CN102650612A - Method for cocaine electrochemical detection and three-sectioned adapter probe used therein - Google Patents
Method for cocaine electrochemical detection and three-sectioned adapter probe used therein Download PDFInfo
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Abstract
The invention provides a three-sectioned adapter probe for cocaine electrochemical detection and a method utilizing the three-sectioned adapter probe for cocaine electrochemical detection. The three-sectioned adapter probe achieves specific recognition of cocaine, and includes a sulfhydryl decorative probe (1), a redox group methylene blue decorative probe (2) and a non-decorative probe (3).
Description
Technical field
The present invention relates to field of biological detection, be specifically related to the field that cocaine detects, relate more specifically to utilize the aptamers probe to carry out the field of cocaine Electrochemical Detection.
Background technology
Though the detection technique for drugs has much at present; Like high performance liquid chromatogram-ultraviolet spectral technique, mass-spectrometric technique, high performance liquid chromatogram-electrochemical techniques, Electronic Nose, electron capture, micro-Raman technology (MRS, micro raman spectroscopy), X ray Detection Techniques, thermal neutron tomoscan etc.These technological detection sensitivities are high, but detect complex steps, apparatus expensive, and test procedure is complicated, and cost is higher, needs the professional technique talent, are not suitable for on-site quick screening and use.
To the actual demand of drugs fighting work, need that exploitation is highly sensitive, selectivity good, portable, energy consumption is low, easy-operating intelligent drugs fast detecting product, to reach on-the-spot, sensitive, testing goal fast.The domestic and international at present major technique over against quick field screening of drug addict and the detection of liquid poisoning article is various based on immunoreactive product, mainly contains EIA enzyme immunoassay and immune colloidal gold technique.The former relates to enzyme reaction, detects complex steps, and reagent stability is poor, can not very surely preserve usually.The latter is easy, and fast, but False Rate is high, can only carry out qualitative test; Sensitivity is lower, is difficult to carry out quality control, even same series-produced reagent; Also be difficult to guarantee the homogeneity of each reagent, therefore generally can only be used for qualitative test, can not carry out quantitative test.
With respect to antibody, aptamers has many-sided remarkable advantage.Aptamers (aptamer) is the functional living being molecule that application prospect is arranged very much of a new generation.Aptamers is system's molecular evolution method (the SystematicEvolution of Ligands by EXponential enrichment through index concentration from the DNA of～1014 random series or RNA storehouse; Be called for short SELEX) screen; Can combine with target molecules specifically; Thereby discern target molecules (Ellington AD, Szostak JW (1990) Nature 346:818-822.Tuerk C, Gold L (1990) Science249:505-510.) specifically.Aptamers can be used to comprise the detection of the various biomolecule of protein, micromolecule, heavy metal ion, cell and virus etc., has great application prospect.Compare with traditional antibody, aptamers has significant superiority (table 1) in many aspects.Aptamers stability is high, cheap, the different batches product quality is identical, and has suitable with antibody even better selectivity and compatibility.
Table 1: the comparison of aptamers and antibody
Utilize aptamers to carry out the method that cocaine detects at present, mainly be to use one section aptamers (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 aptamers is carried out cocaine 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, galvanochemistry or nm of 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.
Specific to the electrochemical detection method of cocaine, and a kind of method that is to use one section aptamers (B.R.Baker, et.al, J.Am.Chem.Soc.2006,128,3138-3139).In the method, a terminal modified sulfydryl of on gold electrode, assembling that is useful on of aptamers is modified with oxidizing reducing group methylene blue (Methylene blue) on the other end.Cocaine can cause the structure phase change of cocaine aptamers; Thereby make the nearly electrode surface of a termination that is modified with the oxidizing reducing group methylene blue of cocaine aptamers; Thereby cause the increase of electrode surface electric current, realize the detection by quantitative of cocaine, the detection sensitivity of this method is 10 μ M.Second kind be to use two-part aptamers probe (J.Am.Chem.Soc., Vol.131, No.21,2009,6944-6945.), one section probe is assembled on the electrode through sulfydryl, and with another segment mark oxidizing reducing group methylene blue.When cocaine exists; The probe that the oxidizing reducing group methylene blue is modified has been brought to electrode surface through forming complex compound with the probe of cocaine on electrode surface; Thereby cause the increase of electrode surface electric current, the sensitivity that detects has been improved an one magnitude (1 μ M).
