CN105784810B - A kind of non-marked type aptamer sensor and the detection method to bisphenol-A - Google Patents
A kind of non-marked type aptamer sensor and the detection method to bisphenol-A Download PDFInfo
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Abstract
A kind of non-marked type aptamer sensor and the detection method to bisphenol-A, base material of the method for the invention using the porous nano-Au of bigger serface as immobilized nucleic acids aptamers, the complementary dna chain for being hybridized load aptamer forms double-stranded DNA, electrochemical probe methylene blue is finally embedded in, constructs measure bisphenol-A non-marked type aptamer electrochemical sensor.The sensitization of porous nano-Au of the present invention, the indicative function of methylene blue electrochemical probe, can with it is sensitive, accurate, specifically detect bisphenol-A.The present invention only needs to change aptamer and complementary dna chain, it is possible to achieve the detection to other substrates.The present invention is applied to electrochemical nucleic acid aptamer sensor method measure bisphenol-A.
Description
Technical field
The present invention relates to a kind of non-marked type aptamer sensor and the detection method to bisphenol-A, the invention belongs to
Chemical sensitisation and technical field of electroanalytical chemistry detection.
Background technology
Bisphenol-A is one of environmental estrogens class compound, can enter in organism and imitate, hinders, disturbs or change
Organism autologous hormones act on, and cause body endocrine system disorder, hinder nervous system transmission, reduce body's immunity,
Even result in malformation and canceration, the health of serious threat human body and existence.Therefore, tool is fast and accurately detected to bisphenol-A
There is important realistic meaning.
Aptamer is in vitro by exponential enrichment Fas lignand system evolution technology filter out one section by 20 ~ 60
The single-stranded or double-stranded oligonucleotide that base is formed, there is high-affinity, be easy to modification and functionalization, can with part efficiently,
The features such as single-minded combination.Electrochemical nucleic acid aptamer sensor based on aptamer, according to whether being produced using label
Detection signal, marking type and non-marked type can be divided into；The labeling process of marking type aptamer sensor is complicated, it is high to spend,
And the binding affinity of aptamers and target molecule can be influenceed to a certain extent.Therefore, build simple, inexpensive and sensitive
Non-marked type electrochemical nucleic acid aptamer sensor has great importance.
Porous metals nano material has bigger specific surface area than common metal nano material, can preferably improve molecule
Fixed efficiency.Porous nano-Au is Typical Representative therein, and it has big specific surface area, good biocompatibility and conduction
Property, the performance of sensor can effectively be strengthened by using it for the preparation of sensor.
The content of the invention
The object of the present invention is to the problem of being existed in use according to marking type aptamer sensor, the present invention carries
For a kind of base material using porous nano-Au as immobilized nucleic acids aptamers, using the methylene blue being embedded in double-stranded DNA as electrochemistry
A kind of non-marked type aptamer sensor and the detection method to bisphenol-A prepared by probe.
Realize the technical scheme is that, the present invention using electrodeposition process prepare porous nano-Au fitted as immobilized nucleic acids
The base material of part, bigger serface and good biocompatibility using porous nano-Au are steady by substantial amounts of aptamer
Surely it is supported on electrode surface.Double-stranded DNA is caused to unwind using specific binding effect of the aptamer to bisphenol-A, so as to
Make the signal of the methylene blue electrochemical probe in double-stranded DNA reduce, using aptamer modified electrode as working electrode, ginseng
It is Ag/AgCl electrodes than electrode, auxiliary electrode is platinum electrode, forms three-electrode system, realizes the highly sensitive detection to bisphenol-A.
The present invention includes the preparation method of non-marked type aptamer sensor and the nucleic acid using the measure bisphenol-A
Detection method of the aptamer sensor to bisphenol-A.
A kind of preparation method step of non-marked type aptamer sensor of the present invention is as follows：
（1）Clean glass-carbon electrode is placed in the HAuCl containing 0.04 ~ 0.12 mol/L4With 0.002 ~ 0.006mol/L
Pb (CH3COO)2In solution, in the s of -0.2 ~ -0.7 V constant potentials electro-deposition 20 ~ 120, the glass that porous nano-Au is modified is made
Carbon electrode.
（2）Porous nano-Au modified electrode is sequentially placed into 6 in the bisphenol-A aptamer containing 0.3 ~ 1.5 μm of ol/L
20~80 min in~24 h, 0.5 ~ 1.5 mmol/L 6- sulfydryl hexanols, 20 in 0.3 ~ 1.5 μm of ol/L complementary DNA~
80 min, the glass-carbon electrode of double-stranded DNA/porous nano-Au modification is made.
