CN106093158A - Glutathion detection biosensor, its preparation method and detection method thereof - Google Patents
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Abstract
The present invention relates to a kind of glutathion detection biosensor, its preparation method and detection method thereof.This sensor is to be modified with DNA double chain probe on the surface of gold electrode, the first probe in this double-chain probe is that 5 ' the terminal modified Probe DNA1 having sulfydryl are scheduled on gold electrode surfaces by gold gold mercapto keying, second probe is the Probe DNA 2 of 3 ' terminal modified methylene blue signaling molecules, the second described probe and the first probe portion hybridization complementary pairing, thus form DNA double chain probe；The base sequence of the first described probe is: 5 ' SH CCCCCTCTATACCGTACCTTTTTT 3 '；The base sequence of the second described probe is: 5 ' GGTACGGTATAGAG MB 3 '.Sensor of the invention can detect that the range of linearity of glutathion is 0.5 nM to 50 μM delicately, detection is limited to 0.14 nM, and high specificity, target can be efficiently differentiated and compare aminoacid and albumen with other, also can in buffer and serum sample the differentiation target molecules of high specific, and system is applied to the detection of cell GSH-PX activity by us.
Description
Technical field
The present invention relates to a kind of DNA biosensor, particularly a kind of glutathion detection biosensor, it prepares
Method and detection method thereof.
Background technology
Glutathion, chemical entitled N-L-gamma-glutamyl-L-cysteinyl, i.e. by glutamic acid, cysteine, glycine
Three part compositions, are present in all of cell, are the important active substances in body.The while that glutathion (GSH) being a kind of
There is glutamyl and the bioactive tripeptide compound of sulfydryl, be also that the interior one maximum with content in eukaryotic cell of human body is non-
Protein sulfhydryl material.Glutathion has a lot of biological functions, and it promotes calcareous suction in amino acid whose absorption and transhipment
Receive, improve immunity of organisms, maintain synthesis and the cell normal growth of DNA, participate in high ferritin and synthesize, removing free radical,
And the aspect such as internal detoxification processes has irreplaceable effect；Pharmacologically, GSH has a convulsion, suppression epilepsy and
Analgesic acts on；Additionally, research finds, the content of GSH also relates to the major disease such as cancer and heart disease to be developed
Process.Just because of GSH, the healthy of people being had the biggest impact, the detection research to GSH has caused people
Pay close attention to the most widely.
The glutathion detection method that presently, there are mainly has high performance liquid chromatography, Electrochemiluminescince, enzyme parameters
And differential thermal analysis etc., but these methods or operate complex, or detection sensitivity is the highest, and be difficult to apply in situ
Analyze and detection in real time.In this, electrochemistry becomes a popular technique of detection glutathion.Nowadays, DNA molecular is one has
The element of profit is used for building effective biosensor, provides a lot of choosing for molecular recognition, signal labelling and signal amplification etc.
Select.Wherein, DNA base causes increasing concern with the selective binding of metal ion in DNA analysis.Such as, Hg2 + Can be with T-T(thymus pyrimidine) high-affinity mates, and C-C (cytosine) can catch Ag specially+.Because DNA base and gold
Belong to interionic and have the adhesion of high specific, be widely used for the structure of ion detection, gate, even cause DNA cloning
Reaction.Recently, selectively formed T-Hg2+-T has turned out as regulatory factor mediated dna strand replacement reaction, thus can be become
One molecular tool with potential significance.
Summary of the invention
An object of the present invention is to provide a kind of glutathion detection biosensor.
Preparation method prepared by two these sensors of offer of the purpose of the present invention
The three of the purpose of the present invention are to provide the method using this sensor detection glutathion.
The present invention is to use electrochemical techniques to glutathion specificity and the detection of susceptiveness.For realizing object above,
Mechanism of the present invention is as follows:
Probe DNA1 5 ' terminal modified have sulfydryl can with self assembly on the surface of gold electrode, 3 ' terminal modified methylene blues letter
Probe DNA 2 and the Probe DNA1 partial hybridization complementary pairing of number molecule, thus form DNA double chain probe.So, methylene
Base indigo plant signaling molecule responds out stronger electrochemical signals after gold electrode.In the presence of mercury ion, helper
5 ' the ends of DNA have 6 T(thymus pyrimidines) short sequence as " foothold " district, 6 can held with the 3 ' of Probe DNA1
T-sequence passes through mercury ion high specific identification T-T, then forms T-Hg2+-T structure.Now, helper DNA by former with
The Probe DNA 2 of Probe DNA1 partial hybridization complementary pairing substitutes, and strand replacement reaction occurs.Meanwhile, methylene blue signal
Molecule is away from responding out more weak electrochemical signals after gold electrode.But, due to glutathion and Hg2+ Between complexation power phase
Than T-T and Hg2+ Between active force higher, therefore, glutathion chelating Hg2 +Can suppress T-Hg2 +-T complex
The strand replacement reaction of mediation.In this case, glutathion can safeguard that DNA double chain probe is on the surface of gold electrode.Different
The glutathion of concentration can respond different electrochemical signals by following the tracks of methylene blue signal probe, thus realizes quantitatively inspection
Survey the purpose of glutathion.