The patent that we applied on November 3rd, 2009: a kind of method (application number 200910198075.4) that improves the cocaine detection sensitivity has proposed to utilize the syllogic aptamers probe of unmodified to carry out the method for the cocaine nm of gold color detection of homogeneous phase, and this method will be brought up to 0.5 μ M by 2.5 μ M based on the sensitivity of the nm of gold color detection of the cocaine of a section or two-part aptamers.After this patented invention has confirmed one section aptamers probe is divided into three sections first, still can form and have the double-stranded DNA structure with the cocaine self assembly.
The above adopts the syllogic aptamers probe of unmodified to carry out the method for cocaine color detection, and the method that adopts a section or two sections aptamers to carry out the cocaine Electrochemical Detection exists following deficiency:
1) the syllogic aptamers probe of employing unmodified carries out the method for cocaine color detection; Only confirmed not pass through mark the aptamers probe can with cocaine generation self assembly; This discovery is merely able to be applied to the detection technique that need not probe mark of other limited kinds, has significant limitation.
2) to carry out the detection sensitivity of method of cocaine Electrochemical Detection not high for one section or two sections aptamers of above-mentioned employing, is respectively 10 μ M and 1 μ M.
3) to carry out the standard deviation of method of cocaine Electrochemical Detection too big for two sections aptamers of above-mentioned employing, and the standard deviation that 1 μ M and 10 μ M cocaines are detected is respectively 12.5% and 20%, and in error range, overlaps, and is difficult to accurately quantitatively.
4) one section or two sections aptamers of above-mentioned employing are carried out the method for cocaine Electrochemical Detection, and the variation of the increase of its current signal and cocaine concentration is not simple function (such as linearity or a logarithm), are difficult to accurately quantitatively.
Summary of the invention
The present invention is directed to the problems of the prior art, its purpose is to provide a kind of syllogic aptamers probe that utilizes to improve cocaine Electrochemical Detection sensitivity of method.
A kind of raising cocaine Electrochemical Detection sensitivity of method of the present invention is characterized in that: the aptamers probe that is used for the cocaine detection is fragmented into three sections.Wherein one section is labeled oxidizing reducing group; One section is labeled sulfydryl, and is attached to the surface of gold electrode; One section does not have mark.
The present invention also promptly utilizes three sections aptamers probes of mark to improve cocaine Electrochemical Detection sensitivity of method, it is characterized in that: utilize and use three sections aptamers probe to detect cocaine.
Cocaine detection method of the present invention is characterized in that:
This method comprises the steps: that (1) encapsulates the probe of sulfydryl modification in three sections aptamers probes on the electrode; (2) probe of the oxidizing reducing group methylene blue being modified, the 3rd section probe of unmodified mix with cocaine solution and buffer solution; (3) carry out Electrochemical Detection.Specifically comprise: (i) the cocaine detector probe is synthetic: chemosynthesis is used for three sections probes of cocaine detection and carries out purifying and sign respectively;
The (ii) configuration of cocaine detector probe storage sample: three sections probes behind the purifying are configured to the dense WS (such as 100-200 μ M) respectively, and packing also is kept at-20 degrees centigrade;
The (iii) modification of detecting electrode: the good electrode of will polishing be immersed in contain an amount of sulfydryl modification probe (such as 5-50 μ L; 100 μ M) with the middle regular hour (such as 30 minutes) of the buffer solution (such as 10mM, 1M NaCl PBS) of going back original reagent reduction (is 5-50 μ M TCEP (tris-(2-carboxyethyl) phosphine hydrochloride) such as ultimate density) of debita spissitudo; Use the deionized water rinsing electrode then, in the buffer solution of the 6-mercaptohexanol of 1mM, soaked 2 hours then.Electrode is used deionized water rinsing then, is immersed in the buffer solution subsequent use.