（3）The methylene blue that the glass-carbon electrode of double-stranded DNA/porous nano-Au modification is placed in 0.1~0.4 mmol/L is molten
5~25min is soaked in liquid, using the electrostatic interaction between methylene blue and double-stranded DNA, is embedded into double-stranded DNA, obtains Asia
The glass-carbon electrode of methyl blue/double-stranded DNA/porous nano-Au modification, the modified electrode are that the aptamer for determining bisphenol-A passes
Sensor.
The present invention is as follows to the detection method of bisphenol-A based on non-marked type aptamer sensor：
After the present invention is specifically bound using the bisphenol-A aptamer in aptamer sensor and bisphenol-A,
Cause double-stranded DNA to unwind and make to be embedded in the reduction of the methylene blue concentration in double-stranded DNA, be i.e. the electric current of electrochemical probe methylene blue
Signal reduces, and realizes the detection to bisphenol-A, is working electrode by foregoing aptamer sensor, reference electrode Ag/
AgCl electrodes, auxiliary electrode are platinum electrode, form three-electrode system, you can realize the detection to bisphenol-A.
The range of linearity of aptamer sensor of the present invention measure bisphenol-A is the ng/mL of 100.0 fg/mL ~ 100.0,
Detection is limited to 32.9 fg/mL.Four aptamer sensors are prepared using same root glass-carbon electrode, determine it to bisphenol-A
Response current, its relative standard deviation is 2.0%, using the aptamer sensor of the parallel preparation of 4 glass-carbon electrodes to bis-phenol
The relative standard deviation of A measure is 3.3%, illustrates that the electrode has good reappearance.The sensor is placed in 4 DEG C of environment
Its stability is investigated, after one week, still retains more than the 90% of response current value, shows that the electrode has good stability.
The operation principle of the present invention is the electrode surface that the present invention modifies in porous nano-Au, will be adapted to by golden sulfide linkage
Body is fixed, then complementary dna chain and aptamers are combined to form into double-stranded DNA, finally will be embedding as the methylene blue of electrochemical probe
Enter in double-stranded DNA, establish a kind of non-marked type aptamer sensor detection bisphenol-A of substrate for induction displacement complementary dna chain
Method.
The invention has the advantages that the double-strand that the present invention is formed by the glassy carbon electrode surface modified in porous nano-Au
Embedded methylene blue prepares aptamer sensor in DNA, due to the signal amplification and methylene blue of porous nano-Au
Electrochemical probe effect, there is provided non-marked type aptamer sensor that is a kind of simple and delicately detecting bisphenol-A
Method.
The present invention is applied to non-marked type aptamer sensor measure bisphenol-A.
Brief description of the drawings
Fig. 1 is that porous nano-Au of the present invention and methylene blue electrochemical probe prepare aptamer sensor flow chart element
Figure；
Fig. 2 is the scanning electron microscope (SEM) photograph of the glass-carbon electrode that porous nano-Au is modified in the present invention；
Fig. 3 is the cyclic voltammogram of the different modifying electrode in pH 7.0 PBS solution,（a）Methylene blue/double-stranded DNA/
Porous nano-Au/glass-carbon electrode,（b）Bisphenol-A of the methylene blue/double-stranded DNA/porous nano-Au/glass-carbon electrode in 1.0 ng/mL
After middle incubation；
Fig. 4 is differential pulse of the methylene blue/double-stranded DNA/porous nano-Au/glass-carbon electrode to the bisphenol-A of various concentrations
Volt-ampere response diagram；
Fig. 5 is calibration curve of the current-responsive to bisphenol A concentration of aptamer mark sensor.
Embodiment
Below by specific embodiment, the present invention is described in detail, and following examples contribute to those skilled in the art
Member further understands the present invention, but is never limited in protection scope of the present invention.
Embodiment 1
Aptamer sensor is prepared based on porous nano-Au and methylene blue electrochemical probe, as shown in Figure 1.
（1）Prepare 0.04 mol/L HAuCl4With 0.002 mol/L Pb (CH3COO)2Solution, by clean glass carbon electricity
Pole is placed in above-mentioned solution, and in the s of -0.2 V constant potentials electro-deposition 120, the glass-carbon electrode that porous nano-Au is modified is made.
