CN102998292B - Fluorescence detection method for detecting cocaine by use of oligonucleotide and graphene oxide - Google Patents
Fluorescence detection method for detecting cocaine by use of oligonucleotide and graphene oxide Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 17
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- 239000003550 marker Substances 0.000 claims description 5
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- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 238000001228 spectrum Methods 0.000 claims description 3
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- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
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- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 240000006890 Erythroxylum coca Species 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a fluorescence detection method for detecting cocaine by use of oligonucleotide and graphene oxide. The method comprises the following steps of: adding a dye-marked oligonucleotide probe into a buffer solution; adding graphene oxide as a solid-phase carrier so as to adsorb the oligonucleotide probe onto the surface of the graphene oxide and form an oligonucleotide/graphene oxide compound, and quenching a fluorescence signal of the dye; adding a complementary probe for reaction, wherein a double-strand secondary structure is formed through the base combination effect between the complementary probe and the oligonucleotide probe so as to recover fluorescence; and adding cocaine for reaction, wherein three stem-like secondary structures are formed through the specific effect between the cocaine and the complementary probe so that the oligonucleotide probe is re-combined to the surface of the graphene oxide and the fluorescence is quenched. According to the invention, the aim of cocaine detection is achieved by detecting the change of fluorescence intensity in the solution; and the detection method is simple and quick, and has relatively high specificity and sensitivity.
Description
Technical field
The present invention relates to the detection method of cocaine, especially relate to a kind of fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine.
Technical background
Cocaine is the topmost alkaloid of one of separating from coca leaf, belongs to central nervous excitation agent, is also a kind of potent local anesthetic.Cocaine belongs to one of common drugs, sucks cocaine and can produce strong psychologic dependence and health is caused to very big injury, causes social safety problem.Therefore find the heat subject that quick, easy, highly sensitive method detects cocaine current research, significant.
If publication number is CN 101948907 A " a kind of method that improves detection sensitivity of cocaine ", use be nanogold particle, can observe change color, and can be with the existence of measuring cocaine by optics and electrochemical signal.The advantage of this patent is directly to detect by change color, and galvanochemistry photochemistry all can, but detectability is not high, is 0.5 μ mol/L.
Along with scientists receives increasing concern to the research of nucleic acid, particularly about the research of the aspect such as identification, order-checking of biological heredity molecule mechanism and oligonucleotide molecules.The novel analytical approach that is directed at present nucleic acid is developed rapidly in fields such as life science, environment measuring, drug toxicity screening and disease molecules diagnosis.Scientist finds that the adaptive physical efficiency of cocaine in nucleic acid is specific and forms the secondary structure compound containing 3 stem shape structures with cocaine, and the detection that this feature is cocaine provides new approach.Therefore, some oligonucleotide probes with function of specific connecting are developed in succession, and have obtained good effect.
In recent years, graphene oxide is learned and electrical properties with its special mechanics, quantum, paid attention to by physics and material educational circles.Because it has the advantages such as excellent electric property, thermal conductivity is good, specific surface area is large, also for the detection of biomolecule provides new thinking.At present, along with the successful Application of Single Walled Carbon Nanotube in bio-sensing, graphene oxide also becomes the object of sensory field favor gradually.Due to the special nature of graphene oxide, make that testing process is simple to operate, reaction fast, greatly reduced like this step of experiment, can improve significantly sensitivity and the specificity of detection simultaneously.
Summary of the invention
The object of the invention is to have for existing cocaine detection method the problem that detecting instrument costliness, sensitivity are lower etc., a kind of fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine is provided, it has compared with high specific and sensitivity.
