CN101948907B - Method for improving detection sensitivity of cocaine - Google Patents
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Abstract
The invention relates to a method for improving the detection sensitivity of cocaine. The method is characterized in that a nucleic acid aptamer for cocaine detection is broken into three sections. The method comprises the following steps of: (1) mixing the cocaine and three sections of nucleic acid aptamer probes to ensure that the detected cocaine and the aptamer probes form a compound; and (2) performing fluorescence detection, electrochemical detection or color detection. As the length of the three sections of nucleic acid probes is obviously shorter than that of one or two sections of nucleic acid probes, the interaction among probes and between the probes and other components in a detection system capable of causing background noise is greatly reduced. Thus the detection sensitivity of the cocaine is greatly improved. Taking the nanogold color detection of the cocaine for example, the sensitivity of the color detection of the cocaine is improved by one magnitude order by the three sections of nucleic acid probes. The method can be popularized to the detection of various materials comprising small molecules and heavy metal ions.
Description
Technical field
The present invention relates to a kind of method of utilizing three sections nucleic acid probes to improve detection sensitivity of cocaine, belong to the nanometer biotechnology field.
Background technology
Illicit drugs inspection recent years take Cocaine as representative makes great progress, and the technology of especially utilizing aptamer to detect has caused great concern.Aptamer is from～10
14The DNA of stochastic sequence or the RNA storehouse in system's molecular evolution method (Systematic Evolution of Ligands by EXponential enrichment by index concentration, be called for short SELEX) screen, can be combined with target molecules specifically, thereby identify specifically target molecules (Ellington AD, Szostak JW (1990) Nature 346:818-822.Tuerk C, Gold L (1990) Science 249:505-510.).Aptamer can for the detection of the various biomolecules that comprise protein, small molecules, heavy metal ion, cell and virus etc., have great application prospect.
Utilizing at present aptamer to carry out the method that Cocaine detects, mainly is to use one section aptamer (M.N.Stojanovic, et.al, J.Am.Chem.Soc.2001,123,4928-4931.; B.R.Baker, et.al, J.Am.Chem.Soc.2006,128,3138-3139), or use and to be separated into detection (Stojanovic, the M.N. that two sections aptamer carries out Cocaine with one section; De Prada, P.; Landry, D.W.J.Am.Chem.Soc.2000,122,11547-11548.; J.Zhang, et.al, small 2008,4, No.8,1196-1200; R.Freeman, et.al, J.Am.Chem.Soc.2009,131,5028-5029; Analyst, 2009,134,653-656; X.Zhou, et.al., J.Am.Chem.Soc.2009,131,6944-6945).Mainly be to utilize fluorescent quenching, enzymatic color reaction, electrochemistry or nanometer gold color reaction to carry out the detection of Cocaine on concrete detection method.The detection sensitivity of these methods is in the scope of 0.1 μ M-500 μ M.
Take the nanometer gold color detection as example, utilize in the detection technique of nanometer gold existing, using maximum is the optical property of utilizing the nanometer gold surface plasma resonance, and self-existent nanometer gold is red, and the nanometer gold that flocks together is pewter or purple.That is to say, prior art must cause the nanometer gold gathering directly or indirectly when carrying out biological detection and environment measuring, perhaps the nanometer gold of assembling is disperseed again, thereby cause the variation of nanometer gold color, the absorbing wavelength of visible light produces the movement up to 300nm.This colour-change can directly detect by an unaided eye, and need not any complex apparatus, also can use spectrograph to carry out detection by quantitative.
The existing method of the gathering of nanometer gold that causes can be divided into two large classes.First kind method is to detect thing as linking agent nano particle to be bound up, thereby causes gathering.Pass through the crosslinked method for congregating that causes between particle such as what DNA induced.These class methods need to be with single stranded DNA, the DNA enzyme, and RNA enzyme or aptamer are connected to the surface of nano material.Experimental procedure is relatively loaded down with trivial details, DNA, and the DNA enzyme, the consumption of RNA enzyme or aptamer is large.The Equations of The Second Kind method is noncrosslinking nanoparticle aggregate method, and this method utilizes nano particle adding the reduction that detects the thing rear stability.The reduction of this stability is because nano grain surface is used for improving the dna molecular of stability of nano gold or the destruction of ATP, replaces or consumption; Perhaps since with the interactional solution of unmodified nano particle in can stable nanoparticles ATP or the minimizing of single stranded DNA.These class methods are used the nm gold particles of unmodified mostly.