（2）Porous nano-Au modified electrode is sequentially placed into 24 h in 0.3 μm of ol/L bisphenol-A aptamers, 0.5
80 min in mmol/L 6- sulfydryl hexanols, 80 min in 0.3 μm of ol/L complementary DNA, double-stranded DNA/porous nano-Au is made
The glass-carbon electrode of modification.
（3）The glass-carbon electrode of double-stranded DNA/porous nano-Au modification is placed in 0.1 mmol/L methylene blue solution and soaked
25 min are steeped, obtain the glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification, the modified electrode is to determine bis-phenol
A aptamer sensor.
Embodiment 2
Aptamer sensor is prepared based on porous nano-Au and methylene blue electrochemical probe, as shown in Figure 1.
（1）Prepare 0.08 mol/L HAuCl4With 0.004 mol/L Pb (CH3COO)2Solution, by clean glass carbon electricity
Pole is placed in above-mentioned solution, and in the s of -0.5 V constant potentials electro-deposition 70, the glass-carbon electrode that porous nano-Au is modified is made.
（2）Porous nano-Au modified electrode is sequentially placed into 12 h in 1.0 μm of ol/L bisphenol-A aptamers, 1.0
60 min in mmol/L 6- sulfydryl hexanols, 60 min in 1.0 μm of ol/L complementary DNA, double-stranded DNA/porous nano-Au is made
The glass-carbon electrode of modification.
（3）The glass-carbon electrode of double-stranded DNA/porous nano-Au modification is placed in 0.2 mmol/L methylene blue solution and soaked
10 min are steeped, obtain the glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification, the modified electrode is to determine bis-phenol
A aptamer sensor.
Embodiment 3
Aptamer sensor is prepared based on porous nano-Au and methylene blue electrochemical probe, as shown in Figure 1.
（1）Prepare 0.12 mol/L HAuCl4With 0.006 mol/L Pb (CH3COO)2Solution, by clean glass carbon electricity
Pole is placed in above-mentioned solution, and in the s of -0.7 V constant potentials electro-deposition 20, the glass-carbon electrode that porous nano-Au is modified is made.
（2）Porous nano-Au modified electrode is sequentially placed into 6 h, 1.5 mmol/ in 1.5 μm of ol/L bisphenol-A aptamers
20 min in L 6- sulfydryl hexanols, 20 min in 1.5 μm of ol/L complementary DNA, double-stranded DNA/porous nano-Au modification is made
Glass-carbon electrode.
（3）The glass-carbon electrode of double-stranded DNA/porous nano-Au modification is placed in 0.4 mmol/L methylene blue solution and soaked
5 min are steeped, obtain the glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification, the modified electrode is to determine bisphenol-A
Aptamer sensor.
Embodiment 4
The glass-carbon electrode that the porous nano-Au that embodiment 2 obtains is modified is scanned into Electronic Speculum to characterize.
The ESEM of the glass-carbon electrode of porous nano-Au modification, which characterizes, sees Fig. 2, can be in obvious branch with porous gold from Fig. 2
Shape, and arrange densification in electrode surface.
Embodiment 5
The aptamer sensor that embodiment 2 is obtained is used for electro-chemical test：
（1）The cyclic voltammetry of different modifying electrode.
Respectively by the glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification and in 1.0 ng/mL bisphenol-A
The glass-carbon electrode of methylene blue/double-stranded DNA after incubation/porous nano-Au modification is working electrode, reference electrode Ag/AgCl
Electrode, auxiliary electrode are platinum electrode；Bottom liquid is 0.2 mol/L PBS（pH 7.0）；Sweep speed is 0.1 V/s.Cyclic voltammetric
Figure is shown in Fig. 3, it can be seen from figure 3 that methylene blue/double-stranded DNA/glass-carbon electrode of porous nano-Au modification, there is a pair of methylene blues
Redox peaks；The glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification is incubated in 1.0 ng/mL bisphenol-A
After educating, bisphenol-A is combined with aptamer makes double-stranded DNA unwind, and the methylene blue concentration in embedded double-stranded DNA also accordingly drops
It is low, therefore the redox peaks of methylene blue diminish.
