal protein A, immunoglobulin (Ig), toxin, glycoprotein, enzyme, antibiotic, hormone,The non-covalent combination such as bovine serum albumin(BSA), thereby become very useful instrument in basic research and experiment.
But the qualitative or half-quantitative detection of gold nano grain often cannot realize highly sensitive detection. At gold nano grainSurface markers fluorescence probe is to improve effective means of detection sensitivity. And gold nano grain is a kind of good glimmeringOptical quenching agent, compared with common quencher, the Stern-Volmer quenching constant of nm of gold exceeds several orders of magnitude, because ofThis is difficult to detect the fluorescence of labelled molecule, thereby has greatly limited the application of mark fluorescence probe on gold nano grain.
Summary of the invention
The object of this invention is to provide a kind of method of fluorescence labeling gold nano grain.
Method provided by the invention, for G-tetra-serobila DNA moleculars, cyanine dyes being marked to gold nano grain surface,To fluorescence labeling gold nano grain;
Described G-tetra-serobila DNA moleculars are strand, and its nucleotides sequence is classified GGYGGZGGMGG as, wherein: Y, Z and MRepresent independently one or more any bases;
Described cyanine dyes is the compound shown in formula I,
Wherein: R1For C1-C6Alkyl, phenyl or alkyl (be also C herein1-C6) replace phenyl;
R2、R3、R4And R5Independently selected from H or C1-C6Alkyl, or R2And R3The carbon atom being connected with themForm together the ring structure of 5 yuan to 7 yuan, or R4And R5Together with the carbon atom connecting with them, form 5 yuan to 7The ring structure of unit;
R6And R7For C1-C6The C that alkyl or sulfonic group replace1-C6Alkyl;
Y is counter ion, according to R6And R7Institute charged difference and difference, if R6And R7For alkyl, Y is halogenAnion; If R6And R7Only have one with sulfonate radical, without Y as counter ion; If R6And R7All with sulfonic acidRoot, Y is triethylamine cation;
X1，X2Independently selected from C, O, S, Se or Te.
In said method,
Described C1-C6Alkyl be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group,Amyl group, isopentyl, n-hexyl or isohesyl;
Described 5 yuan is the saturated or unsaturated ring structure that contains or do not contain N or S atom to 7 ring structures;
Described Y is selected from fluorine, chlorine, bromine, iodine anion or triethylamine cation;
Described R1Be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, differentAmyl group, n-hexyl, isohesyl, phenyl, aminomethyl phenyl or 3,5-dimethylphenyl; R2、R3、R4And R5Independently selected from firstBase, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, isopentyl, n-hexyl or differentHexyl.
Above-mentioned with the method that cyanine dyes is marked at gold nano grain surface by G-tetra-serobila DNA moleculars be following 1) or 2):
1) method shown in comprises the steps: cyanine dyes solution, G-tetra-serobila DNA molecular solution A and gold nanoParticle solution is mixed, and obtains fluorescence labeling gold nano grain; Wherein, the G-tetra-serobila DNA moleculars in described solution AFor the strand G-tetra-serobila DNA moleculars of strand G-tetra-serobila DNA moleculars or 5 ' or 3 ' end sulfydryl modification;
2) method shown in comprises the steps: mixed to G-tetra-serobila DNA molecular solution B and gold nano grain solutionClose, obtain fluorescence labeling gold nano grain; Wherein, the G-tetra-serobila DNA moleculars in described solution B are 5 ' or 3 'The strand G-tetra-serobila DNA moleculars of end mark cyanine dyes.
