CN109613095A - Terminal enzyme (DNA) electrochemica biological sensor preparation method and application based on i-motif change of configuration - Google Patents
Terminal enzyme (DNA) electrochemica biological sensor preparation method and application based on i-motif change of configuration Download PDFInfo
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- CN109613095A CN109613095A CN201910096693.1A CN201910096693A CN109613095A CN 109613095 A CN109613095 A CN 109613095A CN 201910096693 A CN201910096693 A CN 201910096693A CN 109613095 A CN109613095 A CN 109613095A
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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
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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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
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses the terminal enzyme (DNA) electrochemica biological sensor preparation method and applications based on i-motif change of configuration, specific step is as follows: sulfydryl DNA probe being fixed to the naked gold electrode handled well first, mercaptoethanol processing is added dropwise then to replace the fixed capture probe of the non-Au-S key of electrode surface.Then to the TdT reaction solution that preparation is added dropwise on electrode, 1h is placed at 37 DEG C.After rinsing electrode, silver ion solution is added dropwise in electrode surface, aldrich mixture is added dropwise again after being incubated at room temperature a period of time, incubation at room temperature a period of time obtains sensor, detects its electrochemical response in PBS (0.1M, pH7.0) electrolyte solution.Advantage is good specificity, high sensitivity, detection speed is fast, result is accurate and reliable, at low cost.
Description
Technical field
The present invention relates to a kind of electrochemica biological sensor and its detection methods, more particularly, to based on i-motif configuration
The terminal enzyme (DNA) electrochemica biological sensor preparation method and application of variation, belongs to functional biological material and biosensor technique
Field.
Background technique
Deoxynucleotide terminal enzyme (DNA) (TdT) be one kind be not required to template can catalytic deoxidation nucleotide be integrated to DNA
The archaeal dna polymerase of 3 '-OH of molecule.Correlative study shows that activity of the TdT in biosystem changes as histocyte is abnormal,
It can be by TdT content in detection specific organization's cell to which certain diseases are diagnosed and be treated.Traditional TdT detection method
Rely primarily on gel electrophoresis analysis, but gel electrophoresis analysis operating process is complicated, time-consuming, somewhat expensive, reproducibility are poor, and
The result of sxemiquantitative can only be provided.In recent years, some bio-sensing systems have been emerged in large numbers for TdT Activity determination, up to the present
Still possess biggish research space and meaning.Therefore, a kind of TdT detection easy, sensitive, inexpensive, selectivity is good is developed
Means are imperative.
DNA molecular rich in cytimidine (C) passes through the C base and non-protonated C base of protonation in acid condition
Form C-CH+Base-pair, the C-CH of antiparallel arrangements+Base-pair, which is alternately arranged, forms stable parallel double spiral shells with insertion mutually
Chain is revolved, two double-strands are because hydrogen bond forms four helical structures, i.e. i-motif structure.Under room temperature and neutrallty condition, silver ion
(Ag (I)) is embedded in C base and forms C-Ag (I)-C compound, and induction richness C DNA molecular forms i-motif structure.Research shows that
When chemotherapy, controlling gene transcription, i-motif is a kind of attractive pharmaceutical target molecule, and is had in nanotechnology
Very big application potential, i-motif structure cause the great interest of researcher.Currently, it has not been found that being based on i-motif configuration
The TdT bio-sensing architectural study of variation has biggish application prospect.
Terminal enzyme (DNA) electrochemica biological sensor preparation method is devised the present invention is based on i-motif change of configuration and is answered
With the sensor has enzyme effect using TdTT, and cytosine triphosphate (dCTP) is introduced the 3 ' end-OH of DNA primer, is formed rich
C long-chain, insertion Ag (I) form the compound-mediated i-motif structure of C-Ag (I)-C afterwards.Gentian violet (GV) is a kind of triphenyl
Methane class dyestuff can be stacked to i-motif body structure surface and generate electrochemical signals, and the present invention is adjusted by TdT activity change
The length of rich C chain, influences i-motif and is formed, and then influences the amount of insertion GV, realizes that TdT is living using electrochemical signals change
Property detection and its inhibitor screening.