The present invention utilizes oligonucleotides and graphene oxide to detect the fluorescence detection method of cocaine, and step is in the following order carried out successively:
1) oligonucleotide probe of dye marker is joined in buffer solution, solution is carried out to fluoroscopic examination, record the fluorescent emission bands of a spectrum of oligonucleotide probe;
2) join in above-mentioned solution graphene oxide as solid phase carrier, make oligonucleotide probe be adsorbed onto the surface of graphene oxide, form oligonucleotides/graphene oxide compound, by the fluorescence signal quencher of dyestuff;
3) in oligonucleotides/graphene oxide compound, add complementary probe to react, by the base combination of complementary probe and oligonucleotide probe, form Double-chain two stage structure, thereby making oligonucleotide probe depart from and be free in solution from the surface of graphene oxide is recovered fluorescence, at excitation wavelength 480～490nm, under emission wavelength 490～650nm, carry out fluoroscopic examination, described complementary probe comprises one section of single-chain nucleic acid, with oligonucleotide probe base complementrity, without fluorescence labeling;
4) in double-chain probe/graphene oxide compound, add cocaine to react, by the specific effect of cocaine and complementary probe, form the secondary structure containing 3 stem shape structures, make oligonucleotide probe be attached to again the surface of graphene oxide, thereby make fluorescence signal cancellation, at excitation wavelength 480～490nm, under emission wavelength 490～650nm, carry out fluoroscopic examination.
In step 1) of the present invention, be fluorescein FAM for the dyestuff of mark, dye marker is at the 5' of oligonucleotide probe end, and the base composition of described oligonucleotide probe is 5'-CTGTTCCTTTTAGGAAGTTACTTCACCCAG-3 '; Buffer solution used is 0.02mol/L Tris-HCl, 0.1mol/L NaCl, 0.005mol/L KCl, 0.005mol/L MgCl
2, pH=7.4.Carrying out fluorescence detection method is that emission wavelength sweep limit is 490～650 nm taking 480～490nm as excitation wavelength.
Step 2 of the present invention) in, the temperature that described oligonucleotide probe is adsorbed onto graphene oxide surface is room temperature, the time of fluorescent quenching is 1～3 minute.
In step 3) of the present invention, the base composition of described complementary probe is 5 '-GACAAGGAAAATCCTTCAATGAAGTGGGTC-3 ', in oligonucleotides/graphene oxide compound, adding the temperature that complementary probe is reacted is room temperature, and the time that oligonucleotide probe departs from from graphene oxide surface is 10～20 minutes.
In step 4) of the present invention, in double-chain probe/graphene oxide compound, adding the temperature that cocaine reacts is room temperature, cocaine and complementary probe specific effect form the secondary structure containing 3 stem shape structures, and the time that makes oligonucleotide probe again be attached to graphene oxide surface is 10～20 minutes.
The present invention is first used for adsorbing fluorescein-labeled oligonucleotide probe taking graphene oxide as solid phase carrier, because graphene oxide surface has the chemical group that can carry out with oligonucleotides non-covalent bond combination, can make fluorescein-labeled oligonucleotide probe be adsorbed onto the surface of graphene oxide, thereby form oligonucleotides/graphene oxide compound, now, between fluorescein and graphene oxide, there is efficient energy and shift, the effective quencher of the oxidized Graphene of fluorescence of fluorescein.In the time adding complementary probe, complementary probe can be combined with oligonucleotide probe base and be formed duplex structure, make oligonucleotide probe depart from the surface of graphene oxide, now distant between fluorescein base group and graphene oxide, effective energy can not occur to be shifted, the fluorescence signal that is marked at the fluorescein of oligonucleotide probe end is restored, and can detect that stronger fluorescence signal recovers.In the time there is cocaine in solution, cocaine can form the secondary structure containing 3 stem shape structures with complementary probe specific binding, makes oligonucleotide probe again be attached to graphene oxide surface, makes fluorescence generation cancellation.
Beneficial effect of the present invention: the present invention reaches by detecting the variation of fluorescence intensity in solution the object that detects cocaine, and detection method is easy, quick, has compared with high specific and sensitivity, has great importance in cocaine context of detection.According to fluorescence detection method of the present invention, the concentration of minimum detectable cocaine is 6nmol/L.Divide the period of the day from 11 p.m. to 1 a.m when containing other non-target alkaloid such as theophylline, caffeine in system, still can realize the highly sensitive detection to cocaine.Therefore, the present invention is expected to should be in actual sample the detection of cocaine provides a kind of easy, method fast.