In said method,
1) in the method shown in,
The strand G-tetra-serobila DNA moleculars that described G-tetra-serobila DNA moleculars are 5 ' end sulfydryl modification, its nucleotidesSequence is sequence 1, and described cyanine dyes is the compound shown in following formula II:
Or described G-tetra-serobila DNA moleculars be 5 ' end sulfydryl modification strand G-tetra-serobila DNA moleculars, its nucleosidesAcid sequence is sequence 2, and described cyanine dyes is the compound shown in following formula III:
Or described G-tetra-serobila DNA moleculars be 3 ' end sulfydryl modification strand G-tetra-serobila DNA moleculars, its nucleosidesAcid sequence is sequence 3, and described cyanine dyes is the compound shown in following formula IV:
Or described G-tetra-serobila DNA moleculars are strand G-tetra-serobila DNA moleculars, its nucleotides sequence is classified sequence 4 as, instituteStating cyanine dyes is the compound shown in following formula V:
2) in the method shown in,
The strand G-tetra-serobila DNA moleculars that described G-tetra-serobila DNA moleculars are 5 ' end mark cyanine dyes, Qi ZhongjingDyestuff is Cy3, and the nucleotides sequence of strand G-tetra-serobila DNA moleculars is classified the sequence 5 in sequence table as; Described 5 ' endThe structural formula of the strand G-tetra-serobila DNA moleculars of mark cyanine dyes is shown in following formula IX:
Formula IX, wherein oligo is strand G-tetra-serobila DNA moleculars,
The structural formula of described Cy3 is shown in following formula VI:
Or the described G-tetra-serobila DNA moleculars strand G-tetra-serobila DNA moleculars that are 5 ' end mark cyanine dyes, whereinCyanine dyes is Cy3.5, and the nucleotides sequence of strand G-tetra-serobila DNA moleculars is classified the sequence 6 in sequence table as; Described 5 'The structural formula of the strand G-tetra-serobila DNA moleculars of end mark cyanine dyes is shown in following formula X:
Formula X, wherein oligo is strand G-tetra-serobila DNA moleculars,
The structural formula of described Cy3.5 is shown in following formula VII:
In said method,
The solvent of described G-tetra-serobila DNA molecular solution A or described G-tetra-serobila DNA molecular solution B is that pH value isThe cushioning liquid of 5.0-8.2; It is 5,6,6.5,7.0 that the cushioning liquid that described pH value is 5.0-8.2 is specially pH valueOr 8 cushioning liquid;
Described cushioning liquid be following any: Tris-HCl buffer solution, sodium hydrogen phosphate-potassium phosphate buffer,Potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, sodium phosphate-dibastic sodium phosphate buffer solution, citric acid-sodium citrate buffer solution,Tris-HCI buffer, triethanolamine buffer solution, imidazoles-hydrochloride buffer, glycylglycine bufferingLiquid, 2-amino-2-methyl-1-propanol buffer solution, potassium phosphate-potassium hydrogen phosphate buffer solution, barbital sodium-hydrochloride buffer,Boric acid-borate buffer solution, glycine-sodium hydrate buffer solution, borax-sodium hydrate buffer solution or sodium phosphate buffer;
The solvent of described cyanine dyes solution is methyl alcohol.
In said method,
1) in the method shown in, the G-tetra-serobila DNA moleculars in described solution A, described cyanine dyes and described JennerThe mol ratio of rice grain is 2-4:4-7:1;
2) in the method shown in, the G-tetra-serobila DNA moleculars in described solution B and described gold nano grain moleThan being 2-10:1.
In said method,
1) in the method shown in, the G-tetra-serobila DNA moleculars in described solution A, described cyanine dyes and described JennerThe mol ratio of rice grain is 3:6:1 or 4:4:1 or 4:7:1 or 2:6:1;
2) in the method shown in, the G-tetra-serobila DNA moleculars in described solution B and described gold nano grain moleThan being 2:1 or 10:1.
In said method,
1) in the method or 2), described in be mixed into and dark leave standstill 0.5 hour-2 hours, described mixing is speciallyDark standing 1 or 2 hour.
The fluorescence labeling gold nano grain being prepared by said method is also the scope of protection of the invention.
The G-of the utilization provided by the invention tetra-serobilas connection fluorescence probe mark gold nano grain sides that experiment showed, of the present inventionMethod tool has the following advantages: G-tetra-serobilas can effectively be isolated the fluorescence quenching of gold nano grain to fluorescence probe, and energySignificantly strengthen the fluorescence intensity of fluorescence probe, the detector probe of high fluorescent is provided; G-tetra-serobila knots in the methodStructure, probe molecule structure all have diversity, are expected to meet the demand of different detection architecture; The method reaction system becomesDivide simply, preparation process is simple, controlled, has ensured the stability of probe.
Brief description of the drawings
Fig. 1 is the fluorescence spectrum figure that embodiment 1 and comparative example 1 and comparative example 2 draw.