Therefore, just it is able to detect that the glutathion of very low concentrations by the sensitive change of electrochemical signals, participates in Fig. 1.
The present invention constructs strand replacement reaction based on dimercurion mediation and detects glutathion.GSH molecule
In have 3 can dissociate proton and 10 atoms that may participate in coordination, and sulfydryl (-SH) be in GSH molecular structure one important
Functional group, is research biomolecule and the comparatively ideal model of Action of Metal Ions.Metallic element especially heavy metal element has
The characteristic of strong close S element, can combine with the sulfydryl in GSH molecule, thus the chela of metal ion in direct function cells
Mixture, is further used for the electrochemical behavior research of GSH and metal ion.GSH to the binding ability of metallic element and metal from
The soft or hard of son itself is relevant.Therefore, the most soft mercury ion has stronger complexing power, and role is the brightest
Aobvious.
According to above-mentioned mechanism, the present invention adopts the following technical scheme that
A kind of glutathion detection biosensor, it is characterised in that this sensor is to be modified with DNA on the surface of gold electrode
Double-chain probe, the first probe in this double-chain probe is that 5 ' the terminal modified Probe DNA1 having sulfydryl are scheduled on by gold mercapto keying
Gold electrode surfaces, the second probe is the Probe DNA 2 of 3 ' terminal modified methylene blue signaling molecules, the second described probe with
First probe portion hybridization complementary pairing, thus form DNA double chain probe；The base sequence of the first described probe is: 5 '-
SH-CCCCCTCTATACCGTACCTTTTTT-3’；The second described probe base sequence be: 5 '-
GGTACGGTATAGAG- MB-3’。
A kind of method preparing above-mentioned glutathion detection biosensor, it is characterised in that the method concrete
Step is: be immersed in the reactant liquor of modified electrode by pretreated gold electrode, room temperature modification reaction 16h～24h；Often
The reactant liquor of the modified electrode described in 100ml is by 5 μ L, Probe DNA 1, the 5 μ L of 10 μMs, the Probe DNA 2 of 10 μMs
With 5 μ L, the TCEP of 100 mM, blend together uniform solution to be diluted to final volume be 100 μ L with the fixing buffer of 85 μ L
System is constituted, then heated at constant temperature 5 min through 95 DEG C, naturally cools to room temperature；Described fixing buffer is: 10 mM
Tris-HCl, 1 mM EDTA, 0.1 M NaCl, its pH=7.4.
The preprocess method of above-mentioned gold electrode is: by gold electrode sand paper and containing granular size be 1.0 μm, 0.3 μm
After grinding and buffing on the chamois leather of the aluminium powder of 05 μm, the ultrasonic 3min in dehydrated alcohol and ultra-pure water respectively by electrode, so
Afterwards with the Piranha of 50 μ L, the i.e. mixed liquor of the volume ratio of concentrated sulphuric acid hydrogen peroxide=3 1, purify gold electrode, remove residual
Impurity；Again through the H of 0.5 M2SO4Activation.
The detection method of a kind of glutathion, uses three electrode detection systems, and wherein saturated calomel electrode is as reference electricity
Pole, platinum filament uses above-mentioned glutathion detection biosensor as auxiliary electrode, working electrode gold electrode, and its feature exists
Concretely comprising the following steps in the method:
A. prepare reaction system, every 100ml reaction system contains the Hg of 10 μ L 100 μMs2+ With 5 μ L, 10 μMs
helper DNA；Being joined by glutathion in this reaction system, the concentration making reaction system GSH-PX activity is: 0.5
NM～50 μMs, under 37 DEG C of constant temperature, incubation 30min；The sequence of described helper DNA is: 5 '-
TTTTTTGGTACGGTATAGAG-3’；
B. gold electrode is immersed in the reaction system of step a gained 1～1.5h with at electrode surface generation strand replacement reaction；
Under anaerobic environment, then by three electrode detection systems, the electrochemical method of differential pulse voltammetry is used to characterize methylene
The electrochemical signals of base cyan molecule, thus detect the concentration of glutathion..
