Summary of the invention
The objective of the invention is defective at above-mentioned prior art, provide a kind of and can reach multiple real-time quantitative fluorescence check and analysis, thereby improved the specificity that detects greatly, reliability, fluorescence dye that is used for oligonucleotide and protein labeling of homogeneity and sensitivity and uses thereof.
The present invention also provides a kind of preparation method of above-mentioned fluorescence dye.
The technical scheme taked of the present invention is to achieve these goals: a kind of fluorescence dye that is used for oligonucleotide and protein labeling is provided, and the core texture feature that described fluorescence dye is formed is as follows:
Another technical scheme of the present invention provides the preparation method of above-mentioned fluorescence dye, mainly comprises the steps:
(1), will be with the succinate of amino fluorescence dye that shorter maximum absorption and emission wavelength are arranged and the activated fluorescence dye that more apneusis receipts and emission wavelength are arranged 1-100 in molar ratio: 1 ratio reaction obtains reaction product; Maybe will be with the succinate of amino fluorescence dye that more apneusis receipts and emission wavelength are arranged and the activated fluorescence dye that shorter maximum absorption and emission wavelength are arranged 1-100 in molar ratio: 1 ratio reaction obtains reaction product; Described reaction is any a group with above-mentioned two groups of materials, be dissolved in dimethyl formamide or the dimethyl sulfoxide (DMSO) according to described ratio, add triethylamine, at normal temperature or be lower than 60 ℃, react, the reaction process added the water-soluble night of weakly alkaline yellow soda ash of pH7-10 after 1-24 hour in reactant, promptly obtained reaction product in 1-24 hour 60 ℃ of reactions again; Maybe will be in molar ratio with the succinate of amino fluorescence dye that shorter maximum absorption and emission wavelength are arranged and the activated fluorescence dye that more apneusis receipts and emission wavelength are arranged, be dissolved in water-soluble night of weakly alkaline yellow soda ash and the mixing of pH7-10, at normal temperature or be lower than 60 ℃, react and promptly obtained reaction product in 1-48 hour.
(2), reaction product adopts dialysis, the method for crossing post, ultrafiltration or high-pressure liquid phase is removed byproduct of reaction and unreacted chemicals, promptly obtain a kind of novel and effective and absorb laser energy and at the fluorescence dye of longer wavelength emission than hyperfluorescence at shorter wavelength.
The structure of the fluorescence dye that shorter maximum absorption and emission wavelength are arranged that described band is amino is:
Short wavelength's fluorescence dye:
The structure of the succinate of the described fluorescence dye that more apneusis receipts and emission wavelength arranged is:
The constitutional features that the described fluorescence dye that shorter maximum absorption emission wavelength arranged has is: this fluorescence dye is for absorbing than the amidized of light laser energy or through succsinic acid activatory fluorescein at shorter wavelength, be single isomer or its isomer mixture, absorbing stronger laser energy wavelength is 460-520nm.
The constitutional features that the described fluorescence dye that more apneusis receipts and emission wavelength arranged has is: this fluorescence dye is for can be the amidized of longer wavelength absorbs and release strength is higher fluorescence or through succsinic acid activatory fluorescence dye, at red and infrared wavelength emitting fluorescence, be single isomer or its isomer mixture, the higher wavelength of fluorescence of release strength is 550-750nm.
Fluorescence dye of the present invention is used for oligonucleotide and protein labeling after the succsinic acid activation.
The present invention utilizes the principle of energy transformation and transmission successfully to prepare the fluorescence dye that can be used for multicolored fluorescing system, method by chemical coupling has fluorescence molecule DY-630 or synthetic composite fluorescence molecule of Bodipy-635 that maximum fluorescence discharges the fluorescein that has maximum absorption at the 480-520nm wavelength with at the 650nm wavelength, makes this fluorescence molecule that ten times raising arranged in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.This method is particularly useful for fluorescein and absorbs laser energy under the 460-520nm wavelength, and the method that shifts by intramolecular energy is passed to another intramolecularly and had the 535-700nm wavelength under the absorption energy and discharge the stronger fluorescence molecule of 550-750nm wavelength fluorescent.When the fluorescence molecule of so a plurality of different wave lengths is marked on the different nucleic acid fragment or protein molecular, just can reach multiple real-time quantitative check and analysis, thereby improve the specificity, reliability, homogeneity and the sensitivity that detect greatly.This method is particularly suitable for human STR-PCR (chain reaction of STR archaeal dna polymerase) DNA composite amplification gene type, in gene and the protein chip product.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but not as a limitation of the invention.
The core texture feature that fluorescence dye is formed is as follows: new fluorescence molecule is connected to form by the fluorescence molecule of short excitation wavelength and the fluorescence molecule of long excitation wavelength.