Fig. 2 is the fluorescence spectrum figure that embodiment 2 draws.
Fig. 3 is the fluorescence spectrum figure that embodiment 3 draws.
Detailed description of the invention
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The DNA single chain that can form G-tetra-serobilas used in following embodiment is all limited purchased from the raw work biotechnology in ShanghaiCompany, is synthesized by the said firm.
Gold nano grain solution used in following embodiment is all purchased from Beijing Oneder Hightech Co., Ltd..
In following embodiment, cyanine dyes used is the compound shown in formula I,
Wherein: R1For C1-C6Alkyl, phenyl or the phenyl that replaces of alkyl; R2、R3、R4And R5Independently selected from HOr C1-C6Alkyl, or R2And R3Together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan,Or R4And R5Together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan; R6And R7For C1-C6AlkaneThe C that base or sulfonic group replace1-C6Alkyl; Y is counter ion, according to R6And R7Institute charged difference and difference, ifR6And R7For alkyl, Y is halide anion; If R6And R7Only have one with sulfonate radical, without Y as insteadIon; If R6And R7All, with sulfonate radical, Y is triethylamine cation; X1，X2Independently selected from C, O, S,Se or Te.
C1-C6Alkyl be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group,Isopentyl, n-hexyl or isohesyl;
5 yuan is the saturated or unsaturated ring structure that contains or do not contain N or S atom to 7 ring structures;
Y is selected from fluorine, chlorine, bromine, iodine anion or triethylamine cation;
R1Be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, isopentyl,N-hexyl, isohesyl, phenyl, aminomethyl phenyl or 3,5-dimethylphenyl; R2、R3、R4And R5Independently selected from methyl, secondBase, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, isopentyl, n-hexyl or isohesyl.
Embodiment 1, G-tetra-serobilas/cyanine dyes mark gold nano grain
The G-tetra-serobila DNA moleculars that can form that use are in the present embodiment the strand G-tetra-of 5 ' end sulfydryl modificationSerobila DNA molecular, sequence is that (nucleotides sequence is classified sequence table as to SH-5 '-AGGGTTAGGGTTAGGGTTAGGG-3 'In sequence 1); The cyanine dyes using is the compound 1 shown in formula II:
Compound 1
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: the strand G-tetra-serobila DNA of 5 ' the end sulfydryl modification with above-mentioned sequence are dividedSon be dissolved in pH value 7.0 containing the Tris-HCl buffer solution of 10mMNaCl (formula: by NaCl according to final concentrationIt is in 10mM, the pH value Tris-HCl buffer solution that is 7.0 that 10mM is dissolved in concentration) in, be prepared into DNA concentrationBe the DNA mother liquor of 2 μ mol/L, for subsequent use.
2) preparation of cyanine dyes mother liquor: take a certain amount of cyanine dyes, with methyl alcohol dissolving, compound concentration is 200 μ mol/LCyanine dyes mother liquor.
3) G-tetra-serobilas/cyanine dyes mark gold nano grain: get step 2) in cyanine dyes mother liquor 60 μ L and step 1)Middle DNA mother liquor 3mL, after both mix, then to add concentration be 17 μ mol/L, the gold nano grain that particle diameter is 50nmSolution 120 μ L, place 2 hours the wherein molar concentration of G-tetra-serobilas, cyanine dyes and gold nano grain in shady placeThan being about 3:6:1 for 3:6:1.02(), obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) G-tetra-serobilas/cyanine dyes mark gold nano grain solution (compound 1+G-tetra-chains of obtainingBody+gold grain) be placed under XRF the fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. ResultAs shown in Figure 1, can be observed high strength fluorescence.
Comparative example 1
Getting concentration is 17 μ mol/L, and gold nano grain mother liquor 120 μ L that particle diameter is 50nm add pH value 7.0Containing the 10mMTris-HCl buffer solution 3.06mL of 10mMNaCl. Place after 2 hours, in fluorescence light in shady placeUnder spectrometer, detect the fluorescence intensity of solution (gold grain). Result as shown in Figure 1, without fluorescence.