The method of using the unmodified nanometer gold to detect is present detection technique the most simply, cheaply and fast in all detection techniques, and the method has been used for comprising the detection to the multiclass biomolecules of DNA, small molecules and heavy metal ion at present.These class methods are based on the double-stranded DNA structure than golden poor this phenomenon of single stranded DNA stabilized nanoscale more.The method that the use unmodified nanometer gold of reporting detects Cocaine is utilized two sections aptamer probes exactly, when existing, Cocaine forms the supramolecular structure with double-stranded DNA structure with Cocaine, therefore this structure can be used for the detection of Cocaine than the ability of the nucleic acid probe stabilized nanoscale gold of strand.
The method that one section or two sections aptamers of the above various employing carry out the Cocaine detection exists following deficiency:
1) detection method of using one section aptamer to detect Cocaine is to utilize aptamers to be combined the structure phase change that causes aptamers with Cocaine, the aptamers that at first screens by the SELEX technology approximately only has 1% can the structure phase change arranged when target is combined, and this has just greatly limited the versatility of aforesaid method to other target detection; About common 30 bases of the length of aptamers (such as the aptamers of Cocaine and the aptamers of Adenosine), even longer, the chance that self forms like this secondary structure formation diploid, polyploid will significantly increase, thereby attenuating detection sensitivity, especially the Cocaine that carries out in the desmoenzyme reaction detects, and can cause very high background signal.
2) aptamer that the detection method of using two sections aptamers to detect Cocaine will be grown has been divided into two sections, detecting no longer needs aptamers generation structure phase change, this has just enlarged the general applicability of method, the chance that also self is formed secondary structure formation diploid, polyploid has reduced some, thereby has improved detection sensitivity.Yet in two sections nucleic acid probes long one section length still have about 25 bases, and this section nucleic acid probe also has alone the ability (seeing embodiment) in conjunction with Cocaine, so also can cause background signal, makes the sensitivity of detection.
(small 2008 for J.Zhang, et.al when 3) using the unmodified nanometer gold to carry out the detection of Cocaine, 4, No.8,1196-1200), when using two sections aptamer probe in detecting, the method is in detection sensitivity, according to one times of calculating of blank sample standard deviation, 7 μ M (J.Zhang, et.al, small 2008,4, No.8,1196-1200).The sensitivity that detects is high not enough, and the color of nanometer gold is large not enough with the gradient of Cocaine change in concentration, and the standard deviation that detects is larger.These deficiencies mainly are because the single-chain nucleic acid aptamers of relatively growing is stablized the indifferent of unmodified nanometer gold, and the ability difference that forms double-stranded DNA Stability Analysis of Structures nanometer gold with aptamer and Cocaine is not very large.It is larger to produce the degree of assembling under specific salts concentration, thereby causes the large shortcoming of experimental error, and this just directly has influence on the sensitivity of detection.
Summary of the invention
The present invention is directed to the problems of the prior art, its purpose is to provide a kind of method of utilizing three sections nucleic acid probes to improve detection sensitivity of cocaine.
A kind of method that improves detection sensitivity of cocaine of the present invention is characterized in that: the aptamer probe that is used for the Cocaine detection is fragmented into three sections.
The present invention also namely utilizes three sections nucleic acid probes to improve the method for detection sensitivity of cocaine, it is characterized in that: utilize the aptamer probe with three sections to detect Cocaine.