(iiii) Electrochemical Detection of cocaine: electrochemical measurement adopts square wave voltammetry (Square WaveVoltammetry).Use conventional electrochemical workstation (to measure such as CHI 630b voltage stabilizer (CHinstruments, Austin, TX)).The electrode of preparation is a detecting electrode (iii), and platinum filament is to electrode, and the Ag/AgCl electrode is a reference electrode.Scanning electromotive force, potential scan gradient, amplitude and frequency that square wave voltammetry is measured are optimized according to testing result.With the detecting electrode of (iii) preparation, electrode and reference electrode are inserted the concentration (such as 0.1-10 μ M) of the probe that the oxidizing reducing group methylene blue of the unmodified probe (such as 0.1-10 μ M) that contains an amount of concentration, an amount of concentration modifies and the cocaine of variable concentrations.Under the electric potential scanning condition that configures, scan, obtain series A C voltammogram.Under different cocaine concentration, the standard working curve of cocaine Electrochemical Detection is drawn in the variation of the current signal at oxidizing reducing group methylene blue reduction potential place in the AC voltammogram.When unknown sample is measured,, obtain the concentration of cocaine in the unknown sample through this standard working curve according to the variation of current signal.
In this method, one section can selectivity the aptamers probe of identification cocaine be designed with chemosynthesis be three sections aptamers probes, wherein one section is labeled oxidizing reducing group, one section is labeled sulfydryl, and is attached to the surface of gold electrode, one section does not have mark.These three sections probes can with cocaine generation self assembly, make the aptamers probe of the oxidizing reducing group mark in the solution be attached to electrode surface, cause the increase of current signal.The concentration of cocaine is high more, and the increase of current signal is just many more, therefore can be used for the detection of cocaine.Because the length of three sections aptamers probes significantly reduces than the aptamers probe of a section that uses in the prior art or two sections, between probe, and other component can cause the interaction of background noise greatly to reduce in probe and the detection architecture.And since three sections aptamers probes with the structure phase change need not take place when cocaine combines; The structure phase change can combine with cocaine and still need take place for one section or two sections aptamers probes; Therefore it is littler that the cocaine that adopts three sections aptamers probes to carry out electrode surface detects the time space steric hindrance, makes in the low concentration surveyed area current signal growth of cocaine faster relatively.Experiment shows that the remolding sensitivity of the cocaine Electrochemical Detection of using syllogic aptamers probe of the present invention uses the prior art of two-part or one-part form aptamers probe to improve one to two one magnitude.And the denary logarithm of the increase of current signal and cocaine concentration presents good linear relationship, and the standard deviation of measurement makes things convenient for detection by quantitative significantly less than prior art, improves the accuracy that detects.This method can be generalized to other micromolecular detection.
The present invention utilizes syllogic aptamers probe to detect cocaine, has following technique effect:
(1) the present invention utilizes oxidizing reducing group and the sulfydryl syllogic aptamers probe of mark respectively, carries out the cocaine Electrochemical Detection on the electrode surface.The length of the syllogic aptamers probe that is designed in this method is very short, and can it also have the ability that forms compound with the cocaine self assembly behind mark be unknown in the prior art.Through the present invention, confirmed that first the syllogic aptamers probe behind the mark still has the ability with cocaine generation self assembly.
(2) remolding sensitivity with the cocaine Electrochemical Detection uses the prior art of two-part or one-part form probe to improve one to two one magnitude, reaches 0.1 μ M.
(3) reduced the standard deviation of measuring.Utilizing the standard deviation of syllogic probe in detecting 1 μ M of the present invention and 10 μ M cocaines is respectively 3.6% and 4.1%, and Billy has reduced 3-5 doubly with the standard deviation of two-part aptamers probe in detecting 1 μ M and 10 μ M cocaines.The measuring error that significantly reduces helps improving the accuracy of detection.
(4) variation of current signal becomes good linear relationship with the denary logarithm of cocaine concentration: I=11.866log10C+12.303 (r2=0.9984), and wherein C is the volumetric molar concentration of cocaine, and unit is μ M, and I is the percentage change of current signal.Can carry out accurately quantitatively.
