CN104458682B - A kind of multi-functional oligonucleotide chain is used for the fluorescence detection method of many kinds of substance - Google Patents
A kind of multi-functional oligonucleotide chain is used for the fluorescence detection method of many kinds of substance Download PDFInfo
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- CN104458682B CN104458682B CN201410613314.9A CN201410613314A CN104458682B CN 104458682 B CN104458682 B CN 104458682B CN 201410613314 A CN201410613314 A CN 201410613314A CN 104458682 B CN104458682 B CN 104458682B
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Abstract
A kind of multi-functional oligonucleotide chain is used for the fluorescence detection method of many kinds of substance, and measured matter includes complementary strand, fibrin ferment, the Hg of oligonucleotide chain2+With four kinds of different materials of L cysteines, comprise the following steps：(1) solution is prepared：a.Tris‑HNO3Cushioning liquid；B. graphene oxide dispersion；(2) fluorescence probe is preheated and is slowly cooled to room temperature；(3) the standard measured matter of various concentrations is mixed with fluorescence probe solution, be incubated at room temperature；(4) graphene oxide dispersion and cushioning liquid are added, fluorescence emission spectrum is measured, according to the fluorescence signal intensity for detecting, the standard curve of measured matter is set up；(5) sample solution is reacted instead of measured matter, according to the fluorescence signal intensity and corresponding standard curve of detection, obtained measured matter content in sample solution.Advantage of the present invention is：Sensitivity high interference is small, and selectivity is good, can reach quick detection complementary strand, fibrin ferment, Hg2+With four kinds of purposes of different material of L cysteines.
Description
Technical field
The present invention relates to the research that multiprobe realizes many kinds of substance detection, specifically a kind of multi-functional oligonucleotide
Chain is used for complementary strand cTBA, fibrin ferment, Hg2+With the fluorescence detection method of Cys many kinds of substance.
Background technology
Graphene oxide is a kind of Novel Carbon Nanomaterials, and its thickness is only single carbon atom particle size, but with wide
Wealthy two dimensional surface, graphene oxide backbone carbon atoms are all with sp2Form hydridization so that graphene oxide contains abundant π
Electronics.Have been reported that discovery, graphene oxide has superpower glimmering as fluorescent receptor to the fluorescent dye of single stranded DNA end mark
Optical quenching ability.Its principle is that the nucleotide base in single stranded DNA is adsorbed in graphite oxide by the hydrophobic forces of pi-pi accumulation
In alkene plane, simultaneously there is fluorescence resonance energy in the fluorescent dye FAM of DNA ends of effectively having furthered and the distance of graphene oxide
FRET effects are shifted, then the fluorescence of dyestuff is quenched；And work as the single stranded DNA of dye marker and the single stranded DNA of its complete complementary
After forming double-stranded DNA, the hydrophily active force of the exposed phosphate backbones in outside is occupied an leading position, and double-stranded DNA is inconjunction with FAM disengagings
Surface of graphene oxide, the distance between fluorogenic donor FAM and fluorescent receptor graphene oxide become remote, and FRET effects disappear.Profit
This phenomenon is used, the detection ssDNA of high selectivity high sensitivity can be successfully realized, and the difference of single base can be recognized.
However, the research reported can only all realize that oligonucleotide chain detects a kind of target, when needing detection
During different target, can only be realized by changing the sequence of oligonucleotide chain.
The content of the invention
Based on this present situation, the present invention is developed and a kind of can be used for the multi-functional oligonucleotide of many kinds of substance fluoroscopic examination
The oligonucleotide chain of chain, i.e., one realizes complementary strand, fibrin ferment, Hg2+With four kinds of detections of different material of Cys.Its inspection
Survey principle similar with above-mentioned prior art, have the single stranded DNA of FAM as fluorescence probe using end mark, this probe is named as
FAM-TBA.When FAM-TBA and graphene oxide is comprised only in system, FAM-TBA is freely crimped, and is adsorbed in graphene oxide
Surface, FRET effects are produced, and fluorescence is quenched.As addition complementary strand, fibrin ferment or Hg2+Afterwards, FAM-TBA forms double-strand respectively
Tetra- serobilas of DNA, G-/antithrombin complex and " T-Hg2+- T " structures, FAM-TBA departs from surface of graphene oxide, fluorophor
The distance between FAM and graphene oxide are zoomed out, and hinder the generation of FRET effects, and the fluorescence of system is restored, fluorescence
The intensity of recovery respectively with the complementary strand cTBA, fibrin ferment and Hg for introducing2+Amount be closely related.When the introducing Guang ammonia of L- half in system
During sour Cys, the sulfhydryl-group activity and Hg of Cys2+Complexing, by it from " T-Hg2+Removed in-T " structures, FAM-TBA recovers nature volume again
Curved state, oxidized graphenic surface absorption rapidly, FRET effects are produced again, and fluorescence is quenched, now fluorescent quenching journey
Spend and be closely related with the amount of the Cys for introducing, be capable of achieving the detection of Cys.