Comparative example 2
Get cyanine dye compound 1 mother liquor 60 μ L, the Tris-HCl buffer solution 3.0mL containing 10mMNaCl of pH value 7.0,Then adding concentration is 17 μ mol/L, the 120 μ L gold nano grain solution that particle diameter is 50nm. Place 2 in shady placeAfter hour, under XRF, detect the fluorescence intensity of solution (compound 1+ gold grain). Result is as Fig. 1 instituteShow, without fluorescence.
Compared with comparative example, G-tetra-serobilas/cyanine dyes mark gold nano grain solution that the present embodiment obtains can be observedHigh strength fluorescence, illustrates labeling method success.
Embodiment 2, G-tetra-serobilas/cyanine dyes mark gold nano grain
The G-tetra-serobila DNA moleculars that can form that use are in the present embodiment the strand G-tetra-of 5 ' end sulfydryl modificationSerobila DNA molecular, sequence is that (its nucleotides sequence is classified sequence as to SH-5 '-TTGGGTTGGGTTGGGTTGGG-3 'Sequence 2 in table); The cyanine dyes using is compound 2 shown in following formula III:
Compound 2
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: the strand G-tetra-serobila DNA of 5 ' the end sulfydryl modification with above-mentioned sequence are dividedSodium hydrogen phosphate-potassium phosphate buffer that son is dissolved in the 10mM of pH value 6.5 (dissolves successively 2.4g in waterADSP and 4.6g potassium dihydrogen phosphate, be diluted to 100ml, mixes) in, be prepared into DNA concentration and beThe DNA mother liquor of 4 μ mol/L, for subsequent use.
2) preparation of cyanine dyes mother liquor: take a certain amount of cyanine dyes, with methyl alcohol dissolving, compound concentration is 200 μ mol/LCyanine dyes mother liquor.
3) G-tetra-serobilas/cyanine dyes mark gold nano grain: get step 2) in cyanine dyes mother liquor 60 μ L and step 1)Middle DNA mother liquor 3mL, after both mix, then to add concentration be 25 μ mol/L, the gold nano grain that particle diameter is 10nmSolution 120 μ L, place 2 hours the wherein molar concentration of G-tetra-serobilas, cyanine dyes and gold nano grain in shady placeThan for 4:4:1, obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution and be placed under XRF,The fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. Result as shown in Figure 2, can be observed high strength glimmeringLight.
Embodiment 3, G-tetra-serobilas/cyanine dyes mark gold nano grain
The G-tetra-serobila DNA moleculars that can form that use are in the present embodiment the strand G-tetra-of 3 ' end sulfydryl modificationSerobila DNA molecular, sequence is that its nucleotides sequence of 5 '-GGGCCAGGGAGCGGGGCGGAGGGGG-3 '-SH(is classified asSequence 3 in sequence table); The cyanine dyes using is following formula IV compound 3:
Compound 3
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: the strand G-tetra-serobila DNA of 3 ' the end sulfydryl modification with above-mentioned sequence are dividedSon is dissolved in the Tris-HCl buffer solution of 10mM of pH value 8.0, is prepared into the DNA that DNA concentration is 10 μ mol/LMother liquor, for subsequent use.
2) preparation of cyanine dyes mother liquor: take a certain amount of cyanine dyes, with methyl alcohol dissolving, compound concentration is 200 μ mol/LCyanine dyes mother liquor.
3) G-tetra-serobilas/cyanine dyes mark gold nano grain: get step 2) in cyanine dyes mother liquor 200 μ L and steps1) DNA mother liquor 2.6mL in, after both mix, then to add concentration be 30 μ mol/L, the gold nano that particle diameter is 5nmParticle solution 200 μ L, in shady place place 1 hour, wherein G-tetra-serobilas, cyanine dyes and gold nano grain moleConcentration ratio is that 13:20:3(is about 4:7:1), obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution and be placed under XRF,The fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. Result as shown in Figure 3, can be observed high strength glimmeringLight.
Embodiment 4, G-tetra-serobilas/cyanine dyes mark gold nano grain
The nucleotides sequence that can form G-tetra-serobila DNA moleculars using is in the present embodiment classified as5 '-ATGTGGGTCATTGTGGGTGGGTGTGGCA-3 ' (sequence 4 in sequence table); Under the cyanine dyes using isState the compound 4 shown in formula V:
Compound 4
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: the G-tetra-serobila DNA molecular strand G-tetra-serobila DNA with above-mentioned sequence are dividedSon be dissolved in pH value 6.0 containing the Tris-HCl buffer solution of 50mMKCl (Tris-HCl buffer solution be concentration 10mM,PH value is 6.0) in, be prepared into DNA concentration and be the DNA mother liquor of 0.5 μ mol/L, for subsequent use.