The preparation feedback principle of fluorescence dye of the present invention is as follows:
The energy transformation method is synthesized infrared wavelength fluorescence dye composition principle
Perhaps
Infrared wavelength fluorescence molecule synthesis mechanism:
Long wavelength's fluorescence dye DY-630 succinate and amination short wavelength fluorescein are dissolved in the dimethyl formamide, under the katalysis of triethylamine, connect and to form one and new can discharge the stronger fluorescence molecule of fluorescence at 480-520nm wavelength efficient absorption energy and at the 650nm wavelength
Embodiment 1
Amination short wavelength's fluorescein, wavelength 460-520nm is single isomer and 0.1mmole long wavelength's fluorescence dye DY-630 succinate 1: 1 in molar ratio, is dissolved in the 100 μ l dimethyl formamides.Add 10 μ l triethylamines and under its katalysis, react, after 1 hour reaction, add the water-soluble night of pH7-10 weakly alkaline yellow soda ash of 200 μ l and mix at normal temperature.Again 60 ℃ of reactions 1 hour.After reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor (can heat, but should be lower than 60 ℃) behind concentrating under reduced pressure, is dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence dye of wanting promptly obtained.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 2
Amination short wavelength's fluorescein is isomer mixture and 0.1mmole long wavelength's fluorescence dye DY-630 succinate 20: 1 in molar ratio, is dissolved in the 200 μ l dimethyl formamides.Add 20 μ l triethylamines under its katalysis,, react, after 12 hours reaction, add water-soluble night of pH7-10 weakly alkaline yellow soda ash and the mixing of 500 μ l below 60 ℃.Again 60 ℃ of reactions 24 hours.After reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor is dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5% behind concentrating under reduced pressure (can heat, but should be lower than 60 ℃), and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence molecule of wanting promptly obtained.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 3
Amination short wavelength's fluorescein, wavelength 460-520nm is isomer mixture and 0.1mmole long wavelength's fluorescence dye DY-630 succinate 100: 1 in molar ratio, is dissolved in the 1000 μ l dimethyl sulfoxide (DMSO).Add 50 μ l triethylamines and under its katalysis,, react, after 24 hours reaction, add the water-soluble night of pH7-10 weakly alkaline yellow soda ash of 1000 μ l and mix below 60 ℃.Again 60 ℃ of reactions 12 hours.After reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor is dissolved in the solution of methyl alcohol in methylene dichloride of 25ml 5% behind concentrating under reduced pressure (can heat, but should be lower than 60 ℃), and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 2000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band light green fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence molecule of wanting promptly obtained.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 4
Amination short wavelength's fluorescein, wavelength 460-520nm is isomer mixture and 0.1mmole long wavelength's fluorescence dye DY-630 succinate 10: 1 in molar ratio, is dissolved in water-soluble night of 100 μ l pH7-10 weakly alkaline yellow soda ash and mixes.At normal temperatures, react, after 24 hours reaction, high speed centrifugation is removed precipitation, supernatant liquor is behind concentrating under reduced pressure, be dissolved in the solution of methyl alcohol in methylene dichloride of 25ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1500ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band light green fluorescence component, behind concentrating under reduced pressure (can heat, but be lower than 60 ℃), promptly obtain the fluorescence molecule of wanting.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 5
Amination short wavelength's fluorescein, wavelength 460-520nm is isomer mixture and 0.01mmole long wavelength's fluorescence dye DY-630 succinate 100: 1 in molar ratio, is dissolved in water-soluble night of 200 μ l pH7-10 weakly alkaline yellow soda ash and mixes.At 25 ℃-60 ℃, react. after 1 hour reaction, high speed centrifugation is removed precipitation, supernatant liquor is behind concentrating under reduced pressure, be dissolved in the solution of methyl alcohol in methylene dichloride of 25ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band light green fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence molecule of wanting promptly obtained.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 6
Amination short wavelength's fluorescein, wavelength 460-520nm is isomer mixture and 0.001mmole long wavelength's fluorescence dye DY-630 succinate, 100: 1 in molar ratio, is dissolved in water-soluble night of 1000 μ l pH7-10 weakly alkaline yellow soda ash and mixing.Be lower than 60 ℃, react, after 48 hours reaction, high speed centrifugation is removed precipitation, supernatant liquor is behind concentrating under reduced pressure, be dissolved in the solution of methyl alcohol in methylene dichloride of 25ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 2000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band light green fluorescence component, behind concentrating under reduced pressure (can heat, but be lower than 60 ℃), promptly obtain the fluorescence molecule of wanting.This fluorescence molecule has had ten times raising in the fluorescence efficiency of 650nm wavelength after the 488nm wavelength absorption.