Summary of the invention
Good, high sensitivity that technical problem to be solved by the invention is to provide a species specificity, detection speed is fast, result is quasi-
The true terminal enzyme (DNA) electrochemica biological sensor preparation method and application reliable, at low cost based on i-motif change of configuration.
The technical scheme of the invention to solve the technical problem is: the end based on i-motif change of configuration turns
Move enzyme electrochemica biological sensor preparation method and application, the specific steps are as follows:
(1) prepared by sensor
The preparation of a.Electrode 1
The sulfydryl DNA probe solution that 2.5~7.5 μ L concentration are 0.1~1 μM is added drop-wise to clean and activated naked gold electricity
Pole (being labeled as Au) surface, 4 DEG C of refrigerators place 8~16h, then handle 20~60min with 0.1~1.0mM mercaptoethanol (MCH),
The fixed capture probe of the non-Au-S key of electrode surface is replaced by the Au-S covalent bond of MCH and the surface Au, distilled water is slowly
Electrode is rinsed, Electrode 1 is labeled as.
The preparation of b.Electrode 2
The TdT reaction solution that total volume is 5~15 μ L is prepared, wherein containing 2~6 μ L distilled water, 0.5~1.5 5 × TdT of μ L
Buffer, the TdT that the dCTP and 0.5~1.5 μ L concentration that 0.5~1.5 μ L concentration is 5~15mM are 5~15U/mL are uniformly mixed
Afterwards, drop places 0.5~1.5h in 1 surface Electrode at 27~42 DEG C, and distilled water slowly rinses electrode, is labeled as
Electrode 2。
The preparation of c.Electrode 3
It is 0.5~1.5 μM of AgNO by 2.5~7.5 μ L concentration3Solution is added drop-wise to 2 surface Electrode, incubation at room temperature
15~30min, distilled water slowly rinse electrode, are labeled as Electrode 3.
The preparation of 4 sensor of d.Electrode
It is that 0.5~1.5mM GV solution is added drop-wise to 3 surface Electrode by 2.5~7.5 μ L concentration, room temperature reaction 15~
30min, distilled water slowly rinse electrode, be labeled as Electrode 4, be placed on phosphate buffer solution (PBS, 0.1M, pH
7.0) electrochemical response is detected in.
(2) TdT Activity determination
In the preparation process of Electrode 2, change TdT concentration, prepared for Electrode 2, then such as step
(1) a series of sensors are prepared, for detecting the electrochemical response of various concentration TdT.
The sulfydryl DNA probe sequence that the present invention uses is (5 ' -3 '): SH-TGT AGT AGC.
Using the above-mentioned terminal enzyme (DNA) electrochemica biological sensor based on i-motif change of configuration, pass through square wave volt-ampere
Method, setting potential range are -0.6~-0.78V, amplitude 25mV, electrification of the detection sensor in PBS (0.1M, pH 7.0)
Response is learned, a series of corresponding peak current size of various concentration TdT is obtained, establishes the quantitative relationship between current-responsive and TdT,
According to quantitative relationship between the two, TdT content in sample to be tested is determined.
Inventive principle: the present invention is a kind of novel electrochemical Biosensors, under the conditions of existing for the substrate dCTP, TdT
3 ' the ends-OH on catalysis sulfydryl DNA is single-stranded carry out extension polymerization, obtain rich C DNA long-chain, and the long-chain and Ag (I) are logical
It crosses C-Ag (I)-C effect combination and forms i-motif structure, i-motif body structure surface is stacked to by GV and generates electrochemical signals,
Construct a kind of simple, quick, highly sensitive, high selection, label-free TdT activity assays.