The present invention relates to a kind of syllogic aptamers probe that utilizes and improve cocaine Electrochemical Detection sensitivity of method.In the method, one section cocaine aptamers has been cut into three sections, and wherein one section probe is assembled on the electrode through sulfydryl, and with another segment mark oxidizing reducing group methylene blue.When cocaine exists; The probe that the oxidizing reducing group methylene blue is modified is through the 3rd section probe of unmodified and the probe formation complex compound of cocaine and the sulfydryl modification on electrode surface; The oxidizing reducing group methylene blue has been brought to electrode surface; Thereby cause the increase (Fig. 1) of electrode surface electric current, the sensitivity that detects has been improved an one magnitude (0.1 μ M)
The inventive method can be used for detecting protein, enzyme, DNA, micromolecule, metallic ion and bacterium etc.Other characteristics of the present invention and advantage can embody through following embodiment.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.
Particular content is following:
1. the syllogic aptamers probe that is used for the Electrochemical Detection cocaine; Said syllogic aptamers probe is at the specific recognition cocaine; Comprise probe 1, have oxidizing reducing group with sulfydryl modification; The probe 2 that preferred oxidizing reducing group methylene blue is modified and the probe 3 of unmodified, said probe 1,2 and 3 form complex compound with cocaine when having cocaine.
2. above 1 described syllogic aptamers probe; Wherein said probe 1,2 and 3 sequence are respectively 5 '-HS-C6-TTTGTTCTTCAAT; 5 '-GGGAGTCAAGAAC-MB and 5 '-AGTGGGACGACA, wherein MB is the oxidizing reducing group methylene blue, HS is a sulfydryl.3. the method for an Electrochemical Detection cocaine is characterized in that using according to above 1 or 2 described syllogic aptamers probes, may further comprise the steps:
(1) said probe 1 is encapsulated on the gold electrode, form detecting electrode;
(2) said probe 2 is mixed with blank or testing sample with said probe 3, form blank potpourri or testing sample potpourri; With
(3) with said detecting electrode, electrode and reference electrode are inserted in said blank potpourri or the testing sample potpourri; Carry out Electrochemical Detection, represent to contain cocaine in the said testing sample during current signal that the current signal that produces when testing sample produces greater than blank.
4. above 3 described methods is characterized in that:
In said step (1), said gold electrode is immersed in contains said probe 1 and go back in the reaction buffer solution of original reagent; Use the deionized water rinsing electrode then, and be immersed in the reaction buffer solution that contains 1mM6-mercaptohexanol; Electrode is used deionized water rinsing then; Be immersed in the reaction buffer solution subsequent use; The amount of wherein said probe 1 is preferably 5-50 μ L, 100 μ M, and the said original reagent of going back is preferably the TCEP that ultimate density is 5-50 μ M (tris-(2-carboxyethyl) phosphine hydrochloride); Reaction buffer solution is preferably 10mM, 1M NaCl PBS.
5. above 3 or 4 described methods is characterized in that:
In said step (3), Electrochemical Detection adopts square wave voltammetry, wherein uses the platinum filament conduct to electrode, and the Ag/AgCl electrode is electrode as a reference; Scanning electromotive force, potential scan gradient, amplitude and frequency that square wave voltammetry is measured can be optimized according to testing result; Wherein preferably, the scanning electromotive force is that the potential scan gradient is 0.001V from 0 to-0.4V with respect to the Ag/AgCl electrode; Amplitude 0.05V, frequency 60Hz; Said reaction buffer solution is 10mM, 1M NaCl PBS, and pH 7.0; The concentration of said probe 3 is preferably 0.1-10 μ M, and the concentration of said probe 2 is preferably 0.1-10 μ M.
6. the method for an electrochemical quantitative detection cocaine is characterized in that using according to above 1 or 2 described syllogic aptamers probes, may further comprise the steps:
(1) said probe 1 is encapsulated on the gold electrode, form detecting electrode;
(2) said probe 2 is mixed respectively with the cocaine standard items of a series of variable concentrations or the testing sample of unknown concentration with said probe 3, form the cocaine standard items potpourri and the testing sample potpourri of a series of variable concentrations;
(3) with said detecting electrode, electrode and reference electrode are inserted in the cocaine standard items potpourri of a series of variable concentrations of preparation in the step (2); Carry out Electrochemical Detection, produce the standard working curve of the variation drafting cocaine Electrochemical Detection of current signal according to the cocaine standard items of different cocaine concentration; With
(4) with in said detecting electrode, the testing sample potpourri to preparation in electrode and the reference electrode inserting step (2); Carry out Electrochemical Detection, the standard working curve of drafting is confirmed the cocaine concentration in the said testing sample in the variation of the current signal that produces according to said testing sample and the step (3).
