CN101659997B - Fluorescence detection method for distinguishing single stranded nucleotide from double stranded nucleotide - Google Patents
Fluorescence detection method for distinguishing single stranded nucleotide from double stranded nucleotide Download PDFInfo
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- CN101659997B CN101659997B CN2009101804485A CN200910180448A CN101659997B CN 101659997 B CN101659997 B CN 101659997B CN 2009101804485 A CN2009101804485 A CN 2009101804485A CN 200910180448 A CN200910180448 A CN 200910180448A CN 101659997 B CN101659997 B CN 101659997B
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- pyrene
- oligomerization
- fluorescence
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Abstract
The invention provides a fluorescence analysis new method for distinguishing ss DNA from ds DNA based on oligomeric pyrene ramification, comprising the steps: (1) synthesizing the oligomeric pyrene ramification with water solubility by means of chemistry and electrochemistry, wherein a synthesizing line is simper and product purification is easier; and (2) dissolving fluorescence conjugated oligomeric pyrene ramification into Tris-HCL buffer solution, and preparing into the solution with a certain concentration. Based the different fluorescence signal resume of the conjugated oligomeric pyrene ramification induced by the ss DNA and the ds DNA, the ss DNA can be conveniently distinguished from the ds DNA. Compared with the methods for detecting the DNA based on conjugated polymer, the detection system has no expensive fluorescence marked DNA probe, thereby having low detection cost and better practical application foreground.
Description
Technical field
The present invention relates to the fluorescence detection method of a kind of differentiation strand (ssDNA) and double chain nucleotide (dsDNA), belong to technical field of material.
Technical background
In the research of bio-sensing system, the detection of DNA, protein, enzyme, biological micromolecule and analysis are very important research contents.Wherein the quantitative analysis of DNA and RNA and specific recognition have crucial meaning in fields such as genomics, virusology, molecular biology, medical diagnosis on disease, transgenation and gene transmission.Yet, DNA is carried out fast, sensitive, detect cheaply, with identification on still have big difficulty and challenge, and these technology DNA detection analysis key one ring in actual applications just.
In recent years domestic and international research work proves that when chemistry that detects trace and biological target molecules, the sensitivity of fluorescent conjugated polymer will be far above corresponding fluorescent small molecule.Shift and relative stronger light capture ability with molecule interchain energy in the unusual strong molecular chain that its this character comes from that it is distinctive, produced along conjugated backbone delocalized πDian Zi.One of method is by measuring the super cancellation signal of fluorescence of sensing system, detecting the DNA target molecule of lower concentration in the aqueous solution.But this class sensing system mostly needs the polymerized unit conjugated system of expensive fluorescently-labeled dna probe and polymkeric substance little, is unfavorable for the interactional generation of molecule interchain πDian Zi.In the known references, yet there are no utilization and contain the condensed ring structural polymer recovers to detect and distinguish strand (ssDNA) and double chain nucleotide (dsDNA) by the fluorescence of sensing system report at present.
The present invention adopts the derivative of water-soluble oligomeric pyrene as fluorescent probe, fluorescence by the sensing system recovers, directly effectively detect and distinguished ssDNA and dsDNA in the water medium, exempted because of using the expensive fluorescently-labeled dna probe that has to cause detecting problems such as cost rising.
Summary of the invention
Technical problem to be solved by this invention provides the novel method of a kind of ssDNA of differentiation and dsDNA.The preparation process simple controllable of method of the present invention, and have good repeatability.Biggest advantage can directly effectively detect and distinguish ssDNA and dsDNA in the water medium, exempts because of using the expensive fluorescently-labeled dna probe that has to cause detecting problems such as cost rising.
The chemical structural formula of institute of the present invention synthetic oligomer fluorescent probe molecule is:
Oligomer title: oligomerization (N
1, N
1, N
1, N
4, N
4, N
4-hexamethyl-2-(pyrenyl-1-methanoyl) butyl-1,4-dimethyl amine bromide synthetic route is as follows:
Compound 1 compound 2 compounds 3 compounds 4
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the method for distinguishing the fluoroscopic examination of ssDNA and dsDNA comprises following concrete steps:
1) compound 1 is synthetic: (200mg 0.813mmol) and the 20mL methylene dichloride, and adds a N, and the N dimethyl formamide is as catalyzer to add pyrene formic acid in the round-bottomed flask of 50mL.Mixing solutions cools off with ice-water bath.