The purpose of the present invention is just being directed to an oligonucleotide chain in the prior art can only realize an inspection for target
The problem of survey, and a kind of fluorescence detection method of multi-functional oligonucleotide chain for many kinds of substance is provided.The detection of selection is right
As being respectively complementary strand, fibrin ferment, Hg2+With Cys Cys.It is well known that the single base of many diseases of the mankind and gene
Mutation is related, thus the detection of the DNA of single base mismatch is particularly important.And fibrin ferment, it is a kind of serine protease,
Wound can substantial amounts of generation, generation hemoglutination is combined with blood platelet, but also result in thrombosis under pathological conditions, because
This its detection has great importance to biomedical diagnostic.Further, Hg2+There is serious corrosiveness to intestines, kidney and brain,
Hg2+There is very strong affinity with the cooperation base containing sulphur atom in human body, the sulfydryl agllutination of protein, enzyme and film can be caused.It
Severe inflammation symptom can be caused in human body alimentary canal, generally within a few hours i.e. occur abdominal cramps and accompany by nausea,
Vomiting and bloody diarrhea etc..Therefore, recognize and detection Hg2+Tool is of great significance.Finally, Cys are a kind of tools
Have the amino acid of physiological function, be constitutive protein matter 20 several amino acids in the only amino with reproducibility group sulfydryl
Acid, therefore, identification and detection Cys tool are of great significance.The present invention can be by a kind of fluorescence probe, not
Same detection architecture and application field realizes the detection of target analytes.
The purpose of the present invention in the following manner realize by technical scheme：
A kind of multi-functional oligonucleotide chain be used for many kinds of substance fluorescence detection method, be to complementary strand, fibrin ferment,
Hg2+With tetra- kinds of fluoroscopic examinations of material of Cys, following steps are specifically included：
1) solution is prepared：a、 Tris-HNO3Cushioning liquid；B, graphene oxide dispersion；
2）100 μM of fluorescence probe solution is heat-treated, is heated 5-10 minutes at 90-100 DEG C, progressively cool to room
Temperature；
3）By the complementary strand of various concentrations, fibrin ferment, Hg2+Mix with fluorescence probe solution respectively with Cys standard liquids, room
Temperature is lower to be incubated half an hour；
4）Graphene oxide dispersion and cushioning liquid are added in above-mentioned solution, the final volume for making to treat test sample is 200
μ L, graphene oxide concentration is 130 μ g mL-1, fluorescence probe concentration is 0.2 μM, and half an hour is reacted at room temperature；Measurement solution
Fluorescence emission spectrum, set up fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution2+Standard curve and Cys between；
5）Sample solution is replaced into standard liquid, above-mentioned reaction is carried out, the fluorescence signal intensity band of the solution that will be detected
Enter standard curve, obtain complementary strand, fibrin ferment, Hg in sample solution2+With the content of Cys.
Described complementary strand cTBA, fibrin ferment, Hg2+Concentration with Cys Cys standard liquids is respectively：0.005,
0.01,0.04,0.07,0.1,0.3,0.5,0.7 the cTBA with 1 μM；0.01, 0.05, 0.1, 0.5, 1, 2,
3,4,5,6,7,8,9,10,15 and 20 μ g mL-1Fibrin ferment；0.5,1,3,5,8,10,20,30 He
50 μM of Hg2+；10,9,8,7,6,5,4,3,2,1 and 0.5 μM of Cys.
Described Tris-HNO3Cushioning liquid composition is：20 mM Tris, 100 mM NaNO3, pH=8.0。
Described fluorescence probe sequence is 5'FAM-GG TTG GTG TGG TTG G-3'.
Instrument for fluorescence measurement is Hitachi F-4600 sepectrophotofluorometers.
Described fluorescence spectral measuring condition：Excite and be respectively 5.0 nm and 10.0nm with transmite slit width, voltage is
700 V, excitation wavelength is 480 nm, launch wavelength sweep limits 500-580 nm, and sample cell is the quartz cuvette of 0.60 mL
Ware.
In the present invention, complementary strand, fibrin ferment, Hg2+It is as follows with the method that four kinds of materials of Cys are detected respectively：
（1）The detection of complementary strand, as shown in Figure 1.By fluorescence probe solution the pre-heat treatment, 5-10 is heated at 90-100 DEG C
Minute, it is slowly cooled to room temperature.The cTBA solution of various concentrations is mixed with fluorescence probe solution, half an hour is incubated at room temperature.