Compared with the prior art, the advantages of the present invention are as follows: the present invention constructs the end based on i-motif change of configuration
Transferase electrochemica biological sensor preparation method and application.It is tied firstly, passing through Au-S with Au using the sulfydryl on single stranded DNA
It closes, the 3 ' ends-OH of TdT catalytic dna carry out polymerization extension, prepare richness C DNA long-chain, utilize the Ag for causing i-motif to be formed
(I) and it is stacked to the GV on the surface i-motif, sensor is successfully prepared, by square wave voltammetry detection sensor to various concentration
The electrochemical response of TdT.Obviously, in concentration a certain range, target concentration is bigger, and current-responsive is more obvious.Experimental result
Show that size of current is in a linear relationship in a certain range with target concentration, realizes the detection to object.Its advantage exists
In:
(1) highly sensitive.Present invention experiment show that the current-responsive of sensor is y to the linearly related equation of TdT concentration
=3.49lgCTdT+ 11.31, R2=0.9982, detection is limited to 0.0005U/mL, thus illustrates that sensor can realize high spirit to TdT
Quick detection.
(2) high specific.Other common enzymes such as pyrophosphatase, acetylcholinesterase, choline oxidase, histone acetyl
It is noiseless to TdT Activity determination system to change enzyme, protein kinase or alkaline phosphatase.
(3) result is accurate.The rate of recovery is between 90%~110%.
(4) sensor can be used for the screening of micromolecular inhibitor, such as sodium pyrophosphate, IC50For 0.192mM.
(5) it prepares few, at low cost with detection method reagent dosage.The present invention need to only consume a small amount of material and reagent can be real
Now to the highly sensitive high specific detection of TdT.
In conclusion the present invention is detection of the electrochemica biological sensor based on i-motif change of configuration to TdT, tool
Have the advantages that high sensitivity, selectivity it is good, easy to operate, analysis it is quick, easily operated, it can be achieved that low concentration TdT detection
And its screening of micromolecular inhibitor, it has a good application prospect.
Detailed description of the invention
Fig. 1 is the feasibility Experiment figure of inventive sensor；
Fig. 2 is inventive sensor to whether there is or not the electrochemical response figures of TdT；
Fig. 3 is electrochemical response figure of the inventive sensor to various concentration TdT；
Fig. 4 is selective lab diagram of the inventive sensor to TdT；
Fig. 5 is school of the inventive sensor to various concentration sodium pyrophosphate to the current-responsive of TdT inhibiting effect to concentration
Directrix curve figure.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
The preparation of 1 sensor of embodiment
The preparation of a.Electrode 1
The sulfydryl DNA probe solution that 5 μ L concentration are 0.1 μM is added drop-wise to clean and activated naked gold electrode (to be labeled as
Au) surface, 4 DEG C of refrigerators place 12h, then handle 30min with 1.0mM mercaptoethanol (MCH), pass through the Au-S of MCH and the surface Au
Covalent bond and replace the fixed capture probe of the non-Au-S key of electrode surface, distilled water slowly rinses electrode, is labeled as
Electrode 1。
The preparation of b.Electrode 2
The TdT reaction solution that total volume is 10 μ L is prepared, wherein containing 6 μ L distilled water, 1 μ L 5 × TdT buffer, 1 μ L is dense
The TdT that the dCTP and 1 μ L concentration that degree is 10mM are 10U/mL, after evenly mixing, drop are transferred in the surface Electrode1 at 37 DEG C
1h is set, distilled water slowly rinses electrode, is labeled as Electrode 2.
The preparation of c.Electrode 3
It is 1 μM of AgNO by 5 μ L concentration3Solution is added drop-wise to 2 surface Electrode, is incubated at room temperature 15min, and distilled water is slow
Buffering washes electrode, is labeled as Electrode 3.
The preparation of 4 sensor of d.Electrode
It is that 1mM GV solution is added drop-wise to 3 surface Electrode by 5 μ L concentration, reacts at room temperature 15min, distilled water slowly rushes
Wash electrode, be labeled as Electrode 4, the detection electrochemistry in phosphate buffer solution (PBS, 0.1M, pH 7.0) that is placed on ring
It answers.Electrochemical response of the 1~Electrode of the above Electrode 4 and Au in PBS (0.1M, pH 7.0) is detected,
See Fig. 1.It can be seen that Electrode 4 is compared to other electrodes, electrochemical response is it is obvious that prove our sensor assembling
Success, and electrochemical properties can preferably be presented.