Cocaine detection method of the present invention is characterized in that:
The method comprises the steps: that (1) mix Cocaine and three sections aptamer probes (unmarked, fluorescent mark, electrochemical reaction oxidizing reducing group mark or other mark), and determined Cocaine and aptamers probe form mixture; Wherein three sections aptamers mix according to equimolar ratio, with Cocaine solution and the mixed concentration of buffered soln in the scope of 0.5 μ M-50 μ M; (2) carry out fluoroscopic examination, Electrochemical Detection or color detection.Specifically use the Cocaine method for detecting color of unmodified nanometer gold, it is characterized in that: the method comprises the steps: that (1) mix Cocaine with three sections aptamer probes, and Cocaine and aptamers probe form mixture; (2) reaction soln is mixed with nano-Au solution; The mol ratio of reaction soln amplifying nucleic acid probe and nanometer gold generally should be greater than 100: 1; (3) add an amount of salts solution (such as the phosphate solution of sodium chloride-containing), change the existence that comes sxemiquantitative to judge Cocaine by solution colour.The add-on of salts solution comes titration to determine can distinguish the absorbancy (at the ratio of 520nm with 650nm place absorbancy) that contains blank sample and contain the sample nanometer gold, increases gradually the consumption of salts solution, determines optimum value.Being present in of Cocaine noly can absorb quantitative analysis by UV, visible light.
The present invention utilizes three sections nucleic acid probes to detect Cocaine, has following technique effect:
1, three sections nucleic acid probes are compared length with single hop or two sections nucleic acid probes and are significantly reduced, and are 13 bases such as the length the longest of in an embodiment three sections nucleic acid probes; And the longest of two sections nucleic acid probes is 24 bases; The length of single hop nucleic acid probe is 30 bases.The remarkable minimizing of chain length can significantly reduce in the system to form the chance of the molecular structure of not expected, such as dimer or polymer.Especially in the detection system of amplifying based on enzyme, reduce the chance (B.Shlyahovsky of probe and the non-specific hybridization of signal amplifying probe, et al.J.AM.CHEM.SOC.2007,129,3814-3815), reduce the interaction of the background noise that other component causes in probe and the detection system, thereby can improve significantly the sensitivity of detection.
2, three sections nucleic acid probes can be stablized the unmodified nanometer gold better than single hop or two sections nucleic acid probes of same concentrations, thereby can improve the color gradient of Cocaine color detection, and the error that can reduce to test, and then improve the sensitivity that detects.
3, one section that grows in two sections nucleic acid probes used in the prior art has the ability of being combined with Cocaine alone, can cause like this reduction of system detection sensitivity.And in three sections nucleic acid probes any one section or two sections all can not be combined with Cocaine, and this has just eliminated above-mentioned shortcoming.
The present invention relates to a kind of the utilization in the method that three sections nucleic acid probes improve detection sensitivity of cocaines, when Cocaine exists, the mixture that three sections nucleic acid probes and Cocaine self-assembly formation have the double-stranded DNA structure.This mixture is stablized the ability of the nanometer gold of unmodified than the probe of single stranded DNA, thereby realizes Cocaine be need not the color detection of any equipment.If adopt fluorescence molecule, oxidizing reducing group or other signal output group to carry out the nucleic acid probe of mark, the method can also be carried out the detection of fluoroscopic examination, Electrochemical Detection or other signal output form of Cocaine.The inventive method can be used for detecting protein, enzyme, DNA, small molecules, metal ion and bacterium etc.Characteristics of the present invention and advantage can embody by the following examples.It is pointed out that following examples only are used for illustrating, can carry out various variations and modification within the scope of the invention.
Description of drawings
Fig. 1 is transmission electron microscope (TEM) image (Fig. 1 a and b) and the UV, visible light absorption spectrum (Fig. 1 c) of unmodified 13nm gold grain used in the present invention.Sample dilutes 10 times before measuring the UV, visible light absorption, charateristic avsorption band is at 519nm.
Fig. 2. be to use one-part form nucleic acid probe (probe 1 or probe 2), two-part nucleic acid probe (probe 2 and probe 3) and syllogic nucleic acid probe of the present invention (probe 3, probe 4 and probe 5) to carry out the schematic diagram of Cocaine nanometer gold color detection.
Fig. 3. be nano-Au solution with etc. volumetric molar concentration be used for ultraviolet-visible absorption spectroscopy after one-part form nucleic acid probe (probe 1), two-part nucleic acid probe (probe 2 and probe 3) and syllogic nucleic acid probe of the present invention (probe 3, probe 4 and probe 5) that Cocaine detects mix and add salts solution.Illustration is the color of viewed different mixtures.