Graphene oxide dispersion and cushioning liquid are added, the final volume for making to treat test sample is 200 μ L, and graphene oxide concentration is 130
µg mL-1, fluorescence probe concentration is 0.2 μM, is reacted at room temperature half an hour, measures fluorescence emission spectrum.Investigating the sensor
In to the experiment of cTBA selectivity, basic step is consistent with the step of detecting cTBA, unlike, with other widows of same concentrations
Polynucleotide chain replaces cTBA, and by sample, the special of the sensor is judged in the detection of fluorescent emission intensity at 520 nm
Property.
（2）The detection of fibrin ferment, as shown in Figure 2.By fluorescence probe solution the pre-heat treatment, 5-10 is heated at 90-100 DEG C
Minute, it is slowly cooled to room temperature.The thrombin solution of various concentrations is mixed with fluorescence probe solution, half is incubated at room temperature small
When.Graphene oxide dispersion and cushioning liquid are added, the final volume for making to treat test sample is 200 μ L, and graphene oxide concentration is
130 µg mL-1, fluorescence probe concentration is 0.2 μM, is reacted at room temperature half an hour, measures fluorescence emission spectrum.Investigating the biography
Sensor is consistent the step of basic step is with detection fibrin ferment in the experiment of blood coagulation enzyme selectivity, the difference is that, use same concentrations
Other oroteins replace fibrin ferment, by sample at 520 nm fluorescent emission intensity detection judge the sensor spy
The opposite sex.
（3）Hg2+Detection, as shown in Figure 3.By fluorescence probe solution the pre-heat treatment, 5-10 points is heated at 90-100 DEG C
Clock, is slowly cooled to room temperature.By the Hg of various concentrations2+Solution mixes with fluorescence probe solution, and half an hour is incubated at room temperature.Add
Graphene oxide dispersion and cushioning liquid, the final volume for making to treat test sample is 200 μ L, and graphene oxide concentration is 130 μ g
mL-1, fluorescence probe concentration is 0.2 μM, is reacted at room temperature half an hour, measures fluorescence emission spectrum.Investigating the sensor pair
Hg2+Selectivity experiment in, basic step with detection Hg2+The step of it is consistent, unlike, with other metals of same concentrations
Ion replaces Hg2+, by sample, the specificity of the sensor is judged in the detection of fluorescent emission intensity at 520 nm.
（4）The detection of Cys, as shown in Figure 4.To fluorescence probe solution the pre-heat treatment, 5-10 points is heated at 90-100 DEG C
Clock, is slowly cooled to room temperature.By Hg2+Solution mixes with fluorescence probe solution, and the Cys solution of various concentrations, room temperature are added afterwards
It is lower to be incubated 0.5 hour, add graphene oxide dispersion and cushioning liquid so that treat the final volume of test sample for 200 μ L, oxygen
Graphite alkene concentration is 130 μ g mL-1, fluorescence probe concentration is 0.2 μM, Hg2+It is 10 μM, reacts at room temperature half an hour, surveys
Amount fluorescence emission spectrum.In experiment of the sensor to Cys selectivity is investigated, basic step is consistent with the step of detecting Cys,
Unlike, replace Cys with other amino acid of same concentrations, by the detection to sample fluorescent emission intensity at 520 nm
Judge the specificity of the sensor.
The advantage of the invention is that：Sensitivity is high, and interference of other control samples to measured matter is small, is capable of achieving to tested
The selective enumeration method of material, complementary strand, fibrin ferment, Hg are realized so as to reach an oligonucleotide chain2+With Cys four
Plant the detection of different material.
Brief description of the drawings
Fig. 1 is concentration and fluorescence intensity graph of a relation and the interference experiment of complementary strand.Left figure：cTBA (a-i: 0.005,
0.01,0.04,0.07,0.1,0.3,0.5,0.7 with 1 μM) fluorescence emission spectrogram of compound；Right figure：Single base and double alkali
The check experiment of base mispairing.
Fig. 2 is concentration and fluorescence intensity graph of a relation and the interference experiment of fibrin ferment.Left figure：Fibrin ferment (a-p: 0.01,
0.05,0.1,0.5,1,2,3,4,5,6,7,8,9,10,15 with 20 μ g mL-1) fluorescence emission spectrum
Figure；Right figure：The check experiment of other oroteins.