The present invention constructs a kind of biosensor detecting glutathion, and strand replacement reaction based on mercury ion mediation is real
Show the Sensitive Detection to glutathion.The method can also provide technical support for the clinical monitoring of glutathion, has wide
General application prospect.
Accompanying drawing explanation
Fig. 1 is the Method And Principle figure of the present invention.
Fig. 2 is the square wave voltammogram of electrochemical signals under three circumstances.
Fig. 3 is the electrochemical signals variation diagram that obtains in the case of the glutathion of variable concentrations exists, and embedded figure is paddy Guang
Sweet peptide concentration linear relationship chart in the range of 0.5 nM to 5 μM and between electrochemical signals changing value.
Fig. 4 is the electrochemical signals response block diagram obtained under different types of amino acid and protein existence condition.
Fig. 5 is HeLa cell extract and the detection of serum GSH-PX activity.
Fig. 6 is Virus monitory statistical table.
Detailed description of the invention
Embodiment one: the strand replacement reaction of mercury ion mediation
First, gold electrode 1 μm, the aluminium powder polishing of 0.3 μm and 0.05 μm.Then, gold electrode is in ethanol and double steaming
In water each ultrasonic 5 minutes.With Piranha (H2SO4H2O2=3 1) purify 5 minutes, distilled water wash away after with 0.5 M H2SO4's
Electrochemical purification is to remove any remaining impurity.After nitrogen dries up, the standby DNA's of gold electrode is fixing.DNA hybridization system
In have DNA probe 1 and the DNA probe 2 of 500 nM, first 95oUnder C, within 5 minutes, to be cooled to room temperature the most miscellaneous in heating
Hand over.Then, 1 is processed with the MCH of 1 mM after DNA mixed liquor tips upside down on and at room temperature places on the gold electrode handled well 16 hours
Hour, in order to obtain good double chain DNA probe monofilm at the electrode surface.The gold electrode of DNA double chain probe modification exists
37oIt is immersed under the conditions of C containing 10 μMs of Hg2+With in the reactant liquor of 300 nM helper DNA 1 hour there is strand displacement
Reaction.
Embodiment two: methylene blue signaling molecule response electrochemical signals realizes the research of detection glutathion
Former and Probe DNA1 partial hybridization complementary pairing Probe DNA 2 is substituted by the helper DNA in above-mentioned solution,
There is strand replacement reaction.Meanwhile, methylene blue signaling molecule is away from responding out more weak electrochemical signals after gold electrode.In order to examine
Survey glutathion, before electrode submergence, in the above-mentioned solution comprising mercury ion and auxiliary DNA, add the paddy Guang of variable concentrations
Sweet peptide.But, due to glutathion and Hg2+ Between complexation power compare T-T and Hg2+ Between active force higher, therefore, paddy
Guang sweet peptide chelating Hg2 +Can suppress T-Hg2 +The strand replacement reaction that-T is compound-mediated.In this case, paddy Guang
Sweet peptide can safeguard DNA double chain probe on the surface of gold electrode, methylene blue signaling molecule still near gold electrode rear surface ring
Stronger electrochemical signals should be gone out.Therefore the glutathion of variable concentrations can be by using the square wave of electrochemical analyser 660c
Volt-ampere technical measurement analyzes the electrochemical signals that the response of methylene blue signaling molecule is different.One traditional three-electrode system is used for
In electro-chemical test, gold electrode is as working electrode, and saturated calomel electrode is as reference electrode, and platinum filament is as auxiliary electrode.?
Before test, all of electrolyte all passes through High Purity Nitrogen and forms bubble in the solution and within least 20 minutes, make thorough anoxia, it is ensured that institute
There is the anaerobic environment of experiment.