7. above 6 described methods is characterized in that the standard working curve of said cocaine Electrochemical Detection satisfies following relation: I=11.866log10C+12.303 (r
2=0.9984), wherein C is the volumetric molar concentration of cocaine, and unit is μ M, and I is the percentage change of current signal.
8. above 6 or 7 described methods is characterized in that:
In said step (1), said gold electrode is immersed in contains said probe 1 and go back in the reaction buffer solution of original reagent; Use the deionized water rinsing electrode then, and be immersed in the reaction buffer solution that contains 1mM6-mercaptohexanol; Electrode is used deionized water rinsing then; Be immersed in the reaction buffer solution subsequent use; The amount of wherein said probe 1 is preferably 5-50 μ L, 100 μ M, and the said original reagent of going back is preferably the TCEP that ultimate density is 5-50 μ M (tris-(2-carboxyethyl) phosphine hydrochloride); Reaction buffer solution is preferably 10mM, 1M NaCl PBS.
9. each described method among the above 6-8 is characterized in that:
In said step (3) and (4), Electrochemical Detection adopts square wave voltammetry, wherein uses the platinum filament conduct to electrode, and the Ag/AgCl electrode is electrode as a reference; Scanning electromotive force, potential scan gradient, amplitude and frequency that square wave voltammetry is measured can be optimized according to testing result; Wherein preferably, the scanning electromotive force is that the potential scan gradient is 0.001V from 0 to-0.4V with respect to the Ag/AgCl electrode; Amplitude 0.05V, frequency 60Hz; Said reaction buffer solution is 10mM, 1M NaCl PBS, and pH 7.0; The concentration of said probe 3 is preferably 0.1-10 μ M, and the concentration of said probe 2 is preferably 0.1-10 μ M; The concentration of the cocaine standard items of said variable concentrations preferably is respectively 0,1 μ M, 10 μ M, 100 μ M, 1000 μ M.
Description of drawings
Fig. 1 is the schematic diagram that syllogic aptamers probe of the present invention (probe 1,2 and 3) carries out the cocaine Electrochemical Detection.
Fig. 2 uses syllogic aptamers probe (probe 1,2 and 3) to carry out the AC voltammogram of cocaine Electrochemical Detection in the one embodiment of the invention, be the reduction peak of methylene blue among the figure.
Fig. 3 uses syllogic aptamers probe (probe 1,2 and 3) to carry out the typical curve of cocaine Electrochemical Detection in the one embodiment of the invention.
Fig. 4 uses selectivity ratios that syllogic aptamers probe (probe 1,2 and 3) carries out the cocaine Electrochemical Detection in the one embodiment of the invention.
Embodiment
Design aptamers probe, and from Biosearch Technologies, (Novato, CA) (the aptamers sequence is seen table 2) bought in customization to Inc..
Table 2: the aptamers probe sequence that uses among the present invention.
MB: oxidizing reducing group methylene blue
HS: sulfydryl
Embodiment 1: the preparation of detecting electrode.
At room temperature will polish good cylinder gold electrode (available from Bio-Logic USA; LLC; TN, USA) be immersed in the probe 1 that contains 1 μ M sulfydryl modification and 5 μ M TCEP (tris-(2-carboxyethyl) phosphine hydrochloride) (available from Sigma-Aldrich, reaction buffer solution (10mM USA); 1M NaCl PBS, pH 7.0) in 30 minutes.TCEP is used for reducing the cystine linkage in the sulfydryl modification nucleic acid.Electrode is used deionized water rinsing then, and (available from Sigma-Aldrich, immersion is 2 hours in reaction buffer solution USA) (pH 7.0 for 10mM, 1M NaCl PBS) containing the 6-mercaptohexanol of 1mM then.Electrode is used deionized water rinsing then, is immersed in 10mM, and is subsequent use in the 1M NaCl phosphate reaction buffer solution.