Fig. 4 (A)-Fig. 4 (C) uses syllogic nucleic acid probe (probe 3,4 and 5) to carry out the photo of UV, visible light absorption spectrogram, typical curve and the nanometer gold color reaction of Cocaine nanometer gold color detection in the one embodiment of the invention.
Fig. 5 (A)-Fig. 5 (C) uses one-part form nucleic acid probe (probe 1) to carry out the photo of UV, visible light absorption spectrogram, typical curve and the nanometer gold color reaction of Cocaine nanometer gold color detection in the one embodiment of the invention.
Fig. 6 (A)-Fig. 6 (C) uses two-part nucleic acid probe (probe 2 and 3) to carry out the photo of UV, visible light absorption spectrogram, typical curve and the nanometer gold color reaction of Cocaine nanometer gold color detection in the one embodiment of the invention.
Fig. 7 (A)-Fig. 7 (C) is respectively the photo that the one section nucleic acid probe (probe 2) in the use two-part nucleic acid probe carries out UV, visible light absorption spectrogram, typical curve and the nanometer gold color reaction of Cocaine nanometer gold color detection in the one embodiment of the invention.
Fig. 8 (A)-Fig. 8 (B) is the ability in conjunction with Cocaine that confirms the one section nucleic acid probe (probe 2) in the two-part nucleic acid probe in the one embodiment of the invention, can reduce photo (A) and the UV, visible light absorption spectrogram (B) of nanometer gold color reaction that uses two sections nucleic acid probes (probe 2 and 3) to carry out the sensitivity of Cocaine nanometer gold color detection.
Describe in detail: " AuNP " or " AuNPs " refers to gold nano grain, normally spherical, typically be of a size of diameter 1-100nm.
Aptamers refers to DNA (perhaps RNA) molecule that can be combined with target molecules in specific manner, and target can be protein, DNA, small molecules or metal ion etc.
Embodiment
The materials and methods that described embodiment uses
HAuCl
4Buy from ACROS ORGANIC.Trisodium citrate is bought from chemical reagents corporation of traditional Chinese medicines group.All nucleic acid probes are bought (nucleotide sequence sees Table one) from TaKaRa (Dalian).The gold grain of 13 nanometers (AuNP) is according to the Trisodium Citrate reduction legal system of classics, and final concentration approximately is 12 ± 1nM.Sample of cocaine is given by Shanghai Applied Physics professor Fan Chunhai of institute of the Chinese Academy of Sciences.
Table one: the nucleic acid probe sequence that uses among the present invention.
| The nucleic acid probe | Sequence |
| Probe | |
| 1 | ?5’-GAC?AAG?GAA?AAT?CCT?TCA?ATG?AAG?TGG?GTC |
| Probe 2 | ?5’-GTT?CTT?CAA?TGA?AGT?GGG?ACG?ACA |
| Probe 3 | ?5’-GGG?AGT?CAA?GAA?C |
| Probe 4 | ?5’-GTT?CTT?CAA?T |
| Probe 5 | ?5’-AGT?GGG?ACG?ACA |
Embodiment 1: the preparation of the nanometer gold of unmodified.
The aqueous solution (25ml, 38.8mM) of trisodium citrate adds in the golden chloric acid HAuCI4 solution that seethes with excitement (250ml, 1mM) fast.Behind the several minutes, the color of the solution is from the light yellow scarlet that becomes.Solution continuation return stirring made and reacts completely in 15 minutes.Then cool to room temperature at leisure.4 degree are preserved.According to the uv-absorbing intensity (Fig. 1 (C)) of nanometer gold at 520nm, prepared nanometer gold concentration be 12 ± 1nM, size is 13nm (sees Fig. 1 (A) and (B)).
Embodiment 2: be used for one-part form nucleic acid probe (probe 1), two-part nucleic acid probe (probe 2 and probe 3) and the syllogic nucleic acid probe of the present invention (probe 3, probe 4 and probe 5) of Cocaine detection to the difference of the stability influence of unmodified nanometer gold.
The nucleic acid probe sample that has same molar ratio below the preparation at first respectively:
One-part form nucleic acid probe (probe 1) sample: the Cocaine of probe 1,3 microlitre of 1 microlitre, 100 μ M detects damping fluid (pH 8.0 for 0.3M NaCl, 50mM Tris-HCl), and the water of 2 microlitres at room temperature mixes.