2) preparation of cyanine dyes mother liquor: take a certain amount of cyanine dyes, with methyl alcohol dissolving, compound concentration is 200 μ mol/LCyanine dyes mother liquor.
3) G-tetra-serobilas/cyanine dyes mark gold nano grain: get step 2) in cyanine dyes mother liquor 23 μ L and step 1)Middle DNA mother liquor 3mL, after both mix, then to add concentration be 38 μ mol/L, the gold nano that particle diameter is 100nmGrain solution 20 μ L, place 1 hour in shady place, and wherein G-tetra-serobilas, cyanine dyes and gold nano grain is mole denseDegree is than being about 2:6:1 for 75:230:38(), obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution and be placed under XRF,The fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. Can be observed high strength fluorescence.
Embodiment 5, G-tetra-serobilas/cyanine dyes mark gold nano grain
The G-tetra-serobila DNA moleculars that use are in the present embodiment the DNA single chain that 5 ' end mark has cyanine dyes Cy3Molecule, its nucleotides sequence is classified 5 '-GGCCAGGGAGCGGGGCGGAGGGGGCGGTCGGG-3 ' (sequence 5) as; Institute makesWith cyanine dyes Cy3 be the compound 5 shown in formula VI:
Compound 5
Above-mentioned 5 ' end mark has the structural formula of DNA single chain molecule of cyanine dyes Cy3 suc as formula shown in IX:
Formula IX, wherein oligo is strand G-tetra-serobila DNA moleculars,
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: the DNA single chain molecule strand G-tetra-that above-mentioned 5 ' end mark is had to cyanine dyes Cy3Serobila DNA molecular be dissolved in pH value 8.0 (concentration is 5mM, pH containing 10mMNaClTris-HCl buffer solutionValue is 8.0Tris-HCl buffer solution) in, being prepared into DNA concentration is the DNA mother liquor of 1 μ mol/L, for subsequent use.
2) G-tetra-serobilas/cyanine dyes mark gold nano grain: get the DNA solution 3mL in step 1), add concentration to be13 μ mol/L, the gold nano grain solution 120 μ L that particle diameter is 17nm, place 1 hour wherein 5 ' in shady placeIt is that 300:156(is about that end mark has the DNA single chain molecule of cyanine dyes Cy3 and the molar concentration rate of gold nano grain2:1), obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution and be placed under XRF,The fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. Result can be observed high strength fluorescence.
Embodiment 6, G-tetra-serobilas/cyanine dyes mark gold nano grain
The DNA single chain of G-tetra-serobilas that use is in the present embodiment the DNA that 5 ' end mark has cyanine dyes Cy3.5Molecule, its nucleotides sequence is classified 5 '-AGGGGCGGGCGCGGGAGGAAGGGGGCGGGAGCGGGGCTG-3 ' (order asRow 6); The cyanine dyes Cy3.5 using is the compound 6 shown in formula VII:
Compound 6
Above-mentioned 5 ' end mark has the structural formula of DNA single chain molecule of cyanine dyes Cy3.5 suc as formula shown in X:
Formula X, wherein oligo is strand G-tetra-serobila DNA moleculars.
(1) process of G-tetra-serobilas/cyanine dyes mark gold nano grain is as follows:
1) preparation of DNA mother liquor: will have above-mentioned 5 ' end mark has the DNA single chain molecule of cyanine dyes Cy3.5 moltenSolution in the 10mM of pH value 5.0 citric acid-sodium citrate buffer solution (in water, dissolve successively 1.92g citric acid and3.22g natrium citricum, is diluted to 1L, mixes) in, be prepared into DNA concentration and be the DNA mother liquor of 5 μ mol/L, standbyWith.
2) G-tetra-serobilas/cyanine dyes mark gold nano grain: get the DNA mother liquor 3mL in step 1), add concentration to be10 μ mol/L, particle diameter is 100nm gold nano grain solution 150 μ L, places 1 hour wherein 5 ' in shady placeIt is 10:1 that end mark has the DNA single chain molecule of cyanine dyes Cy3.5 and the molar concentration rate of gold nano grain, obtains G-Four serobilas/cyanine dyes mark gold nano grain solution.