2 feasibility Experiment of embodiment
In order to prove that the detection to TdT may be implemented in inventive sensor, our bio-sensing is prepared based on embodiment 1
Device.See Fig. 2, when having TdT, sensor electrochemical response in PBS (0.1M, pH7.0) is obvious, and without TdT in the presence of, substantially without
Electrochemical response, it was demonstrated that the sensor can be used for TdT Activity determination.
3 sensor of embodiment is used for TdT Activity determination
Electrochemica biological sensor is prepared based on embodiment 1, when preparing Electrode 2, in TdT reaction solution, is changed
The concentration of TdT, final concentration is successively are as follows: and 0,0.001,0.002,0.005,0.01,0.02,0.05,0.1,0.2,0.5,1,2,5,
10,15U/mL, it is uniformly mixed, drop obtains different Electrode 2 in 1 surface Electrode, places 1h at 37 DEG C.It steams
Distilled water slowly rinses electrode, is then prepared into Electrode 4, detection various concentration TdT preparation gained according to 1 step of embodiment
Electrochemical response of the sensor in PBS (0.1M, pH 7.0) illustrates to increase with TdT concentration, sensing experimental result is shown in Fig. 3
The electrochemical response of device is more obvious, and peak current is in good linear relationship to the logarithm of TdT concentration, and linearly related equation is y=
3.49lgCTdT+ 11.31, R2=0.9982, the range of linearity is 0.001~5U/mL, and detection is limited to 0.0005U/mL, illustrates out
Highly sensitive detection can be achieved to TdT activity in the sensor of hair.
Specific detection of 4 sensor of embodiment to TdT
The concentration of TdT and other enzymes is 1U/mL in selectivity experiment, and the abbreviation of other used enzymes is as follows: burnt phosphorus
Sour enzyme (PPase), acetylcholinesterase (AChE), choline oxidase (ChOx), acetylation of histone enzyme (HAT), protein kinase
(PKA), alkaline phosphatase (ALP).By the sensor preparation step of above-described embodiment 1, when preparing Electrode2, with other phases
Enzyme with concentration replaces TdT, and then Electrode 4 is prepared.As a result as shown in figure 4, being compared with TdT, sensor is to it
His electrochemical response of enzyme is very small, substantially close to blank signal, illustrates that sensor shows preferable selection to the detection of TdT
Property.
The inhibitory effect of 5 TdT inhibitor sodium pyrophosphate (PP) of embodiment detects
By the sensor preparation step of above-described embodiment 1, when preparing Electrode 2, in TdT reaction solution not with addition
With the sodium pyrophosphate (PP) of concentration, concentration is successively are as follows: and 0,0.01,0.02,0.05,0.08,0.1,0.2,0.5,0.8,1,2,5,
8mM, and then Electrode4 is prepared.As a result as shown in figure 5, obtaining the electric current of sensor and the relationship of PP log concentration, meter
Calculate PP to the 503nhibiting concentration IC of TdT50It is good to illustrate that PP has TdT activity substantially close to blank signal for 0.192mM
Inhibitory effect.
Certainly, above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art
The variations, modifications, additions or substitutions that those of ordinary skill makes within the essential scope of the present invention also should belong to protection of the present invention
Range.
Claims (4)
1. the terminal enzyme (DNA) electrochemica biological sensor preparation method and application based on i-motif change of configuration, feature exist
In mechanism is as follows: using sulfydryl and the Au on single stranded DNA by Au-S in conjunction with, the 3 ' ends-OH of TdT catalytic dna are polymerize
Extend, prepare richness C DNA long-chain, using the Ag (I) for causing i-motif to be formed and the GV for being stacked to the surface i-motif, successfully makes
The concentration variation of standby sensor, TdT will affect electrochemical response.
2. the terminal enzyme (DNA) electrochemica biological sensor preparation method and application based on i-motif change of configuration, specific steps
It is as follows:
(1) prepared by sensor
The preparation of a.Electrode 1
The sulfydryl DNA probe solution that 2.5~7.5 μ L concentration are 0.1~1 μM is added drop-wise to clean and activated naked gold electrode
(being labeled as Au) surface, 4 DEG C of refrigerators place 8~16h, then handle 20~60min with 0.1~1.0mM mercaptoethanol (MCH), lead to
It crosses the Au-S covalent bond of MCH and the surface Au and replaces the fixed capture probe of the non-Au-S key of electrode surface, distilled water slowly rushes
Electrode is washed, Electrode 1 is labeled as.