Brief description of the drawings
Fig. 1 is that the present invention utilizes oligonucleotides and graphene oxide to detect the fundamental diagram of the fluorescence detection method of cocaine.
Fig. 2 is differential responses system fluorescence intensity result comparison diagram in testing process.Wherein, a is dye marker oligonucleotide probe; B is oligonucleotides+graphene oxide compound; C is double-chain probe+graphene oxide compound; D is double-chain probe+graphene oxide compound+cocaine; E is that double-chain probe+graphene oxide compound+water is made blank.
Embodiment
Further set forth the present invention below in conjunction with the drawings and specific embodiments.Following examples are only for to further specific descriptions of the present invention, instead of for the restriction to the claimed scope of the present invention.
In following embodiment, fluoroscopic examination instrument is fluorescent sub-photometer (RF-5301, Japan).Fluorescence spectral measuring condition: xenon lamp excites, exciting and launching slit width is 5.0 nm and 10.0 nm, voltage is 950V, response times 2 S, excitation wavelength is 480～490nm, emission wavelength sweep limit 490～650 nm; Measure sample volume 1.60 mL with 3 mL quartz colorimetric utensils; Room temperature.
Graphene oxide used is synthetic according to the method for Hummers in document (W. S. Hummers, R. E. Offeman, J. Am. Chem. Soc. 1958,80,1339-1339).
Tris-HCl buffer solution used consist of 0.02mol/L Tris-HCl, 0.1mol/L NaCl, 0.005mol/L KCl, 0.005mol/L MgCl
2, pH=7.4.
Oligonucleotide probe used and complementary probe be purchased from Shanghai biotechnology company limited,
| Nucleic acid probe title | Base composition |
| Oligonucleotide probe | 5′-CTGTTCCTTTTAGGAAGTTACTTCACCCAG-3′ |
| Complementary probe | 5′-GACAAGGAAAATCCTTCAATGAAGTGGGTC-3′ |
Dyestuff for mark is fluorescein FAM, is marked at the 5' end of oligonucleotide probe.
Embodiment
1) be that the oligonucleotide probe of the fluorescein FAM mark of 1.8 μ mol/L joins in the Tris-HCl buffer solution of 1 mL by 1 μ L concentration, add again the water of 0.6 mL to carry out fluoroscopic examination, the fluorescent emission bands of a spectrum that record oligonucleotide probe, fluorescence intensity result is as shown in a in Fig. 2;
2) and then add 3 μ L graphene oxide solution, at room temperature oligonucleotide probe is adsorbed onto rapidly graphene oxide surface, form oligonucleotides/graphene oxide compound, and by the fluorescence signal quencher of fluorescein FAM, the time of fluorescent quenching is 2 minutes, and under same fluorescence spectral measuring condition, fluorescence intensity result is as shown in the b in Fig. 2;
3) complementary probe that is 1.8 μ mol/L by 1 μ L concentration joins in oligonucleotides/graphene oxide compound, complementary probe and oligonucleotide probe at room temperature carry out base combination, form Double-chain two stage structure, make oligonucleotide probe depart from and be free in solution from graphene oxide surface, the time departing from is 20 minutes, carry out fluoroscopic examination, can observe the recovery phenomenon of fluorescence, under same fluorescence spectral measuring condition, fluorescence intensity result is as shown in the c in Fig. 2;
4) cocaine that is 1 mol/L by 10 μ L concentration joins in nucleic acid probe/graphene oxide compound, cocaine and complementary probe are at room temperature carried out specific combination, form the secondary structure containing 3 stem shape structures, make oligonucleotide probe again be attached to graphene oxide surface, thereby make fluorescent quenching, the time of reaction is 20 minutes, at excitation wavelength 480nm, under emission wavelength 490～650nm, carry out fluoroscopic examination, can observe fluorescent quenching phenomenon, under same fluorescence spectral measuring condition, fluorescence intensity result is as shown in the d in Fig. 2.
