CN109507254A - A kind of electrochemica biological sensor and preparation method and application detecting kanamycins - Google Patents
A kind of electrochemica biological sensor and preparation method and application detecting kanamycins Download PDFInfo
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- CN109507254A CN109507254A CN201811588936.5A CN201811588936A CN109507254A CN 109507254 A CN109507254 A CN 109507254A CN 201811588936 A CN201811588936 A CN 201811588936A CN 109507254 A CN109507254 A CN 109507254A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
Abstract
The present invention relates to a kind of biosensors based on aptamer detection kanamycins, belong to electrochemica biological sensor technical field.Cutting function is identified using havingNt.BbvCIRestriction endonuclease realizes recycling for primer, is exaggerated detection signal, improves the sensitivity of detection；The identification and hydrolysis that the specificity of exonuclease III is utilized realize second step circulation amplification, further improve the sensitivity of detection.Electrochemica biological sensor of the invention can be detected with high specific；The reaction condition of the sensor is mild, and reaction speed is fast；The process costs for making electrode are low, the inexpensive requirement suitable for industrialization, the practical application of the detection of kanamycins and biosensor industrialization suitable for food safety.
Description
Technical field
The present invention relates to a kind of biosensors based on aptamer detection kanamycins, and in particular to one kind is based on
Target induction aptamer conformation change andNt.BbvCIRestriction endonuclease and exonuclease III auxiliary circulation amplification detection card
The electrochemica biological sensor of that mycin belongs to electrochemica biological sensor technical field.
Background technique
Antibiotic has the function of killing and destroying microbial cell structure, worldwide be widely used in each
The preparation of kind antibacterials, but at the same time, residue problem caused by the abuse of antibiotic has become in world wide and is badly in need of
The problem of solution.Antibiotic is excessively used, and bacterium caused by infection can be stimulated to become drug resistance.
Kanamycins (Kanamycin) is a kind of protein biology synthetic inhibitor, is a kind of antibiotic, takes orally for treating
Enteric infection caused by sensitive bacteria and as preparing before intestinal surgery, and it is reduced the effect that enteric bacteria generates ammonia, it is hard to liver
The hepatic coma for changing patients with digestive tract hemorrhage has and centainly prevents from acting on.Intramuscular injection is used for system infections caused by sensitive bacteria, and such as pneumonia loses
Mass formed by blood stasis, urinary tract infections etc..
The method for the detection kanamycins reported at present includes high performance liquid chromatography, capillary electrophoresis, surface etc. from
The methods of daughter resonance method and fluorescence.These methods have testing cost high, and instrumentation is complicated, need professional operator etc.
Disadvantage, therefore, the method for establishing an easy to operate, sensitive high specific, in terms of Food Safety Analysis and environmental monitoring
It is vital to detect kanamycins and its residual.
Summary of the invention
It is all relatively low, at high cost in order to solve the above method specificity for detecting kanamycins in the prior art and sensitivity
The problem of, the present invention provides a kind of easy to operate, specific and high sensitivities, the nucleic acid at low cost based on object induction
Aptamers conformation change andNt.BbvCIThe electrochemical student of restriction endonuclease and exonucleaseⅢ auxiliary circulation amplification detection kanamycins
Object sensor.Another object of the present invention be to provide a kind of above-mentioned electrochemica biological sensor preparation method and detection card that
Application in mycin.
A kind of electrochemica biological sensor detecting kanamycins, comprising: aptamers Aptamer, primer Primer, hair fastener
Primer HAP1, hair fastener primer HAP2, hair fastener primer HAP3, ferroheme, PBS buffer solution, hydrogen peroxide H2O2、Nt.BbvCIInscribe
Enzyme, exonucleaseⅢ, the gold electrode for modifying capture probe CP；
The PBS buffer solution contains K+；
The sequence of the Aptamer is as shown in SEQ No.1；
The sequence of Primer is as shown in SEQ No.2；
The sequence of HAP1 is as shown in SEQ No.3；
The sequence of HAP2 is as shown in SEQ No.4；
The sequence of HAP3 is as shown in SEQ No.5；
The sequence of CP is as shown in SEQ No.6；
5 ' terminal modified-SH of the capture probe CP sequence.
The gold electrode of the modification capture probe CP, is prepared using following methods:
A) gold electrode is successively carried out being polished to mirror surface in 0.3 μm and 0.05 μm of oxidation aluminium paste, is rushed repeatedly with ultrapure water
It washes；
B) gold electrode surfaces by CP solution drop after the treatment, heat preservation are incubated for.
The HAP1 concentration is 1-20 μM；The HAP2 concentration is 1-20 μM；The HAP3 concentration is 1-20 μM；Institute
The concentration for stating CP is 1-20 μM.