Two-part nucleic acid probe (probe 2 and probe 3) sample: the probe 2 of 1 microlitre, 100 μ M, the Cocaine of probe 3,3 microlitres of 1 microlitre, 100 μ M detects damping fluid (0.3M NaCl, 50mM Tris-HCl, pH 8.0), and the water of 1 microlitre at room temperature mixes.
Syllogic nucleic acid probe (probe 3,4 and 5) sample: the probe 4 of probe 3, the 1 microlitres 100 μ M of 1 microlitre, 100 μ M, the probe 5 of 1 microlitre, 100 μ M, at room temperature mix with the Cocaine detection damping fluid (pH 8.0 for 0.3MNaCl, 50mM Tris-HCl) of 3 microlitres.
Get respectively above sample 2 microlitres, add respectively the nanometer gold of 12 ± 1nM of 100 microlitres, then add immediately the 0.6M NaCl phosphate solution of 20 microlitres.One-part form nucleic acid probe (probe 1) sample produces gathering immediately, and two-part nucleic acid probe (probe 2 and probe 3) sample is partly assembled, and syllogic nucleic acid probe (probe 3,4 and 5) sample keeps stable redness (Fig. 3).Then carried out UV-vis absorptiometry (Fig. 3).
Above-mentioned experimental result shows under same molar ratio, and the shortest syllogic nucleic acid probe of nucleic acid probe length can be than the long one-part form of probe length and two-part nucleic acid probe stabilized nanoscale gold better.This just provides a prerequisite for improve Cocaine nanometer gold color detection with the syllogic nucleic acid probe, color gradient that can Effective Raise nanometer gold color detection and reduce experimental error, thus improve the sensitivity that detects.
Embodiment 3: use the syllogic nucleic acid probe to detect Cocaine.
The preparation of Cocaine standard model: the Cocaine solution of (pH 8.0 for 0.15M NaCl, 25mM Tris-HCl) configuration 500 μ M, 200 μ M, 100 μ M, 50 μ M, 20 μ M, 0 μ M concentration in buffered soln.
200 μ M nucleic acid probes, 3,200 μ M probes, 4,200 μ M probes, 5 each 0.5 microlitre, 1.5 microlitres contain the 50mM Tris-HCl damping fluid of 0.3M NaCl, mixing.Get the above-mentioned mixing solutions of 0.5 microlitre, add Cocaine solution 4.5 microlitres of different concns, preserve at normal temperatures 30min.From above-mentioned sample, respectively get 2 microlitres, add respectively the nano-Au solution of 100 microlitres, 12 ± 1nM, preserve 5min under the room temperature, then add the 10mM phosphate buffer soln that 15 microlitres contain 0.2M NaCl.
Carry out UV-vis absorptiometry (Fig. 4 A), and take pictures (Fig. 4 C).Absorbancy according to 520nm and 650nm place is drawn the typical curve (Fig. 4 B) that Cocaine detects.In the application's specification sheets, if no special instructions, the UV-vis absorptiometry of all embodiment all is that experiment finishes to carry out immediately, and photo all is that experiment finishes to take after 2 hours.
The result shows that naked eyes can obviously distinguish the sample of cocaine of 5 μ M and above concentration and the colour-change of blank (Fig. 4 C).The same higher sample of concentration, blueness is darker, distinguishes more obvious.Ultraviolet-visible spectrometry (Fig. 4 A) also can obviously be distinguished 5 μ M sample of cocaine and blank samples.According to one times of calculating of blank sample standard deviation, the detection sensitivity of the method is 0.5 μ M.
Embodiment 4: use the one-part form nucleic acid probe to detect Cocaine.
The preparation of Cocaine standard model: the Cocaine solution of (pH 8.0 for 0.15M NaCl, 25mM Tris-HCl) configuration 500 μ M, 200 μ M, 100 μ M, 50 μ M, 20 μ M, 0 μ M concentration in buffered soln.
