CN103913500A - Electrochemical biosensor based on dendritic polymer formed by TiO2@Fe3O4 and DNA (Deoxyribose Nucleic Acid), preparation method and application of sensor - Google Patents

Electrochemical biosensor based on dendritic polymer formed by TiO2@Fe3O4 and DNA (Deoxyribose Nucleic Acid), preparation method and application of sensor Download PDF

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CN103913500A
CN103913500A CN201410166673.4A CN201410166673A CN103913500A CN 103913500 A CN103913500 A CN 103913500A CN 201410166673 A CN201410166673 A CN 201410166673A CN 103913500 A CN103913500 A CN 103913500A
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dna
tio
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biology sensor
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CN103913500B (en
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王广凤
陈玲
朱艳红
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Anhui Normal University
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Anhui Normal University
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Abstract

The invention relates to an electrochemical biosensor based on a dendritic polymer formed by TiO2@Fe3O4 and DNA (Deoxyribose Nucleic Acid), a preparation method and application of the sensor. The preparation method of the sensor comprises the following steps: synthesizing a TiO2@Fe3O4 nanocompound by using a hydrothermal method; and preparing the electrochemical biosensor based on the dendritic polymer formed by TiO2@Fe3O4 and DNA by using specific binding effect between one-end phosphorylated DNA and the TiO2 and complementary pairing effect of bases between DNA sequences. The sensor is used for detecting sensitivity, specificity and stability of target DNA molecules.