The Aptamer is final concentration of 1 μM preferred；The Primer is final concentration of 1 μM preferred；The ferroheme is excellent
Final concentration of 10 μM of choosing；The H2O2It is final concentration of 10 μM preferred；It is describedNt.BbvCIRestriction endonuclease and exonucleaseⅢ are preferred
Concentration be 500U/mL.
A kind of preparation method of above-mentioned electrochemica biological sensor, comprising the following steps:
(1) Aptamer and Primer are hybridized to probe Probe in PBS buffer solution；
(2) by probe Probe, HAP1, HAP2, HAP3,Nt.BbvCIRestriction endonuclease, exonucleaseⅢ, 10 ×Nt.BbvCIRestriction endonuclease
With constant-temperature incubation after exonucleaseⅢ buffer, the mixing of kanamycins solution；
(3) mixed solution in step (2) is added drop-wise on the gold electrode of modification capture probe CP, then constant-temperature incubation；
(4) haemachrome solution is added drop-wise in step (3) resulting solution, then constant-temperature incubation；
(5) with the electrode in PBS buffer solution rinse step (4)；
(6) H is added into PBS buffer solution2O2, it is uniformly mixed, is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode
Change in electric is measured with differential pulse voltammetry；
(7) standard curve is made according to the electric signal of kanamycins standard solution, calculates regression equation, according to regression equation with it is to be measured
Liquid electric signal calculates the concentration of kanamycins in prepare liquid.
The step (1) specifically comprises the processes of: by 14 μ L aqua sterilisas, 25 × PBS of μ L, 2 10 μM of μ L Aptamer chains
It is mixed with 2 μ L, 10 μM of Primer chains, is incubated for 5min at 95 DEG C, is slowly cooled to room temperature, be stored in spare at -20 DEG C.
Constant-temperature incubation temperature is 37 DEG C in the step (2).
The parameter of measurement change in electric in the step (6) are as follows: 0 to -0.6 V of current potential, 0.05 V of pulse width, scanning
0.06 s of rate.
Above-mentioned electrochemica biological sensor is used to detect kanamycins in terms of Food Safety Analysis and environmental monitoring.
The working principle of above-mentioned electrochemica biological sensor is as follows:
Aptamer:5 '-TGGGGGTTGAGGCTAAGCCGAC-3 '
Primer:5-GGCTTAGCTGAGGACCCCCAAAAA-3
HAP1: 5-ATCACAACGGTCCTCAGCTAGTGATAAA-3
HAP2:5- ACTAACCTCGTAGGGCGGGATGGGTACGAGGTTAGTCCGTTGTGAT-3
HAP3: 5-GGGTAGGATCGAACAATCCTACCCATCACTAGCTGA -3
Capture probe (CP): 5-TGTTCGATCCTAAAATACGAGGTTAGTAAA-SH-3
Aptamer can with Primer by number of base pairs in conjunction with arched probe (Probe).As shown, working as target
In the presence of object kanamycins, kanamycins releases primer (Primer) in conjunction with aptamers.Subsequent primer is opened
Hair clip 1(HAP1),Nt.BbvCIUnder the action of restriction endonuclease, HAP1 is cut off and discharges lower primer 2 (P2) and primer 3(P3),
The circulation of Primer is realized so as to continue to open remaining HAP1.The P2 and P3 released can be each by base
Complementary pairing and hair clip 2(HAP2) and hair clip 3(HAP3) combine.It is flat from 3 ' that exonuclease III can be catalyzed single nucleotide acid
End hydrolyzes step by step.Under the action of exonuclease III, HAP2 and HAP3 can be hydrolyzed into from 3 ' ends miscellaneous with P2, P3
The end of friendship, so that being flat end through postdigestive hair clip 3 ' end, exonuclease III can continue to act on hair clip at this time
Hybridization portion generates trigger1 and trigger2 and simultaneously discharges lower P2 and P3 so that P2 and P3 can with ringing in HAP2 and
HAP3 generates a large amount of trigger1 and trigger2.In addition on gold electrode modify one section can capture trigger1 and
The capture probe of trigger2, the 3 ' ends of trigger1 and the 5 ' ends of trigger2 are containing 9 and 3 guanines respectively
Sequence, in K+Under the conditions of existing for ferroheme, G- tetrad/ferroheme compound can be formed, there is horseradish peroxidating
The effect of object enzyme, to be catalyzed H2O2It decomposes and generates electrochemical signals.In a certain range, through measurement electrochemical signals come between
Connect quantitative detection kanamycins.