In order to verify that just first this experimental system can be carried out in the case of the strand replacement reaction that mercury ion mediation occurs, I
Devise three groups of experiments.As shown in Fig. 2, we have applied this sensitive electrochemical techniques of square wave voltammetry and have proved
The principle of our detection.Highest signal peak (the curve of the double chain DNA probe modified on electrode is obtained at-0.25V place
A), effective electron transmission between methylene blue signaling molecule and electrode surface is indicated.After adding mercury ion, it is thus achieved that
One at a fairly low electrochemical response electric current of electrode surface, the strand replacement reaction owing to mercury ion mediation makes to be modified with Asia
The DNA probe 2 of methyl blue signaling molecule is away from the DNA probe 1 (curve b) of electrode surface.In the presence of glutathion, paddy
The sweet peptide of Guang can chelate the strand replacement reaction of mercury ion suppression mercury ion mediation, therefore obtains one and is similar to modify on electrode
Double chain DNA probe response electrochemical signals (curve c).Square wave voltammetry research demonstrates the feasible of our Cleaning Principle
Property.
Embodiment three: the detection of variable concentrations glutathion
The glutathion of variable concentrations is added in system (0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500
NM, 1 μM, 5 μMs, 10 μMs, 25 μMs, 50 μMs), result is as shown in Figure 3.Glutathion can suppress to pass through T-Hg effectively2+-
The strand replacement reaction of the foothold mediation that T complex is formed, therefore increases modifying on DNA probe 2 at electrode surface
The quantity of methylene blue signaling molecule responds electrochemical signals, and peak point current increases along with the increase of glutathione concentrations.
The embedded figure of Fig. 3 further demonstrated that glutathione concentrations between 0.5 nM to 5 μM with the linear relationship of peak current.Linearly
Equation isI (μA)=0.76467+0.19055×lgC (μM), R2=0.998.The detection limit obtained under three times of noise ratios
It is 0.14 nM.
Embodiment four: biosensor specificity is studied
Control experiment indicates the specificity of our detection method.As shown in Figure 4, in the presence of glutathion, higher peak is obtained
Electric current, when at comparison molecule either other aminoacid or albumen (glutamic acid, phenylalanine, thrombin or bovine serum albumin
In the presence of in vain), only obtain the lowest current value.Additionally, matched group also has the cysteine containing sulfhydryl-group activity equally can also
Distinguishing by our method, the peak current that it is responded is also below glutathion.Chelating between glutathion and heavy metal ion
Effect depends on the binding site of two or more, and such as sulfydryl and carbonyl, this is than only functional group binding sites's
Aminoacid (such as cysteine) to be combined stablizing.Therefore control experiment demonstrates our method and has satisfactory spy
The opposite sex.
Embodiment five: serum and the detection of cell experiment simulation human internal environment
Hyclone have studied our side as an example having interference factor and HeLa cell as in an actual sample
The suitability of method.As shown in Fig. 5, with the glutathion in dilute serum or from HeLa cell extract glutathion sample
Higher peak point current can be obtained during product.Acetyl group maleimide is introduced into as effective glutathion sulfydryl blocking agent
In glutathion sample, find that electrochemical signals significantly reduces.The electrification obtained in serum sample and cell extraction are tested
Learn response and show clearly that this experimental design is given the credit to the existence for glutathion.Additionally, the serum sample of given concentration
This response rate proves between 96.1% and 107%, shows the selectivity that the method is good.See Fig. 6.Therefore, in complexity
The high selectivity of our method has not only been reaffirmed in experiment in biological specimen, also show our method in clinical practice
In there is great potential.
More than test result indicate that, the inventive method achieves the Sensitive Detection of glutathion, and can efficiently differentiate it
He compares aminoacid and protein molecular.This simple, convenient, the sensitive and selective detection means of height, for foothold from now on
The application in DNA analysis of the strand replacement reaction of mediation provides a new thinking, is also expected to be applied to clinical inspection in the future
To meet different detection demands in survey.
Claims (4)
1. a glutathion detection biosensor, it is characterised in that this sensor is to be modified with on the surface of gold electrode
DNA double chain probe, the first probe in this double-chain probe is that 5 ' the terminal modified Probe DNA1 having sulfydryl are determined by gold mercapto keying
In gold electrode surfaces, the second probe is the Probe DNA 2 of 3 ' terminal modified methylene blue signaling molecules, the second described probe
Hybridize complementary pairing with the first probe portion, thus form DNA double chain probe；The base sequence of the first described probe is: 5 '-
SH-CCCCCTCTATACCGTACCTTTTTT-3’；The second described probe base sequence be: 5 '-
GGTACGGTATAGAG- MB-3’。
2. the method preparing glutathion detection biosensor according to claim 1, it is characterised in that should
Method concretely comprise the following steps: pretreated gold electrode is immersed in the reactant liquor of modified electrode, room temperature modification reaction
16h-24h；The reactant liquor of every modified electrode described in 100ml by 5 μ L, Probe DNA 1, the 5 μ L of 10 μMs, 10 μMs
Probe DNA 2 and 5 μ L, the TCEP of 100 mM, blend together uniform solution with the fixing buffer of 85 μ L and be diluted to whole body
Amassing is that the system of 100 μ L is constituted, then heated at constant temperature 5 min through 95 DEG C naturally cools to room temperature；Described fixing buffer
For: 10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, its pH=7.4.