Embodiment 7
Amination short wavelength's fluorescein, wavelength 460-520nm is single isomer and 0.1mmole long wavelength's fluorescence dye Bodipy-635 succinate 1: 1 in molar ratio, is dissolved in the 100 μ l dimethyl formamides.Add 10 μ l triethylamines and under its katalysis, react, after 1 hour reaction, add the water-soluble night of pH7-10 weakly alkaline yellow soda ash of 200 μ l and mix at normal temperature.Again 60 ℃ the reaction 1 hour after, high speed centrifugation is removed precipitation, supernatant liquor (can heat through concentrating under reduced pressure, but should be lower than 60 ℃) after, be dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence dye of wanting promptly obtained.This fluorescence molecule has had the raising more than ten times in the fluorescence efficiency at the 650nm wavelength after the 488nm wavelength absorption.
Embodiment 8
Amination long wavelength's fluorescence dye DY-630 and 0.1mmole short wavelength's fluorescence dye fluorescein succinate 50: 1 in molar ratio are dissolved in the 100 μ l dimethyl formamides.Add 10 μ l triethylamines and under its katalysis, react, after 1 hour reaction, add the water-soluble night of pH7-10 weakly alkaline yellow soda ash of 200 μ l and mix at normal temperature.Again 60 ℃ of reactions 1 hour.After reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor is dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5% behind concentrating under reduced pressure (heat, but should be lower than 60 ℃), and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence dye of wanting promptly obtained.This fluorescence molecule has had the raising more than ten times in the fluorescence efficiency at the 650nm wavelength after the 488nm wavelength absorption.
Embodiment 9
Amination short wavelength's fluorescein, wavelength 460-520nm is single isomer and 0.1mmole long wavelength's fluorescence dye Alexa-633 succinate 2: 1 in molar ratio, is dissolved in the 100 μ l dimethyl formamides.Add 10 μ l triethylamines and under its katalysis, carry out hybrid reaction, after 1 hour reaction, add water-soluble night of pH7-10 weakly alkaline yellow soda ash and the mixing of 200 μ l at normal temperature.Again 60 ℃ of reactions 1 hour.After reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor is dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5% behind concentrating under reduced pressure (can heat, but should be lower than 60 ℃), and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, the fluorescence dye of wanting promptly obtained.This fluorescence molecule has had the raising more than ten times in the fluorescence efficiency at the 650nm wavelength after the 488nm wavelength absorption.
Embodiment 10
Amination short wavelength's fluorescein, wavelength 460-520nm is single isomer and 0.1mmole long wavelength's fluorescence dye Bodipy-635 succinate 5: 1 in molar ratio, is dissolved in the 100 μ l dimethyl formamides.Add 10 μ l triethylamines and under its katalysis, carry out hybrid reaction, after 1 hour reaction, add water-soluble night of pH7-10 weakly alkaline yellow soda ash and the mixing of 200 μ l at normal temperature.Again 60 ℃ of reactions 1 hour.After reaction finishes, high speed centrifugation is removed precipitation, and supernatant liquor is behind concentrating under reduced pressure (can heat, but should be lower than 60 ℃), be dissolved in the solution of methyl alcohol in methylene dichloride of 10ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 1000ml 5-30% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, behind concentrating under reduced pressure, can heat, but be lower than 60 ℃, promptly obtain the fluorescence dye of wanting.This fluorescence molecule has had the raising more than ten times in the fluorescence efficiency at the 650nm wavelength after the 488nm wavelength absorption.
0.1mmole fluorescence dye that obtains through the aforesaid method of implementation column 1 to 10 and succsinic acid are dissolved in the 100 μ l dimethyl formamides with mol ratio 1: 1-100.Add 10 μ l triethylamine alkali and under its katalysis, at normal temperature or be lower than 60 ℃, react.Reaction through 1-24 hour, after reaction finishes, high speed centrifugation is removed precipitation, supernatant liquor (can heat through concentrating under reduced pressure, but be lower than 60 ℃) after, be dissolved in the organic solvent of methyl alcohol in methylene dichloride of 5ml 5%, and separate through silica gel column chromatography with the concentration gradient solution of methyl alcohol in methylene dichloride of 300-500ml5-25% (volume by volume concentration), remove byproduct of reaction and unreacted compound, collect last band look fluorescence component, behind concentrating under reduced pressure (can heat, but be lower than 60 ℃), promptly obtain the fluorescence molecule succinate of the purifying of wanting.This succinate promptly can be used for oligonucleotide and protein labeling, the product behind the mark, and by dialysis, the equal method of post and high pressure liquid of crossing is removed byproduct of reaction and unreacted compound, promptly obtains required fluorescently-labeled oligonucleotide or albumen.When the fluorescence molecule of so a plurality of different wave lengths is marked on the different nucleic acid fragment or protein molecular, just can reach multiple real-time quantitative check and analysis, thereby improve the specificity, reliability, homogeneity and the sensitivity that detect greatly.This method is particularly suitable for human STR-PCR DNA composite amplification gene type, in gene and the protein chip product.
The embodiment of the above, the present invention embodiment a kind of more preferably just, the common variation that those skilled in the art carries out in the technical solution of the present invention scope and replacing all should be included in protection scope of the present invention.