The invention has the following advantages that
1, high sensitivity
The specific recognition of aptamers and kanamycins is utilized in electrochemica biological sensor of the invention, realizes to object
The high specific of kanamycins detects；Cutting function is identified using havingNt.BbvCIRestriction endonuclease realizes following for primer
Ring utilizes, and is exaggerated detection signal, improves the sensitivity of detection；Be utilized exonuclease III specificity identification and
Hydrolysis realizes second step circulation amplification, further improves the sensitivity of detection.
2, reaction condition is mild, speed is fast
The reaction condition of the sensor is mild, and reaction speed is fast；Due to using gold electrode, electrode is easy, minimizes, is portable
Band is used multiple times；The main process of testing principle is to improve reaction speed in homogeneous middle realization, reduce operation
Complexity, realize the quick of object, it is simply, sensitive to detect.
3, probe preparation method is simple, is suitble to industrialization
The preparation method of the application is simple, and performance is stablized, reproducible, the inspection of kanamycins suitable for food safety of electrode
Survey the practical application with biosensor industrialization；The process costs for making electrode are low, the inexpensive requirement suitable for industrialization.
Detailed description of the invention
Fig. 1 is the operation principle schematic diagram of this electrochemica biological sensor；
Fig. 2 is the electric signal curve graph for modifying the gold electrode measurement of various concentration CP；
Fig. 3 is the electric signal curve graph of various concentration HAP1 measurement；
Fig. 4 is the electric signal curve graph of various concentration HAP2 measurement
Fig. 5 is the electric signal figure of serial kanamycins standard solution；
Fig. 6 is the standard curve for measuring kanamycins.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
In embodiment, the buffer of two kinds of enzymes is CutSmart, 50 mM KAc, 20 mM Tris-Ac, 10 mM Mg
(Ac)2, 25 DEG C of 100 μ g/ml BSA(pH, 7.9@).
PBS buffer solution contains Na2HPO4 (10 mM), NaH2PO4 (10 mM), NaCl (140 mM), KCl (1 mM),
MgCl2 (1 mM), CaCl2 (1 mM), pH value 7.4.
Embodiment 1 modifies the preparation of the gold electrode of CP.
A) gold electrode polishing treatment: gold electrode is successively polished in 0.3 μm and 0.05 μm of oxidation aluminium paste
Mirror surface, ultrapure water repeated flushing 5 times；
B) the gold electricity by (final concentration of 1 μM, 5 μM, 10 μM, 15 μM, 20 μM) of 10 μ L CP solution drop after the treatment
Pole surface, 37 DEG C of constant-temperature incubation 2h must modify the gold electrode S1-S6 of CP.
Influence of the 2 difference CP concentration of embodiment to detection kanamycins.
(1) Aptamer and Primer are hybridized to Probe(10 μM of probe in PBS buffer solution)；
(2) by probe Probe, 10 μM of HAP1,10 μM of HAP2,10 μM of HAP3,1U/ μ L Nt.BbvCI restriction endonuclease and 1U/
μ L exonucleaseⅢ, constant-temperature incubation after 10 × cutsmart buffer, the mixing of 5nM kanamycins standard solution；
(3) mixed solution in step (2) is added drop-wise on the gold electrode S1-S6 of modification various concentration CP, then 37 DEG C of constant temperature
It is incubated for 2h；
(4) 5 μ L haemachrome solutions (10 μM) are added drop-wise on gold electrode, then 37 DEG C of constant-temperature incubation 1h；
(5) with electrode 3 times in PBS buffer solution rinse step (4)；
(6) containing H2O2It is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode in the PBS buffer solution of (10 μM)
Curent change, 0 to -0.6 V of current potential, 0.05 V of pulse width, 0.06 s of sweep speed are measured with differential pulse voltammetry.
Using CP concentration as abscissa, using current value as ordinate, make the telecommunications of the gold electrode measurement of modification various concentration CP
Number curve graph, as shown in Figure 2.As shown in Figure 2, the current signal detected increases in 0-10 μM of section with the concentration of CP
And increase, after concentration is more than 10 μM, electric current tends towards stability.
Influence of the 3 difference HAP1 concentration of embodiment to detection kanamycins.