（2）Aptamer sensor is tested the differential pulse voltammetry of bisphenol-A.It is work by aptamer sensor
Electrode, reference electrode are Ag/AgCl electrodes, and auxiliary electrode is platinum electrode；Bottom liquid is 0.2 mol/L PBS（pH 7.0）；Scanning
The V of potential range 0.08~-0.52；Aptamer sensor is placed in differential pulse test bottom liquid, scanning obtains blank electricity
StreamI 0, then aptamer sensor is placed in certain density bisphenol-A solution after incubation again, then scan to obtain electric currentI,
Then the response current of sensor is ΔI= I 0 - I, measurement result is shown in Fig. 4 and Fig. 5.The range of linearity for determining bisphenol-A is 100.0
The ng/mL of fg/mL ~ 100.0, detection are limited to 32.9 fg/mL.The sensor is placed in 4 DEG C of environment, after one week, is still retained
More than the 90% of response current value.
Claims (3)
1. a kind of preparation method of non-marked type aptamer sensor, it is characterised in that methods described step is：
(1) clean glass-carbon electrode is placed in the HAuCl containing 0.04~0.12mol/L4With 0.002~0.006mol/L Pb
(CH3COO)2It is electric in -0.2~-0.7V constant potential 20~120s of electro-deposition, the glass carbon that porous nano-Au modification is made in solution
Pole；
(2) the porous nano-Au modified electrode that (1) obtains is sequentially placed into the bisphenol-A nucleic acid containing 0.3~1.5 μm of ol/L to be adapted to
20~80min in 6~24h in body, 0.5~1.5mmol/L 6- sulfydryl hexanols, 20 in 0.3~1.5 μm of ol/L complementary DNA
~80min, the glass-carbon electrode of double-stranded DNA/porous nano-Au modification is made；
(3) glass-carbon electrode of the double-stranded DNA for obtaining (2)/porous nano-Au modification is placed in 0.1~0.4mmol/L methylene
5~25min is soaked in blue solution, using the electrostatic interaction between methylene blue and double-stranded DNA, is embedded into double-stranded DNA, obtains
To the glass-carbon electrode of methylene blue/double-stranded DNA/porous nano-Au modification, the modified electrode is the nucleic acid adaptation for determining bisphenol-A
Body sensor.
2. the detection of non-marked type aptamer sensor prepared by preparation method according to claim 1 to bisphenol-A
Method, it is characterised in that methods described is as follows：
Acted on using the specific binding to bisphenol-A of signal amplification and aptamers of the porous nano-Au of bigger serface,
The concentration for the methylene blue for causing to be embedded in double-stranded DNA reduces so that the signal of methylene blue reduces, using electrochemistry difference
Pulse voltammetry is used to detect bisphenol-A, is working electrode by aptamer modified electrode, and reference electrode is Ag/AgCl electricity
Pole, auxiliary electrode are platinum electrode, form three-electrode system, you can realize the detection to bisphenol-A.
3. a kind of non-marked type aptamer sensor exists to the detection method of bisphenol-A, its feature according to claim 2
The range of linearity for being used to detecting bisphenol-A in, the aptamer sensor is 100.0fg/mL~100.0ng/mL, test limit
For 32.9fg/mL.
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| CN106841339A (en) * | 2017-02-03 | 2017-06-13 | 吉林大学 | A kind of aptamer sensor for detecting bisphenol-A and preparation method thereof |
| CN108226251B (en) * | 2018-01-17 | 2020-01-21 | 中国检验检疫科学研究院 | Preparation method of disposable bisphenol A aptamer biosensor |
| CN110044985A (en) * | 2019-05-21 | 2019-07-23 | 西南石油大学 | A kind of electrochemical probe and preparation method thereof of bisphenol-A detection |
| CN113295739A (en) * | 2021-04-19 | 2021-08-24 | 济南大学 | Based on hollow Fe2O3The dual-mode electrochemical aptamer sensor and the method for determining acetamiprid |
| CN113406160B (en) * | 2021-05-27 | 2023-10-03 | 山东师范大学 | Photoelectrochemical biosensor and application thereof in methyltransferase activity detection |
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| CN103760332A (en) * | 2014-01-16 | 2014-04-30 | 江南大学 | Method for detecting bisphenol A by utilizing aptamer-based chiral sensor |
| CN104749233A (en) * | 2015-03-31 | 2015-07-01 | 山东大学 | Bisphenol A sensor of nano-porous gold modified based on aptamer and method |
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| CN103760332A (en) * | 2014-01-16 | 2014-04-30 | 江南大学 | Method for detecting bisphenol A by utilizing aptamer-based chiral sensor |
| CN104749233A (en) * | 2015-03-31 | 2015-07-01 | 山东大学 | Bisphenol A sensor of nano-porous gold modified based on aptamer and method |
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