The method of glutathion detection biosensor the most according to claim 2, it is characterised in that described gold electricity
The preprocess method of pole is: by gold electrode sand paper and the chamois leather containing the aluminium powder that granular size is 1.0 μm, 0.3 μm and 05 μm
After upper grinding and buffing, the ultrasonic 3min in dehydrated alcohol and ultra-pure water respectively by electrode, then with the Piranha of 50 μ L,
The i.e. mixed liquor of the volume ratio of concentrated sulphuric acid hydrogen peroxide=3 1, purifies gold electrode, removes the impurity of residual；Again through 0.5 M's
H2SO4Activation.
4. a detection method for glutathion, uses three electrode detection systems, and wherein saturated calomel electrode is as reference electrode,
Platinum filament uses glutathion detection bio-sensing according to claim 1 as auxiliary electrode, working electrode gold electrode
Device, it is characterised in that concretely comprising the following steps of the method:
A. prepare reaction system, every 100ml reaction system contains the Hg of 10 μ L 100 μMs2+ With 5 μ L, 10 μMs
helper DNA；Being joined by glutathion in this reaction system, the concentration making reaction system GSH-PX activity is: 0.5
NM～50 μMs, under 37 DEG C of constant temperature, incubation 30min；The sequence of described helper DNA is: 5 '-
TTTTTTGGTACGGTATAGAG-3’；
B. gold electrode is immersed in the reaction system of step a gained 1～1.5h with at electrode surface generation strand replacement reaction；
C., under anaerobic environment, then by three electrode detection systems, the electrochemical method of differential pulse voltammetry is used to characterize Asia
The electrochemical signals of methyl blue molecule, thus detect the concentration of glutathion.
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| CN106802294A (en) * | 2017-03-09 | 2017-06-06 | 青岛科技大学 | A kind of glutathione fluorescent optical sensor based on controlled release technologies and preparation method thereof |
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| CN106932371A (en) * | 2017-03-09 | 2017-07-07 | 青岛科技大学 | A kind of fluorescence imaging method of glutathion inside cell |
| CN107144618A (en) * | 2017-06-20 | 2017-09-08 | 孙丽洲 | A kind of methods and applications of the protein activation levels of utilization DNA probe part Electrochemical Detection STAT 3 |
| CN113495091A (en) * | 2021-05-28 | 2021-10-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
| CN113528513A (en) * | 2021-08-12 | 2021-10-22 | 基因科技（上海）股份有限公司 | In-situ hybridization probe complex, preparation method and application thereof |
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| CN106802294A (en) * | 2017-03-09 | 2017-06-06 | 青岛科技大学 | A kind of glutathione fluorescent optical sensor based on controlled release technologies and preparation method thereof |
| CN106908429A (en) * | 2017-03-09 | 2017-06-30 | 青岛科技大学 | A kind of method for detecting glutathione |
| CN106932371A (en) * | 2017-03-09 | 2017-07-07 | 青岛科技大学 | A kind of fluorescence imaging method of glutathion inside cell |
| CN106802294B (en) * | 2017-03-09 | 2020-03-10 | 青岛科技大学 | Glutathione fluorescence sensor based on controlled release technology and preparation method thereof |
| CN106932371B (en) * | 2017-03-09 | 2020-03-10 | 青岛科技大学 | Fluorescence imaging method of intracellular glutathione |
| CN106908429B (en) * | 2017-03-09 | 2020-03-10 | 青岛科技大学 | Method for detecting glutathione |
| CN107144618A (en) * | 2017-06-20 | 2017-09-08 | 孙丽洲 | A kind of methods and applications of the protein activation levels of utilization DNA probe part Electrochemical Detection STAT 3 |
| CN113495091A (en) * | 2021-05-28 | 2021-10-12 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
| CN113495091B (en) * | 2021-05-28 | 2024-01-23 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
| CN113528513A (en) * | 2021-08-12 | 2021-10-22 | 基因科技（上海）股份有限公司 | In-situ hybridization probe complex, preparation method and application thereof |
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