The 50mM Tris-HCl damping fluid mixing that the nucleic acid probe 1 of 2 microlitres, 200 μ M and 2 microlitres contain 0.3M NaCl.Get 0.5 microlitre mentioned solution, add Cocaine solution 4.5 microlitres of different concns, preserve at normal temperatures 30min.From above-mentioned sample, respectively get 2 microlitres, add respectively the nano-Au solution of 100 microlitres, 12 ± 1nM, preserve 5min under the room temperature, then add the 10mM phosphate buffer soln that 30 microlitres contain 0.2M NaCl.Carry out immediately UV-vis absorptiometry (Fig. 5 A), and take pictures (Fig. 5 C).Absorbancy according to 520nm and 650nm place is drawn the typical curve (Fig. 5 B) that Cocaine detects.
The result shows with the naked eye can obviously distinguish the sample of cocaine of 50 μ M and above concentration and the colour-change of blank (Fig. 4 C).The same higher sample of concentration, blueness is darker, distinguishes more obvious.And the sample of cocaine of 20 μ M and blank still keep stable redness.Ultraviolet-visible spectrometry (Fig. 5 A) can be distinguished 20 μ M sample of cocaine and blank samples.According to one times of calculating of blank sample standard deviation, the detection sensitivity of the method is 16 μ M.
Embodiment 5: use the two-part nucleic acid probe to detect Cocaine.
The preparation of Cocaine standard model: the Cocaine solution of (pH 8.0 for 0.15M NaCl, 25mM Tris-HCl) configuration 500 μ M, 200 μ M, 100 μ M, 50 μ M, 20 μ M, 0 μ M concentration in buffered soln.
The nucleic acid probe 3 of nucleic acid probe 2, the 1 microlitres 200 μ M of 1 microlitre, 200 μ M contains the 50mM Tris-HCl damping fluid mixing of 0.3M NaCl with 2 microlitres.Get 0.5 microlitre mentioned solution, add Cocaine solution 4.5 microlitres of different concns, preserve at normal temperatures 30min.From above-mentioned sample, respectively get 2 microlitres, add respectively the nano-Au solution of 100 microlitres, 12 ± 1nM, preserve 5min under the room temperature, then add the 10mM phosphate buffer soln that 20 microlitres contain 0.2M NaCl.Carry out immediately UV-vis absorptiometry (Fig. 6 A), and take pictures (Fig. 6 C).Absorbancy according to 520nm and 650nm place is drawn the typical curve (Fig. 6 B) that Cocaine detects.
The result shows with the naked eye can obviously distinguish the sample of cocaine of 20 μ M and above concentration and the colour-change of blank (Fig. 6 C).The same higher sample of concentration, blueness is darker, distinguishes more obvious.And the sample of cocaine of 5 μ M and blank still keep stable redness.Ultraviolet-visible spectrometry (Fig. 6 A) can be distinguished 20 μ M sample of cocaine and blank samples, but can not distinguish 5 μ M sample of cocaine.According to one times of calculating of blank sample standard deviation, the detection sensitivity of the method is 7 μ M.This result is consistent with the result of bibliographical information, is relatively laying a good foundation of experimental data of the present invention.
Embodiment 6: use the long one section nucleic acid probe 2 in the two-part nucleic acid probe to detect Cocaine.
The preparation of Cocaine standard model: the Cocaine solution of (pH 8.0 for 0.15M NaCl, 25mM Tris-HCl) configuration 500 μ M, 200 μ M, 100 μ M, 50 μ M, 20 μ M, 0 μ M concentration in buffered soln.
The 50mM Tris-HCl damping fluid mixing that the nucleic acid probe of 2 microlitres, 200 μ M and 2 microlitres contain 0.3M NaCl.Get 0.5 microlitre mentioned solution, add Cocaine solution 4.5 microlitres of different concns, preserve at normal temperatures 30min.From above-mentioned sample, respectively get 2 microlitres, add respectively the nano-Au solution of 100 microlitres, 12 ± 1nM, preserve 5min under the room temperature, then add the 10mM phosphate buffer soln that 30 microlitres contain 0.2M NaCl.Carry out immediately UV-vis absorptiometry (Fig. 7 A), and take pictures (Fig. 7 C).Absorbancy according to 520nm and 650nm place is drawn the typical curve (Fig. 7 B) that Cocaine detects.