Then, in 10min, progressively be added dropwise to 0.21mL oxalyl chloride (2.44mmol).Reaction 8h after rotary evaporation remove and to desolvate.After adding 50mL toluene afterwards, behind rotary evaporation, obtain the thick product of light amber again.With this product with the mixed solvent (v/v=1/1) of toluene and normal hexane in-20 ℃ of following recrystallization purifying 24h, obtain the crystal of white at last, productive rate 97% (242mg), fusing point: 88-90 ℃;
2) compound 2 is synthetic: with salt of wormwood (88mg, 0.813mmol) and the pyrene formyl chloride (200mg 0.813mmol) is dissolved in the 5mL methylene dichloride and adds a N, and dinethylformamide is as catalyzer.Then, in 5 minutes, progressively drip 0.2mL 1, and 4-two bromo-2-butanols (0.39mL, 1.19mmol).Mixing solutions uses the ice-water bath control reaction temperature at 0 ℃.After the question response cooling, concentrate mixing solutions, use 3 * 40mL, 5% wet chemical (weight ratio) and 3 * 40mL aqueous solution repetitive scrubbing three times then, and it is dry to put into vacuum drying oven.This crude product is that eluent adopts silicagel column to carry out purifying with the chloroform, obtains light yellow viscosity oily matter 303mg, productive rate: 95%;
3) compound 3 is synthetic: the electrochemical polymerization of compound 2 is at EG﹠amp; Carry out on the 273A of G Princeton applied research manufacturing and the electrochemistry synthesis analyzer.Working electrode and counter electrode are that the area that pair of parallel is placed is 1.0cm
2Platinum electrode, the vertical range between them is 1.0cm.Reference electrode is freshly prepd homemade Ag/AgCl.Typical electrolytic solution is 5mmoL
-1The boron trifluoride ether solution of compound 2.Before the polymerization, at first feed high pure nitrogen, electrochemical polymerization 22 hours under the operating voltage of 0.75V (vs.Ag/AgCl) then at electrolytic solution.Reaction peels polymeric membrane, and cleans with anhydrous diethyl ether immediately after finishing from platinum electrode.The product of gained is dry under vacuum condition, obtains the 31mg product, and productive rate is 35%;
4) compound 4 (fluorescence conjugated oligomerization pyrene derivatives) is synthetic: the 2.3mL Trimethylamine 99 is joined in tetrahydrofuran (THF) (3mL) solution of compound 3 (10mg).Mixed solution stirred 50 hours at ambient temperature.Under reduced pressure, solvent is removed then.The solid sample that obtains cleans three times with tetrahydrofuran (THF), and is dry under vacuum condition then, obtains the 11.7mg product, and productive rate is 93%;
5) fluorescence conjugated few pyrene derivatives is dissolved in the Tris-HCl damping fluid, is mixed with finite concentration, promptly can be used for the fluorometric analysis of DNA sample.
In the described step (5), the Tris-HCl damping fluid of use can be weak acid or weakly alkaline.
Compared with prior art, the invention has the advantages that:
1. the present invention has synthesized a kind of new water-soluble oligomerization pyrene fluorescent probe molecule by electrochemical techniques;
2. the present invention passes through the fluorescence recovery of sensing system, directly effectively detects and distinguished ssDNA and dsDNA in the water medium;
3. the present invention need not fluorescently-labeled dna probe, and it is lower to detect cost, and preparation technology is controlled.
Embodiment
Embodiment one
Take by weighing an amount of institute synthetic conjugation oligomerization pyrene derivatives and place the 15mL reagent bottle.With the ultrapure water compound concentration is 10m molL
-1The Tris-HCl damping fluid of pH=7.4.Afterwards, the damping fluid of certain volume added contain in the conjugation oligomerization pyrene derivatives reagent bottle, making its concentration is 3.34 * 10
-6Mol L
-1, and pipette 3 parts of these solution of 5mL as detecting liquid.Then, in this system, drip ssDNA, dsDNA sample to be measured.The final concn of ssDNA, dsDNA is 8 * 10
-7Mol L
-1Conjugation oligomerization pyrene derivatives detects the fluorescence response of liquid to DNA, under 25 ℃, the fluorescence intensity of fluorescence sense system at the 465nm place write down and obtains with FL 4600 type luminoscopes, and excitation wavelength is 340nm.Experimental result is: ssDNA, dsDNA make the recovery value of conjugation oligomerization pyrene derivatives fluorescent signal be respectively 234 and 81, and the former is three times of the latter approximately, shows that fluorescent probe of the present invention can effectively detect and distinguish ssDNA and dsDNA.
Embodiment two
Take by weighing an amount of institute synthetic conjugation oligomerization pyrene derivatives and place the 15mL reagent bottle.With the ultrapure water compound concentration is 10m molL
-1The Tris-HCl damping fluid of pH=7.4.Afterwards, the damping fluid of certain volume added contain in the conjugation oligomerization pyrene derivatives reagent bottle, making its concentration is 3.34 * 10
-6Mol L
-1, and pipette 3 parts of these solution of 5mL as detecting liquid.Then, in this system, drip ssDNA, dsDNA sample to be measured.The final concn of ssDNA, dsDNA is 3 * 10
-5Mol L
-1Conjugation oligomerization pyrene derivatives detects the fluorescence response of liquid to DNA, under 25 ℃, the fluorescence intensity of fluorescence sense system at the 465nm place write down and obtains with FL 4600 type luminoscopes, and excitation wavelength is 340nm.Experimental result is: ssDNA, dsDNA make the recovery value of conjugation oligomerization pyrene derivatives fluorescent signal be respectively 645 and 231, and the former is three times of the latter approximately, shows that fluorescent probe of the present invention can effectively detect and distinguish ssDNA and dsDNA.