(2) detect and analyze
By above-mentioned steps 3) obtain G-tetra-serobilas/cyanine dyes mark gold nano grain solution and be placed under XRF,The fluorescence intensity of the cyanine dyes of certification mark on gold nano grain. Result can be observed high strength fluorescence.
Claims (9)
1. a method for fluorescence labeling gold nano grain, for being marked at gold with G-tetra-serobila DNA moleculars by cyanine dyesNano grain surface, obtains fluorescence labeling gold nano grain;
Described G-tetra-serobila DNA moleculars are strand, and its nucleotides sequence is classified GGYGGZGGMGG as, wherein: Y, Z and MRepresent independently one or more any bases;
Described cyanine dyes is the compound shown in formula I,
Wherein: R1For C1-C6Alkyl, phenyl or the phenyl that replaces of alkyl;
R2、R3、R4And R5Independently selected from H or C1-C6Alkyl, or R2And R3The carbon atom being connected with themForm together the ring structure of 5 yuan to 7 yuan, or R4And R5Together with the carbon atom connecting with them, form 5 yuan to 7The ring structure of unit;
R6And R7For C1-C6The C that alkyl or sulfonic group replace1-C6Alkyl;
Y is counter ion, according to R6And R7Institute charged difference and difference, if R6And R7For alkyl, Y is halogenAnion; If R6And R7Only have one with sulfonate radical, without Y as counter ion; If R6And R7All with sulfonic acidRoot, Y is triethylamine cation;
X1，X2Independently selected from C, O, S, Se or Te;
Described C1-C6Alkyl be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group,Amyl group, isopentyl, n-hexyl or isohesyl;
Described 5 yuan is the saturated or unsaturated ring structure that contains or do not contain N or S atom to 7 ring structures;
Described Y is selected from fluorine, chlorine, bromine, iodine anion or triethylamine cation;
Described R1Be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, differentAmyl group, n-hexyl, isohesyl, phenyl, aminomethyl phenyl or 3,5-dimethylphenyl; R2、R3、R4And R5Independently selected from firstBase, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, amyl group, isopentyl, n-hexyl or differentHexyl;
Described with the method that cyanine dyes is marked at gold nano grain surface by G-tetra-serobila DNA moleculars be following 1) or 2):
1) method shown in comprises the steps: cyanine dyes solution, G-tetra-serobila DNA molecular solution A and gold nanoParticle solution is mixed, and obtains fluorescence labeling gold nano grain; Wherein, the G-tetra-serobila DNA moleculars in described solution AFor the strand G-tetra-serobila DNA moleculars of strand G-tetra-serobila DNA moleculars or 5 ' or 3 ' end sulfydryl modification;
2) method shown in comprises the steps: mixed to G-tetra-serobila DNA molecular solution B and gold nano grain solutionClose, obtain fluorescence labeling gold nano grain; Wherein, the G-tetra-serobila DNA moleculars in described solution B are 5 ' or 3 'The strand G-tetra-serobila DNA moleculars of end mark cyanine dyes.