Adopt fluorescence detection method of the present invention, the concentration of minimum detectable cocaine is 6nmol/L.
Contrast test
Meanwhile, do following test as a comparison.
Detect 2: test with isopyknic water replacement graphene oxide, as blank, other condition is identical with detection 1.
Detect 3: test with the other biological base molecule of isopyknic same concentrations, as negative control, other conditions are identical with detection 1.
Test findings: blank (detecting 2) fluorescence intensity result under same fluorescence spectral measuring condition, as shown in the e in Fig. 2, is more or less the same compared with the fluorescence signal intensity of double-chain probe/graphene oxide compound.Visible, cocaine itself is little on the impact of experiment.
The fluorescence intensity when fluorescence signal intensity of other biological base molecule (detecting 3) exists much larger than cocaine.Visible, the present invention has the specificity of height to the detection of cocaine.
Claims (6)
1. utilize oligonucleotides and graphene oxide to detect a fluorescence detection method for cocaine, step is in the following order carried out successively:
1) oligonucleotide probe of dye marker is joined in buffer solution, solution is carried out to fluoroscopic examination, record the fluorescent emission bands of a spectrum of oligonucleotide probe;
2) join in above-mentioned solution graphene oxide as solid phase carrier, make oligonucleotide probe be adsorbed onto the surface of graphene oxide, form oligonucleotides/graphene oxide compound, by the fluorescence signal cancellation of dyestuff;
3) in oligonucleotides/graphene oxide compound, add complementary probe to react, by the base combination of complementary probe and oligonucleotide probe, form Double-chain two stage structure, thereby making oligonucleotide probe depart from and be free in solution from the surface of graphene oxide is recovered fluorescence, at excitation wavelength 480～490nm, under emission wavelength 490～650nm, carry out fluoroscopic examination, described complementary probe comprises one section of single-chain nucleic acid, with oligonucleotide probe base complementrity, without fluorescence labeling;
4) in double-chain probe/graphene oxide compound, add cocaine to react, by the specific effect of cocaine and complementary probe, form the secondary structure containing 3 stem shape structures, make oligonucleotide probe be attached to again the surface of graphene oxide, thereby make fluorescence signal cancellation, at excitation wavelength 480～490nm, under emission wavelength 490～650nm, carry out fluoroscopic examination.
2. the fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine according to claim 1, it is characterized in that: step 1) in, dyestuff for mark is fluorescein FAM, dye marker is at the 5' of oligonucleotide probe end, and the base composition of described oligonucleotide probe is 5'-CTGTTCCTTTTAGGAAGTTACTTCACCCAG-3 '; Buffer solution used is Tris-HCl buffer solution, and it consists of 0.02mol/LTris-HCl, 0.1mol/LNaCl, 0.005mol/LKCl, 0.005mol/LMgCl2, pH=7.4.
3. the fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine according to claim 1, it is characterized in that: step 1) in carry out fluorescence detection method for taking 480～490nm as excitation wavelength, emission wavelength sweep limit is 490～650nm.
4. the fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine according to claim 1, it is characterized in that: step 2) in, the temperature that described oligonucleotide probe is adsorbed onto graphene oxide surface is room temperature, and the time of fluorescent quenching is 1～3 minute.
5. the fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine according to claim 1, it is characterized in that: step 3) in, the base composition of described complementary probe is 5 '-GACAAGGAAAATCCTTCAATGAAGTGGGTC-3 ', in oligonucleotides/graphene oxide compound, adding the temperature that complementary probe is reacted is room temperature, and the time that oligonucleotide probe departs from from graphene oxide surface is 10～20 minutes.
6. the fluorescence detection method that utilizes oligonucleotides and graphene oxide to detect cocaine according to claim 1, it is characterized in that: step 4) in, in double-chain probe/graphene oxide compound, adding the temperature that cocaine reacts is room temperature, cocaine and complementary probe specific effect form the secondary structure containing 3 stem shape structures, and the time that makes oligonucleotide probe again be attached to graphene oxide surface is 10～20 minutes.
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| CN102998292A (en) | 2013-03-27 |
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