Embodiment three
Take by weighing an amount of institute synthetic conjugation oligomerization pyrene derivatives and place the 15mL reagent bottle.With the ultrapure water compound concentration is 10m molL
-1The Tris-HCl damping fluid of pH=7.4.Afterwards, the damping fluid of certain volume added contain in the conjugation oligomerization pyrene derivatives reagent bottle, making its concentration is 3.34 * 10
-6Mol L
-1, and pipette 3 parts of these solution of 5mL as detecting liquid.Then, in this system, drip ssDNA, dsDNA sample to be measured.The final concn of ssDNA, dsDNA is 8 * 10
-5Mol L
-1Conjugation oligomerization pyrene derivatives detects the fluorescence response of liquid to DNA, under 25 ℃, the fluorescence intensity of fluorescence sense system at the 465nm place write down and obtains with FL 4600 type luminoscopes, and excitation wavelength is 340nm.Experimental result is: ssDNA, dsDNA make the recovery value of conjugation oligomerization pyrene derivatives fluorescent signal be respectively 873 and 312, and the former is three times of the latter approximately, shows that fluorescent probe of the present invention can effectively detect and distinguish ssDNA and dsDNA.
The present invention proposes a kind of fluorometric analysis novel method of distinguishing ssDNA and dsDNA based on the oligomerization pyrene derivatives.The electrochemical polymerization of passing through of oligomerization pyrene derivatives involved in the present invention obtains, and synthetic route is simpler, and productive rate is higher.With existing many different based on conjugated polymers detection DNA method, detection architecture involved in the present invention does not contain expensive fluorescently-labeled dna probe, and this is for having established certain basis based on the oligomerization pyrene derivatives in the application of distinguishing ssDNA and dsDNA.The present invention will provide a kind of new technology and method for the pickup probe that preparation is used to distinguish ssDNA and dsDNA.
Claims (2)
1. distinguish the fluorometric analysis novel method of ssDNA and dsDNA based on the oligomerization pyrene derivatives for one kind, it comprises following concrete steps:
1) compound 1 is synthetic: with N, the N dimethyl formamide reacts pyrene formic acid and oxalyl chloride as catalyzer;
2) compound 2 is synthetic: with N, the N dimethyl formamide is as catalyzer, and with compound 1 and 1,4-two bromo-2-butanols react;
3) compound 3 is synthetic: at EG﹠amp; On the 273A and electrochemistry synthesis analyzer of the applied research manufacturing of G Princeton, compound 2 is carried out electrochemical polymerization;
4) compound 4 (fluorescence conjugated oligomerization pyrene derivatives) is synthetic: compound 3 and Trimethylamine 99 are reacted;
5) fluorescence conjugated few pyrene derivatives is dissolved in the Tris-HCl damping fluid, being mixed with concentration is 3.34 * 10
-6Mol L
-1Solution.This solution promptly can be used for the fluorometric analysis of DNA sample.
2. the fluorometric analysis novel method based on oligomerization pyrene derivatives differentiation ssDNA and dsDNA according to claim 1 is characterized in that: in the described step (3), adopt electrochemical polymerization that compound 2 is carried out polymerization at boron trifluoride ether solution.
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| CN101659997B true CN101659997B (en) | 2011-12-28 |
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| CN102936620B (en) * | 2012-07-16 | 2015-04-01 | 北京科技大学 | Method for detecting nucleotide sequence by using poly(methyl acrylic pyrene-poly (methyl) acrylic dimethylamine ethyl ester copolymer, and product thereof |
| CN105044059B (en) * | 2015-07-13 | 2017-08-29 | 深圳市瀚海基因生物科技有限公司 | A kind of deoxygenation reagent and its application process that can be used for slowing down single molecular fluorescence bleaching phenomenon |
| CN105543399B (en) * | 2016-02-26 | 2019-07-16 | 中国科学院水生生物研究所 | A method of utilizing fluorescence detection microcystis DNA damage |
| CN112326763B (en) * | 2020-09-24 | 2022-10-21 | 福建医科大学附属第一医院 | DNA nano-sieve reproducible electrochemical sensor for clinical sample allelic genotyping |
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Non-Patent Citations (1)
| Title |
|---|
| Youqiang Chen et al.《Fluorescence Detection and Discrimination of ss- and ds-DNA with a Water Soluble Oligopyrene Derivative》.《Sensors》.2009,(第9期),参见4166页第一段，线路图1及其说明，图2附图说明等. * |
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