2. method according to claim 1, is characterized in that:
1) in the method shown in,
The strand G-tetra-serobila DNA moleculars that described G-tetra-serobila DNA moleculars are 5 ' end sulfydryl modification, its nucleotidesSequence is sequence 1, and described cyanine dyes is the compound shown in following formula II:
Or described G-tetra-serobila DNA moleculars be 5 ' end sulfydryl modification strand G-tetra-serobila DNA moleculars, its nucleosidesAcid sequence is sequence 2, and described cyanine dyes is the compound shown in following formula III:
Or described G-tetra-serobila DNA moleculars be 3 ' end sulfydryl modification strand G-tetra-serobila DNA moleculars, its nucleosidesAcid sequence is sequence 3, and described cyanine dyes is the compound shown in following formula IV:
Or described G-tetra-serobila DNA moleculars are strand G-tetra-serobila DNA moleculars, its nucleotides sequence is classified sequence 4 as, instituteStating cyanine dyes is the compound shown in following formula V:
2) in the method shown in,
The strand G-tetra-serobila DNA moleculars that described G-tetra-serobila DNA moleculars are 5 ' end mark cyanine dyes, Qi ZhongjingDyestuff is Cy3, and the nucleotides sequence of strand G-tetra-serobila DNA moleculars is classified the sequence 5 in sequence table as; Described 5 ' endThe structural formula of the strand G-tetra-serobila DNA moleculars of mark cyanine dyes is shown in following formula IX:
Formula IX, wherein oligo is strand G-tetra-serobila DNA moleculars,
The structural formula of described Cy3 is shown in following formula VI:
Or the described G-tetra-serobila DNA moleculars strand G-tetra-serobila DNA moleculars that are 5 ' end mark cyanine dyes, whereinCyanine dyes is Cy3.5, and the nucleotides sequence of strand G-tetra-serobila DNA moleculars is classified the sequence 6 in sequence table as; Described 5 'The structural formula of the strand G-tetra-serobila DNA moleculars of end mark cyanine dyes is shown in following formula X:
Formula X, wherein oligo is strand G-tetra-serobila DNA moleculars,
The structural formula of described Cy3.5 is shown in following formula VII:
3. method according to claim 1, is characterized in that:
The solvent of described G-tetra-serobila DNA molecular solution A or described G-tetra-serobila DNA molecular solution B is that pH value isThe cushioning liquid of 5.0-8.2;
Described cushioning liquid be following any: Tris-HCl buffer solution, sodium hydrogen phosphate-potassium phosphate buffer,Potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, sodium phosphate-dibastic sodium phosphate buffer solution, citric acid-sodium citrate buffer solution,Tris-HCI buffer, triethanolamine buffer solution, imidazoles-hydrochloride buffer, glycylglycine bufferingLiquid, 2-amino-2-methyl-1-propanol buffer solution, potassium phosphate-potassium hydrogen phosphate buffer solution, barbital sodium-hydrochloride buffer,Boric acid-borate buffer solution, glycine-sodium hydrate buffer solution, borax-sodium hydrate buffer solution or sodium phosphate buffer;
The solvent of described cyanine dyes solution is methyl alcohol.
4. method according to claim 3, is characterized in that: the cushioning liquid that described pH value is 5.0-8.2For pH value is 5,6,6.5,7.0 or 8 cushioning liquid.
5. according to the method described in claim 3 or 4, it is characterized in that:
1) in the method shown in, the G-tetra-serobila DNA moleculars in described solution A, described cyanine dyes and described JennerThe mol ratio of rice grain is 2-4:4-7:1;
2) in the method shown in, the G-tetra-serobila DNA moleculars in described solution B and described gold nano grain moleThan being 2-10:1.
6. method according to claim 5, is characterized in that:
1) in the method shown in, the G-tetra-serobila DNA moleculars in described solution A, described cyanine dyes and described JennerThe mol ratio of rice grain is 3:6:1 or 4:4:1 or 4:7:1 or 2:6:1;
2) in the method shown in, the G-tetra-serobila DNA moleculars in described solution B and described gold nano grain moleThan being 2:1 or 10:1.
7. according to the arbitrary described method of claim 1-3, it is characterized in that:
1) in the method or 2), described in be mixed into and dark leave standstill 0.5 hour-2 hours.
8. method according to claim 7, is characterized in that: described in be mixed into and dark leave standstill 1 or 2 hour.
9. by fluorescence labeling gold nano grain that in claim 1-8, arbitrary described method prepares.
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| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
| CN101482508A (en) * | 2009-01-21 | 2009-07-15 | 苏州纳米技术与纳米仿生研究所 | High-sensibility detection method for trace metal ion |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101424642A (en) * | 2008-11-14 | 2009-05-06 | 中国科学院上海应用物理研究所 | Target molecule detecting method based on nanometer aurum and nucleic acid structure |
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Non-Patent Citations (2)
| Title |
|---|
| 菁染料超分子体系识别人体端粒G-四链体：溶液与界面研究;杨千帆等;《中国化学会第26届学术年会分析化学分会场论文集》;20080731;第77页 * |
| 识别人体端粒DNA G-四链体的染料结构设计及光谱研究;杨千帆等;《光谱学与光谱分析》;20081031;第28卷(第10期);第291～292页 * |
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