The preparation of b.Electrode 2
The TdT reaction solution that total volume is 5~15 μ L is prepared, wherein contain 2~6 μ L distilled water, 0.5~1.5 5 × TdT of μ L buffering
Liquid, the TdT that the dCTP and 0.5~1.5 μ L concentration that 0.5~1.5 μ L concentration is 5~15mM are 5~15U/mL, after evenly mixing,
It drips in 1 surface Electrode, 0.5~1.5h is placed at 27~42 DEG C, distilled water slowly rinses electrode, is labeled as
Electrode 2。
The preparation of c.Electrode 3
It is 0.5~1.5 μM of AgNO by 2.5~7.5 μ L concentration3Solution is added drop-wise to 2 surface Electrode, and incubation at room temperature 15~
30min, distilled water slowly rinse electrode, are labeled as Electrode 3.
The preparation of 4 sensor of d.Electrode
It is that 0.5~1.5mM GV solution is added drop-wise to 3 surface Electrode by 2.5~7.5 μ L concentration, room temperature reaction 15~
30min, distilled water slowly rinse electrode, be labeled as Electrode 4, be placed on phosphate buffer solution (PBS, 0.1M, pH
7.0) electrochemical response is detected in.
(2) TdT Activity determination
In the preparation process of Electrode 2, change TdT concentration, prepared for Electrode 2, then as step (1) makes
A series of standby sensors, for detecting the electrochemical response of various concentration TdT.
3. the preparation of sensor described in mechanism and claim 2 according to claim 1, it is characterised in that: for detecting TdT's
Electrochemical method is square wave voltammetry, and potential range is -0.6~-0.78V, amplitude 25mV.
4. various concentration TdT detection and its small point may be implemented in electrochemica biological sensor according to claims 1 to 3
Sub- inhibitor screening, detection are limited down to 0.0005U/mL, the IC of inhibitor50For 0.192mM.
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| CN110672694A (en) * | 2019-10-14 | 2020-01-10 | 宁波大学 | Electrochemical method for detecting uracil-DNA glycosylase activity based on DNA NANOTREE |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
| CN111781262A (en) * | 2020-06-22 | 2020-10-16 | 宁波大学 | Construction and application of dynamic analysis method for terminal transferase activity based on alternating-current impedance technology |
| CN112326757A (en) * | 2020-06-22 | 2021-02-05 | 宁波大学 | Novel electrochemical biosensing method for detecting glucose oxidase and urease and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110672694A (en) * | 2019-10-14 | 2020-01-10 | 宁波大学 | Electrochemical method for detecting uracil-DNA glycosylase activity based on DNA NANOTREE |
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| CN112326757A (en) * | 2020-06-22 | 2021-02-05 | 宁波大学 | Novel electrochemical biosensing method for detecting glucose oxidase and urease and application thereof |
| CN111781262B (en) * | 2020-06-22 | 2022-11-15 | 宁波大学 | Construction and application of dynamic analysis method for terminal transferase activity based on alternating-current impedance technology |
| CN112326757B (en) * | 2020-06-22 | 2022-12-02 | 宁波大学 | Electrochemical biosensing method for detecting glucose oxidase and urease |
| CN111763673A (en) * | 2020-07-09 | 2020-10-13 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
| CN111763673B (en) * | 2020-07-09 | 2023-11-24 | 南方科技大学 | C-quadruplex deoxyribozyme and preparation method and application thereof |
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Application publication date: 20190412 Assignee: Ningbo Science and Technology Innovation Association Assignor: Ningbo University Contract record no.: X2023980033633 Denomination of invention: Preparation and application of electrochemical biosensor based on i-motif configuration changes for terminal transferase Granted publication date: 20210611 License type: Common License Record date: 20230317 |
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