Embodiment 2: the Electrochemical Detection of cocaine.
Electrochemical measurement employing square wave voltammetry (Square Wave Voltammetry) (B.R.Baker, et.al, J.Am.Chem.Soc.2006,128,3138-3139).(TX), the electrode of preparation is a detecting electrode among the embodiment 1 for CHinstruments, Austin, and platinum filament is to electrode, and the Ag/AgCl electrode is a reference electrode to use CHI 630b voltage stabilizer.The scanning electromotive force that square wave voltammetry is measured is from 0 to-0.4V (with respect to the Ag/AgCl electrode), and the potential scan gradient is 0.001V, amplitude 0.05V, frequency 60Hz.Reaction buffer solution is 10mM, 1M NaCl PBS (pH 7.0).The concentration of the probe (probe 2, table 2) that unmodified probe (probe 3, table 2) and oxidizing reducing group methylene blue are modified in all experiments is respectively 0.2 μ M and 0.1 μ M.With the detecting electrode of preparation among the embodiment 1, electrode and reference electrode are inserted in the potpourri of the cocaine (available from Nat'l Pharmaceutical & Biological Products Control Institute) (0,1 μ M, 10 μ M, 100 μ M, 1000 μ M) that contains 0.2 μ M unmodified probe, probe that 0.1 μ M oxidizing reducing group methylene blue is modified and variable concentrations.Under the electric potential scanning condition that configures, scan, obtain series A C voltammogram.Under different cocaine concentration, the standard working curve of cocaine Electrochemical Detection is drawn in the variation of the current signal at oxidizing reducing group methylene blue reduction potential place in the AC voltammogram.When unknown sample is measured,, obtain the concentration of cocaine in the unknown sample through this standard working curve according to the variation of current signal.
When Fig. 2 was illustrated in the cocaine existence, current signal significantly increased.
Fig. 3 shows that electric current continues to increase along with the increase of cocaine concentration, can detect the cocaine of 0.1 μ M to several mM.Particularly very fast along with the increase electric signal of cocaine concentration increases ground at the low concentration region of cocaine, the denary logarithm of current signal percentage change and cocaine concentration is linear.I=11.866log
10C+12.303 (r
2=0.9984), wherein C is the volumetric molar concentration of cocaine, and unit is μ M, and I is the percentage change of current signal.Data point among Fig. 3 and error are mean value and the errors by at least three detecting electrode data measured that prepare respectively.
According to the cocaine examination criteria curve of Fig. 3 and the standard deviation 2.9% of blank sample, the detection limit that can calculate the inventive method is 0.1 μ M.The current signal of 10 μ M cocaines and 1 μ M cocaine can obviously be distinguished, zero lap in error range; 1 μ M cocaine also can obviously be distinguished zero lap in error range with blank current signal; Therefore, improve the accuracy that detects, especially the accuracy of low concentration cocaine detection.The technology of two sections aptamers detections of the available technology adopting of comparing cocaine (J.Am.Chem.Soc., Vol.131, No.21,2009,6944-6945.), the current signal of 10 μ M cocaines and 1 μ M cocaine is overlapping in a large number in error range; 1 μ M cocaine is also overlapping in a large number in error range with blank current signal; Therefore the poor accuracy that detects.
Fig. 4 shows that syllogic aptamers probe possesses high selectivity.The two kinds of analog benzoyl ecgonine (BE) (available from Nat'l Pharmaceutical & Biological Products Control Institute) and the ecgoninemethyl ester (EME) (available from Nat'l Pharmaceutical & Biological Products Control Institute) of cocaine are detected as testing sample.The result shows that the benzoyl ecgonine (BE) of 100mM and ecgonine methyl ester (EME) only produce the change in electric about 10%, and the cocaine of 2mM can produce 47% change in electric.This this method of explanation has high selectivity to cocaine.
Claims (9)
1. the syllogic aptamers probe that is used for the Electrochemical Detection cocaine; Said syllogic aptamers probe is at the specific recognition cocaine; Comprise probe 1, have oxidizing reducing group with sulfydryl modification; The probe 2 that preferred oxidizing reducing group methylene blue is modified and the probe 3 of unmodified, said probe 1,2 and 3 form complex compound with cocaine when having cocaine.