(1) Aptamer and Primer are hybridized to Probe(10 μM of probe in PBS buffer solution)；
(2) by final concentration of 1 μM of probe, HAP1(in step (1), 5 μM, 10 μM, 15 μM, 20 μM), 10 μM
HAP2,10 μM of HAP3,1U/ μ L Nt.BbvCI restriction endonuclease and 1U/ μ L exonucleaseⅢ, 10 ×cutsmartBuffer, 5nM card
In 37 DEG C of constant-temperature incubations after the mixing of that mycin standard solution；
(3) mixed solution in step (2) is added drop-wise on the gold electrode S4 of modification CP, then 37 DEG C of constant-temperature incubation 2h；
(4) 5 μ L haemachrome solutions (10 μM) are added drop-wise on step (3) resulting electrode, then 37 DEG C of constant-temperature incubation 1h；
(5) with electrode 3 times in PBS buffer solution rinse step (4)；
(6) containing H2O2It is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode in the PBS buffer solution of (10 μM)
Curent change, 0 to -0.6 V of current potential, 0.05 V of pulse width, 0.06 s of sweep speed are measured with differential pulse voltammetry.
Using HAP1 concentration as abscissa, using current value as ordinate, make the electricity containing various concentration HAP1 measurement in homogeneous
Signal curve figure, as shown in Figure 3.From the figure 3, it may be seen that the current signal detected is as the concentration of HAP1 is in 0-10 μM of section
Increase and increase, after concentration is more than 10 μM, electric current tends towards stability.
Influence of the 4 difference HAP2 concentration of embodiment to detection kanamycins.
(1) Aptamer and Primer are hybridized to Probe(10 μM in PBS buffer solution)；
(2) by final concentration of 1 μM of probe, 10 μM of HAP1, HAP2(in step (1), 5 μM, 10 μM, 15 μM, 20 μ
M), 10 μM of HAP3,1U/ μ LNt.BbvCIRestriction endonuclease and 1U/ μ L exonucleaseⅢ, 10 ×cutsmartBuffer, 5nM card that
In 37 DEG C of constant-temperature incubations after the mixing of mycin standard solution；
(3) mixed solution in step (2) is added drop-wise on the gold electrode S4 of modification CP, then 37 DEG C of constant-temperature incubation 2h；
(4) 5 μ L haemachrome solutions (10 μM) are added drop-wise on step (3) resulting electrode, then 37 DEG C of constant-temperature incubation 1h；
(5) with electrode 3 times in PBS buffer solution rinse step (4)；
(6) containing H2O2It is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode in the PBS buffer solution of (10 μM)
Curent change, 0 to -0.6 V of current potential, 0.05 V of pulse width, 0.06 s of sweep speed are measured with differential pulse voltammetry.
Using HAP2 concentration as abscissa, using current value as ordinate, make the electricity containing various concentration HAP2 measurement in homogeneous
Signal curve figure, as shown in Figure 3.From the figure 3, it may be seen that the current signal detected is as the concentration of HAP2 is in 0-10 μM of section
Increase and increase, after concentration is more than 10 μM, electric current tends towards stability.
Detection of the embodiment 5 to kanamycins
(1) by Aptamer(10 μM) and Primer(10 μM) Probe(10 μM is hybridized in PBS buffer solution)；
(2) by step (1) probe, HAP1(10 μM), HAP2(10 μM), HAP3(10 μM), 1U/ μ LNt.BbvCIIt is interior
Enzyme cutting and 1U/ μ L exonucleaseⅢ, 10 ×cutsmartBuffer, kanamycins solution (final concentration of 1,5,10,100,500,
1000,5000,10000 pM) or prepare liquid mixing after 37 DEG C of constant-temperature incubation 2h；
(3) mixed solution in step (2) is added drop-wise to together with exonuclease λ on the gold electrode S4 of modification HAP2, then
37 DEG C of constant-temperature incubation 2h；
(4) 5 μ L haemachrome solutions (10 μM) are added drop-wise in step (3) resulting solution, then 37 DEG C of constant-temperature incubation 1h；
(5) with electrode 3 times in PBS buffer solution rinse step (4)；
(6) containing H2O2It is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode in the PBS buffer solution of (10 μM)
Curent change, 0 to -0.6 V of current potential, 0.05 V of pulse width, 0.06 s of sweep speed are measured with differential pulse voltammetry；
(7) standard curve is made according to the electric signal of kanamycins standard solution, as shown in figure 5, calculating regression equation isI (10- 6A) = 0.38524 + 0.43326 lg(C/ pM), related coefficient 0.99402；Work as electric signalI (10-6When A)=1, according to
The concentration of kanamycins is 26pM in regression equation calculation prepare liquid.
Meanwhile we continue on the basis of the concentration of 1 pM to lower Concentration Testing, through detection when concentration is lower than 1 pM
When, the relationship of electric current and concentration is no longer complies with matched curve rule just, i.e., therefore the party can be obtained in the electric current minimum point in figure
The Monitoring lower-cut of method is 0.87 pM.