The result shows with the naked eye can obviously distinguish the sample of cocaine of 100 μ M and above concentration and the colour-change of blank (Fig. 7 C).The same higher sample of concentration, blueness is darker, distinguishes more obvious.And the sample of cocaine of 20 μ M and blank still keep stable redness.Ultraviolet-visible spectrometry (Fig. 7 A) can be distinguished 50 μ M sample of cocaine and blank samples.According to one times of calculating of blank sample standard deviation, the detection sensitivity of the method is 27 μ M.One section ability that still has independent detection Cocaine growing in two sections nucleic acid probes of this description of test, its detectivity are 1/4th of two-section joint detection Cocaines.
Embodiment 7: checking than long-chain probe 2 in conjunction with the impact on two-part nucleic acid probe detection sensitivity of the ability of Cocaine.
Sample of cocaine: (pH 8.0 for 0.15M NaCl, 25mM Tris-HCl) configuration concentration is each 9 microlitre of sample of cocaine of 50 μ M, 20 μ M, 0 μ M in buffered soln.Per 4.5 microlitres are one group, and being divided into is two groups.
The nucleic acid probe 2 of sample A:0.5 microlitre 200 μ M contains the 50mM Tris-HCl damping fluid mixing of 0.3M NaCl with 0.5 microlitre.
The nucleic acid probe 3 of sample B:0.5 microlitre 200 μ M contains the 50mM Tris-HCl damping fluid mixing of 0.3M NaCl with 0.5 microlitre.
The ssDNA probe 3 of nucleic acid probe 2, the 0.5 microlitres 200 μ M of sample C:0.5 microlitre 200 μ M and the 50mM Tris-HCl damping fluid mixing that 1 microlitre contains 0.3M NaCl.
In the Cocaine sample of first group of each concentration, add respectively 0.25 microlitre sample A, in second group of each Cocaine sample, add respectively 0.5 microlitre sample C.Room temperature keeps 5min, adds respectively 0.25 microlitre sample B again in first group of each sample, and second group constant.Keep again 5min under the room temperature.From above sample, respectively get 2 microlitres, add 100 microlitre nano-Au solutions, behind the room temperature 5min, add 20 microlitre phosphate solutions (containing 0.2M NaCl).Then take pictures immediately (Fig. 8 A) and measure UV-vis absorption spectrum (Fig. 8 B).
Above experimental result confirmed in the two-part nucleic acid probe than long probe 2 can reduce the chance that Cocaine and probe 3 are combined again in conjunction with Cocaine alone, thereby cause the reduction of detection sensitivity.From experimental data, probe 2 and 3 together with Cocaine add fashionable, with the naked eye the eye can distinguish the sample of cocaine of 50 μ M and the colour-change of blank (Fig. 8 A, second group).And the sample of cocaine of 20 μ M and blank still keep stable redness.Ultraviolet-visible spectrometry (Fig. 6 A) can clearly be distinguished 20 μ M sample of cocaine and blank samples.Add in the ban probe 2 and Cocaine, when adding probe 3 subsequently, naked eyes can not be distinguished the sample of cocaine of 50 μ M and the colour-change of blank (Fig. 8 A, first group).Ultraviolet-visible spectrometry (Fig. 8 B) can not clearly be distinguished 50 μ M, 20 μ M sample of cocaine and blank samples.
Claims (7)
1. method that improves detection sensitivity of cocaine, it is characterized in that being fragmented into three sections for the aptamer that Cocaine detects, described method comprises the steps: that (1) mix Cocaine with three sections aptamer probes, makes determined Cocaine and aptamers probe form mixture; (2) carry out color detection;
Wherein, 1. described aptamers is DNA or the RNA molecule of being combined with target molecules; Target molecules is protein, DNA, small molecules or metal ion;
2. determined Cocaine and aptamers and probe form the mixture with double-stranded DNA structure;
3. the sequence in described three sections aptamers is 5 '-GGG AGT CAA GAA C, 5 '-GTT CTT CAA T and 5 '-AGT GGG ACG ACA.