2. the described syllogic aptamers of claim 1 probe; Wherein said probe 1,2 and 3 sequence are respectively 5 '-HS-C6-TTTGTTCTTCAAT; 5 '-GGGAGTCAAGAAC-MB and 5 '-AGTGGGACGACA, wherein MB is the oxidizing reducing group methylene blue, HS is a sulfydryl.
3. the method for an Electrochemical Detection cocaine is characterized in that using syllogic aptamers probe according to claim 1 and 2, may further comprise the steps:
(1) said probe 1 is encapsulated on the gold electrode, form detecting electrode;
(2) said probe 2 is mixed with blank or testing sample with said probe 3, form blank potpourri or testing sample potpourri; With
(3) with said detecting electrode, electrode and reference electrode are inserted in said blank potpourri or the testing sample potpourri; Carry out Electrochemical Detection, represent to contain cocaine in the said testing sample during current signal that the current signal that produces when testing sample produces greater than blank.
4. the described method of claim 3 is characterized in that:
In said step (1), said gold electrode is immersed in contains said probe 1 and go back in the reaction buffer solution of original reagent; Use the deionized water rinsing electrode then, and be immersed in the reaction buffer solution that contains 1mM6-mercaptohexanol; Electrode is used deionized water rinsing then; Be immersed in the reaction buffer solution subsequent use; The amount of wherein said probe 1 is preferably 5-50 μ L, 100 μ M, and the said original reagent of going back is preferably the TCEP that ultimate density is 5-50 μ M (tris-(2-carboxyethyl) phosphine hydrochloride); Reaction buffer solution is preferably 10mM, 1M NaCl PBS.
5. claim 3 or 4 described methods is characterized in that:
In said step (3), Electrochemical Detection adopts square wave voltammetry, wherein uses the platinum filament conduct to electrode, and the Ag/AgCl electrode is electrode as a reference; Scanning electromotive force, potential scan gradient, amplitude and frequency that square wave voltammetry is measured can be optimized according to testing result; Wherein preferably, the scanning electromotive force is that the potential scan gradient is 0.001V from 0 to-0.4V with respect to the Ag/AgCl electrode; Amplitude 0.05V, frequency 60Hz; Said reaction buffer solution is 10mM, 1M NaCl PBS, and pH 7.0; The concentration of said probe 3 is preferably 0.1-10 μ M, and the concentration of said probe 2 is preferably 0.1-10 μ M.
6. the method for an electrochemical quantitative detection cocaine is characterized in that using syllogic aptamers probe according to claim 1 and 2, may further comprise the steps:
(1) said probe 1 is encapsulated on the gold electrode, form detecting electrode;
(2) said probe 2 is mixed respectively with the cocaine standard items of a series of variable concentrations or the testing sample of unknown concentration with said probe 3, form the cocaine standard items potpourri and the testing sample potpourri of a series of variable concentrations;
(3) with said detecting electrode, electrode and reference electrode are inserted in the cocaine standard items potpourri of a series of variable concentrations of preparation in the step (2); Carry out Electrochemical Detection, produce the standard working curve of the variation drafting cocaine Electrochemical Detection of current signal according to the cocaine standard items of variable concentrations; With
(4) with in said detecting electrode, the testing sample potpourri to preparation in electrode and the reference electrode inserting step (2); Carry out Electrochemical Detection, the standard working curve of drafting is confirmed the cocaine concentration in the said testing sample in the variation of the current signal that produces according to said testing sample and the step (3).
7. the described method of claim 6 is characterized in that the standard working curve of said cocaine Electrochemical Detection satisfies following relation: I=11.866log
10C+12.303 (r
2=0.9984), wherein C is the volumetric molar concentration of cocaine, and unit is μ M, and I is the percentage change of current signal.
8. claim 6 or 7 described methods is characterized in that:
In said step (1), said gold electrode is immersed in contains said probe 1 and go back in the reaction buffer solution of original reagent; Use the deionized water rinsing electrode then, and be immersed in the reaction buffer solution that contains 1mM6-mercaptohexanol; Electrode is used deionized water rinsing then; Be immersed in the reaction buffer solution subsequent use; The amount of wherein said probe 1 is preferably 5-50 μ L, 100 μ M, and the said original reagent of going back is preferably the TCEP that ultimate density is 5-50 μ M (tris-(2-carboxyethyl) phosphine hydrochloride); Reaction buffer solution is preferably 10mM, 1M NaCl PBS.