Sequence table
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Claims (9)
1. a kind of electrochemica biological sensor for detecting kanamycins characterized by comprising aptamers Aptamer, primer
Primer, hair fastener primer HAP1, hair fastener primer HAP2, hair fastener primer HAP3, ferroheme, PBS buffer solution, hydrogen peroxide H2O2、Nt.BbvCIRestriction endonuclease, exonucleaseⅢ, the gold electrode for modifying capture probe CP；
The PBS buffer solution contains K+；
The sequence of the Aptamer is as shown in SEQ No.1；
The sequence of Primer is as shown in SEQ No.2；
The sequence of HAP1 is as shown in SEQ No.3；
The sequence of HAP2 is as shown in SEQ No.4；
The sequence of HAP3 is as shown in SEQ No.5；
The sequence of CP such as SEQ No.6；
5 ' terminal modified-SH of the shown capture probe CP sequence.
2. electrochemica biological sensor according to claim 1, which is characterized in that the gold of the modification capture probe CP
Electrode is prepared using following methods:
A) gold electrode is successively carried out being polished to mirror surface in 0.3 μm and 0.05 μm of oxidation aluminium paste, is rushed repeatedly with ultrapure water
It washes；
B) gold electrode surfaces by CP solution drop after the treatment, heat preservation are incubated for.
3. electrochemica biological sensor according to claim 1, which is characterized in that the HAP1 concentration is 1-20 μM；Institute
Stating HAP2 concentration is 1-20 μM；The HAP3 concentration is 1-20 μM；The concentration of the CP is 1-20 μM.
4. electrochemica biological sensor according to claim 1, which is characterized in that the preferred final concentration of Aptamer
It is 1 μM；The Primer is final concentration of 1 μM preferred；The ferroheme is final concentration of 10 μM preferred；The H2O2Preferably
Final concentration of 10 μM；It is describedNt.BbvCIRestriction endonuclease and the preferred concentration of exonucleaseⅢ are 500U/mL.
5. a kind of preparation method of electrochemica biological sensor described in claim 1, which comprises the following steps:
(1) Aptamer and Primer are hybridized to probe Probe in PBS buffer solution；
(2) by probe Probe, HAP1, HAP2, HAP3,Nt.BbvCIRestriction endonuclease, exonucleaseⅢ, 10 ×Nt.BbvCIRestriction endonuclease
With constant-temperature incubation after exonucleaseⅢ buffer, the mixing of kanamycins solution；
(3) mixed solution in step (2) is added drop-wise on the gold electrode of modification capture probe CP, then constant-temperature incubation；
(4) haemachrome solution is added drop-wise in step (3) resulting solution, then constant-temperature incubation；
(5) with the electrode in PBS buffer solution rinse step (4)；
(6) H is added into PBS buffer solution2O2, it is uniformly mixed, is to be adopted to electrode with Pt electrode using Ag/AgCl as reference electrode
Change in electric is measured with differential pulse voltammetry；
(7) standard curve is made according to the electric signal of kanamycins standard solution, calculates regression equation, according to regression equation with it is to be measured
Liquid electric signal calculates the concentration of kanamycins in prepare liquid.
6. preparation method according to claim 5, which is characterized in that the step (1) specifically comprises the processes of: 14 μ L go out
Bacterium water, 25 × PBS of μ L, 10 μM of Primer chains of 2 10 μM of μ L Aptamer chains and 2 μ L are mixed, are incubated at 95 DEG C
5min is slowly cooled to room temperature, and is stored in spare at -20 DEG C.
7. preparation method according to claim 5, which is characterized in that constant-temperature incubation temperature is 37 DEG C in the step (2).
8. preparation method according to claim 5, which is characterized in that the ginseng of measurement change in electric in the step (6)
Number are as follows: 0 to -0.6 V of current potential, 0.05 V of pulse width, 0.06 s of sweep speed.
9. claim 1 or the electrochemica biological sensor using the preparation method of claim 5 are used in Food Safety Analysis
Kanamycins is detected with environmental monitoring aspect.
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| CN111474224B (en) * | 2020-04-24 | 2022-04-22 | 山东大学齐鲁医院 | Renewable electrochemical sensor for detecting trace kanamycin and preparation method and application thereof |
| CN112378976A (en) * | 2020-11-12 | 2021-02-19 | 济南大学 | Electrochemical aptamer sensor for ampicillin detection |
| CN112378976B (en) * | 2020-11-12 | 2022-07-29 | 济南大学 | Electrochemical aptamer sensor for ampicillin detection |
| CN113308520A (en) * | 2021-06-23 | 2021-08-27 | 中国地质大学（武汉） | Method and kit for detecting concentration of detection object based on wettability sliding analysis |
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