2. by the method for raising detection sensitivity of cocaine claimed in claim 1, the step that comprises when it is characterized in that color detection is:
(1) Cocaine is mixed with three sections aptamer probes, make determined Cocaine and aptamers probe form mixture; (2) reaction soln is mixed with nano-Au solution; The mol ratio of reaction soln amplifying nucleic acid probe and nanometer gold was greater than 100: 1; (3) phosphate solution of adding sodium chloride-containing changes the existence that Cocaine is judged in sxemiquantitative by solution colour; The add-on of salts solution contains blank sample with differentiation to be determined in the recently titration of 520nm and 650nm place absorbancy with the nanometer gold that contains sample, increases gradually the consumption of salts solution, determines optimum value; Being present in of Cocaine is no by UV, visible light absorption quantitative analysis.
3. by the method for claim 1 or 2 described raising detection sensitivity of cocaines, it is characterized in that described three sections aptamers mix by equimolar ratio.
4. by the method for described raising detection sensitivity of cocaine claimed in claim 2, it is characterized in that detection comprises that step is:
(1) preparation of Cocaine standard model: at 0.15M NaCl, 25mM Tris-HCl, the Cocaine solution of configuration 500 μ M, 200 μ M, 100 μ M, 50 μ M, 20 μ M, 0 μ M concentration in pH 8.0 buffered soln;
Each 0.5 microlitre of (2) 200 μ M nucleic acid probes 3,200 μ M probes 4,200 μ M probes 5 contain the 50mM Tris-HCl damping fluid of 0.3M NaCl, mixing with 1.5 microlitres; Described nucleic acid probe 3,4 and 5 sequence are respectively 5 '-GGG AGT CAA GAA C, 5 ' GTT CTT CAAT and 5 '-AGT GGG ACG ACA;
(3) get 0.5 microlitre step 2 mixing solutions, add Cocaine solution 4.5 microlitres of different concns, preserve at normal temperatures 30min.From above-mentioned sample, respectively get 2 microlitres, add respectively the nano-Au solution of 100 microlitres, 12 ± 1nM, preserve 5min under the room temperature, then add the 10mM phosphate buffer soln that 15 microlitres contain 0.2MNaCl, carry out the UV-vis absorptiometry and take pictures;
(4) draw the typical curve that Cocaine detects according to the absorbancy at 520nm and 650nm place.
5. by the method for described raising detection sensitivity of cocaine claimed in claim 2, it is characterized in that described nanometer gold is for spherical.
6. by the method for raising detection sensitivity of cocaine claimed in claim 5, it is characterized in that described spherical diameter is 1～100nm.
7. by the method for each described raising detection sensitivity of cocaine in the claim 1～4, it is characterized in that at detection sensitivity of cocaine be 0.5 μ M.
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| CN102650612B (en) * | 2011-02-25 | 2014-05-14 | 首都师范大学 | Method for cocaine electrochemical detection and three-sectioned adapter probe used therein |
| CN102967523B (en) * | 2012-11-18 | 2014-09-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for detecting cocaine by using quartz crystal microbalance |
| CN102998292B (en) * | 2012-12-13 | 2014-10-29 | 湖北中烟工业有限责任公司 | Fluorescence detection method for detecting cocaine by use of oligonucleotide and graphene oxide |
| CN102980888A (en) * | 2012-12-18 | 2013-03-20 | 合肥工业大学 | Nucleic acid aptamer probe-based rapid one-step method non-mark type bisphenol-A colorimetric detection method |
| CN104450714B (en) * | 2014-11-27 | 2017-07-21 | 中国农业科学院农业质量标准与检测技术研究所 | Estradiol aptamers fragment and its application in estradiol detection |
| CN105675563B (en) * | 2016-01-20 | 2018-06-26 | 广州阳普医疗科技股份有限公司 | A kind of cocaine fast quantitative measurement method for detecting suitable for Site Detection |
| CN106855571A (en) * | 2016-12-29 | 2017-06-16 | 广州华弘生物科技有限公司 | A kind of antistreptolysin O (ASO) quick diagnosis reagent kit and its application method |
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| Milan N. Stojanovic and Donald W. Landry.Aptamer-Based Colorimetric Probe for Cocaine.《Journal of the American Chemical Society》.2002,第124卷(第33期),9678-9679. * |
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