9. each described method among the claim 6-8 is characterized in that:
In said step (3) and (4), Electrochemical Detection adopts square wave voltammetry, wherein uses the platinum filament conduct to electrode, and the Ag/AgCl electrode is electrode as a reference; Scanning electromotive force, potential scan gradient, amplitude and frequency that square wave voltammetry is measured can be optimized according to testing result; Wherein preferably, the scanning electromotive force is that the potential scan gradient is 0.001V from 0 to-0.4V with respect to the Ag/AgCl electrode; Amplitude 0.05V, frequency 60Hz; Said reaction buffer solution is 10mM, 1M NaCl PBS, and pH 7.0; The concentration of said probe 3 is preferably 0.1-10 μ M, and the concentration of said probe 2 is preferably 0.1-10 μ M; The concentration of the cocaine standard items of said variable concentrations preferably is respectively 0,1 μ M, 10 μ M, 100 μ M, 1000 μ M.
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| CN201110046862.4A CN102650612B (en) | 2011-02-25 | 2011-02-25 | Method for cocaine electrochemical detection and three-sectioned adapter probe used therein |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103630598A (en) * | 2013-12-10 | 2014-03-12 | 重庆医科大学 | Electrochemical detection method of cocaine based on rolling circle amplification and supramolecular aptamer |
| CN104897742A (en) * | 2015-01-22 | 2015-09-09 | 济南大学 | Biosensor used for detecting tetracycline based on nucleic acid aptamer and preparation method thereof |
| CN105675563A (en) * | 2016-01-20 | 2016-06-15 | 广州阳普医疗科技股份有限公司 | Rapid quantitative detection method of cocaine suitable for field detection |
| CN108384830A (en) * | 2018-01-24 | 2018-08-10 | 安徽师范大学 | A kind of preparation method of nanowires of gold DNA biosensor and its quantitative detecting method to DNA concentration |
| CN109116040A (en) * | 2018-08-21 | 2019-01-01 | 江苏大学 | A method of cocaine is detected based on double sulfydryl aptamers |
| CN109709167A (en) * | 2018-11-21 | 2019-05-03 | 上海大学 | The preparation method of detection drugs analog sensor based on three-dimensional gold nano structure |
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| US20080286877A1 (en) * | 2002-04-19 | 2008-11-20 | The Trustees Of Columbia University In The City Of New York | Displacement assay for detection of small molecules |
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| US20080286877A1 (en) * | 2002-04-19 | 2008-11-20 | The Trustees Of Columbia University In The City Of New York | Displacement assay for detection of small molecules |
| CN101948907A (en) * | 2009-10-30 | 2011-01-19 | 中国科学院上海微系统与信息技术研究所 | Method for improving detection sensitivity of cocaine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630598A (en) * | 2013-12-10 | 2014-03-12 | 重庆医科大学 | Electrochemical detection method of cocaine based on rolling circle amplification and supramolecular aptamer |
| CN104897742A (en) * | 2015-01-22 | 2015-09-09 | 济南大学 | Biosensor used for detecting tetracycline based on nucleic acid aptamer and preparation method thereof |
| CN105675563A (en) * | 2016-01-20 | 2016-06-15 | 广州阳普医疗科技股份有限公司 | Rapid quantitative detection method of cocaine suitable for field detection |
| CN108384830A (en) * | 2018-01-24 | 2018-08-10 | 安徽师范大学 | A kind of preparation method of nanowires of gold DNA biosensor and its quantitative detecting method to DNA concentration |
| CN109116040A (en) * | 2018-08-21 | 2019-01-01 | 江苏大学 | A method of cocaine is detected based on double sulfydryl aptamers |
| CN109709167A (en) * | 2018-11-21 | 2019-05-03 | 上海大学 | The preparation method of detection drugs analog sensor based on three-dimensional gold nano structure |
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| CN102650612B (en) | 2014-05-14 |
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