Fig. 3 is Hg2+Concentration and fluorescence intensity graph of a relation and interference experiment.Left figure：Hg2+ (a-i: 0.5, 1, 3,
5,8,10,20,30 and 50 μM) fluorescence emission spectrogram of compound；Right figure：The check experiment of other metal ions.
Fig. 4 is concentration and fluorescence intensity graph of a relation and the interference experiment of Cys.Left figure：Cys (a-k: 10, 9, 8,
7,6,5,4,3,2,1 and 0.5 μM) fluorescence emission spectrogram of compound；Right figure：The check experiment of other amino acid.
Specific embodiment
The present invention is described further by example in detail below：
A kind of multi-functional oligonucleotide chain is used for the fluorescence detection method of many kinds of substance, specifically includes following steps：
1) solution is prepared：a、 Tris-HNO3Cushioning liquid；B, graphene oxide dispersion；
2）100 μM of fluorescence probe solution is heat-treated, is heated 5-10 minutes at 90-100 DEG C, progressively cool to room
Temperature；
3) by the complementary strand of various concentrations, fibrin ferment, Hg2+With Cys standard liquid respectively with fluorescence probe solution
Mixing, is incubated half an hour at room temperature；
4) graphene oxide dispersion and cushioning liquid are added in above-mentioned solution, the final volume for making to treat test sample is 200
μ L, graphene oxide concentration is 130 μ g mL-1, fluorescence probe solution is 0.2 μM, and half an hour is reacted at room temperature；
5) fluorescence emission spectrum of solution is measured, fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution is set up2+With
Standard curve between Cys；
6) four kinds of mark-on solution of unknown concentration are replaced into standard liquid, carries out above-mentioned reaction, the solution that will be detected
Fluorescence signal intensity brings standard curve into, obtains complementary strand cTBA, fibrin ferment, Hg in mark-on solution2+With Cys Cys's
Content is respectively 0.083 μM, 3.12 μ g mL-1, 5.27 μM and 4.12 μM.
Claims (4)
1. a kind of multi-functional oligonucleotide chain is used for the fluorescence detection method of many kinds of substance, it is characterised in that：Be to complementary strand,
Fibrin ferment, Hg2+With four kinds of fluoroscopic examinations of material of Cys, following steps are specifically included：
1）Prepare solution：a、 Tris-HNO3Cushioning liquid；B, graphene oxide dispersion；
2）100 μM of fluorescence probe solution is heat-treated, is heated 5-10 minutes at 90-100 DEG C, be slowly cooled to room temperature；
3）By the complementary strand of various concentrations, fibrin ferment, Hg2+It is mixed with fluorescence probe solution respectively with Cys standard liquid
Close, half an hour is incubated at room temperature, described fluorescence probe sequence is 5'FAM-GG TTG GTG TGG TTG G-3'；
4）Graphene oxide dispersion and cushioning liquid are added in above-mentioned solution, the final volume for making to treat test sample is 200 μ L,
Graphene oxide concentration is 130 μ g mL-1, fluorescence probe concentration is 0.2 μM, and half an hour is reacted at room temperature；Measure the glimmering of solution
Optical emission spectroscopy, sets up fluorescence signal intensity and complementary strand, fibrin ferment, the Hg of solution2+Standard and Cys between is bent
Line；
5）Sample solution is replaced into standard liquid, above-mentioned reaction is carried out, the fluorescence signal intensity of the solution that will be detected brings mark into
Directrix curve, obtains complementary strand, fibrin ferment, Hg in sample solution2+With the content of Cys.
2. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, its feature
It is：Described complementary strand, fibrin ferment, Hg2+Concentration with Cys standard liquid is respectively：0.005, 0.01,
0.04,0.07,0.1,0.3,0.5,0.7 the complementary strand with 1 μM；0.01, 0.05, 0.1, 0.5, 1, 2, 3,
4,5,6,7,8,9,10,15 and 20 μ g mL-1Fibrin ferment；0.5,1,3,5,8,10,20,30 and 50 μ
The Hg of M2+；10,9,8,7,6,5,4,3,2,1 and 0.5 μM of Cys.
3. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, its feature
It is：Described Tris-HNO3Cushioning liquid composition is：20 mM Tris, 100 mM NaNO3, pH=8.0。
4. multi-functional oligonucleotide chain according to claim 1 is used for the fluorescence detection method of many kinds of substance, its feature
It is：Instrument for fluorescence measurement is Hitachi F-4600 sepectrophotofluorometers, and fluorescence spectral measuring condition is：Excite
5.0 nm and 10.0 nm are respectively with transmite slit width, voltage is 700 V, and excitation wavelength is 480 nm, launch wavelength scanning
Scope 500-580 nm, sample cell is the quartz colorimetric utensil of 0.60 mL.
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