Description

A kind of based on TiO 2@Fe 3o 4tree-shaped polymer electrochemical biology sensor, its preparation method and purposes with DNA formation
Technical field
The invention belongs to biosensor technology field, be specifically related to use TiO 2@Fe 3o 4the electrochemica biological sensor of the tree-shaped polymkeric substance assembling forming with DNA and be applied to the detection of target dna molecule.
Background technology
21 century is the century of life science, is an extremely important aspect in life science to the research of DNA, and the research of DNA biology sensor has become focus.There is a lot of research related to this to be devoted to amplify with metal nanoparticle the measured signal of DNA biology sensor.Wherein what is interesting is and find Fe 3o 4magnetic nano-particle, it not only can be widely used in bio-imaging technology, and there is the performance of similar peroxidase activity catalysis, and can be by other nano material functionalization coated catalysts or enzyme, thereby make the function nano compound of preparation can be used to better research in conjunction with its nucleocapsid function.
Summary of the invention
The object of the present invention is to provide a kind of based on TiO 2@Fe 3o 4tree-shaped polymer electrochemical biology sensor with DNA formation, its preparation method and purposes, utilize the base complementrity effect of DNA in target dna molecule and tree-shaped polymkeric substance, at the surface-assembled electrochemica biological sensor of gold electrode, be applied to the detection of target dna molecule.Concrete technical scheme is as follows:
A kind of based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, comprises the steps:
(1) DNA sequence dna: sulfhydrylation: S1, target dna: S2, one end hydroxylation: S3, be dissolved in buffer solution respectively, and save backup;
(2) buffer solution that contains probe S1 is dripped and is coated onto on gold electrode, cultivate, the gold electrode of modifying by Au-S covalent bond effect self assembly S1;
(3) with step (2) simultaneously, in the buffer solution that contains probe S3, add T4PNK, ATP and cultivate this mixed solution, obtain the S3 of phosphorylation;
(4) add the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate, and obtains DNA-TiO 2@Fe 3o 4biological composite;
(5) gold electrode of S1 being modified is put into and is contained S2 and DNA-TiO 2@Fe 3o 4in the buffer solution of biological composite, cultivate, utilize the base complementrity pairing effect between DNA, based on TiO 2@Fe 3o 4the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA is successfully prepared.
Further, TiO described in step (4) 2@Fe 3o 4the preparation of nano composite material comprises the steps:
(4-1) utilize hydrothermal synthesis method, synthesize the Fe of size uniform 3o 4nano particle;
(4-2) by the Fe being successfully prepared 3o 4nano particle utilizes hydrothermal synthesis method at the coated one deck TiO in its surface 2coating;
(4-3) compound substance syncaryon Fe 3o 4living things catalysis performance and shell TiO 2biologic specificity.
Further, in step (1), DNA sequence dna is dissolved in respectively in 0.05M Tris-HCl (pH7.4) buffer solution, and saves backup at 4 ℃.
Further, described in step (2), gold electrode carries out polishing and cleaning before in step (2).
Further, described polishing and cleaning comprise the steps: gold electrode first to carry out polishing with the aluminium powder of 0.3 and 0.5 μ m successively; Put into successively HNO 3: H 2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
Further, in step (2), cultivate 6-10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1; And/or step (3) adds T4PNK, ATP and cultivates this mixed solution 3-5h in the buffer solution that contains probe S3, obtains the S3 of phosphorylation; And/or step (4) adds the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 1-3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
Further, in step (2), cultivate h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1; Step (3) adds T4PNK, ATP and cultivates this mixed solution 5h in the buffer solution that contains probe S3, obtains the S3 of phosphorylation; Step (4) adds the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
A kind of based on TiO 2@Fe 3o 4tree-shaped polymer electrochemical biology sensor with DNA forms, adopts above-mentioned preparation method to prepare.
A kind of above-mentioned based on TiO 2@Fe 3o 4the purposes of tree-shaped polymer electrochemical biology sensor forming with DNA, for the detection to target dna molecule.
Further, the concentration difference of S2, the DNA-TiO of assembling 2@Fe 3o 4the amount difference of biological composite, realizes variable concentrations target dna S2 and quantitatively detects.
Compared with currently available technology, the invention provides TiO 2@Fe 3o 4the electrochemica biological sensor of the tree-shaped polymkeric substance assembling forming with DNA and be applied to the detection of target dna molecule, the present invention uses hydro-thermal method to synthesize TiO 2@Fe 3o 4nano-complex, utilizes one end phosphorylated cdna and TiO 2between specific binding effect, utilize the complementary pairing effect of base between DNA sequence dna, prepare based on TiO 2@Fe 3o 4with the tree-shaped polymer electrochemical biology sensor of DNA, this sensor has been realized the detection to target dna molecule sensitivity, specificity, stability.
Specifically, the preparation method of this biology sensor, the nano-complex using is synthetic simple, consume energy low, cost is low, good biocompatibility, and preparation biology sensor detect amplify the signal of Electrochemical Detection with the catalytic action of nano composite material, by target dna molecule and DNA-TiO 2@Fe 3o 4base complementrity effect between DNA in biological composite, can prepare the sensor of dendrimer structure, the nano-complex more with living things catalysis performance can be assembled on sensor, therefore can realize the detection to target dna molecule, result shows that this biology sensor is satisfactory to the testing result of target dna molecule, the range of linearity detecting is wider, approximately from 10 -10m to 10 -6within the scope of M, have sensitiveer detection, and have highly sensitive, detectability is low, selectivity good, the feature of good stability.In addition, this electrochemica biological sensor is the base complementrity pairing effect utilizing between DNA, by TiO 2@Fe 3o 4the tree-shaped polymkeric substance forming by target dna molecule connection function with DNA biological composite, and this tree-shaped polymkeric substance is assembled on electrode and forms.Electrochemica biological sensor is successfully prepared.Given this method of attachment (such as the base mispairing of cytimidine-silver ion-cytimidine or the specific binding of aptamers and aptamer etc.) that, we can utilize other is by TiO 2@Fe 3o 4form tree-shaped polymkeric substance and be assembled into electrochemica biological sensor with DNA biological composite, realize the detection to specific ion or protein.
Accompanying drawing explanation
Fig. 1 (A) is the Fe of preparation 3o 4nano particle and TiO 2@Fe 3o 4the scanning electron microscope (SEM) photograph of composite nanoparticle.
Fig. 1 (B) is the Fe of preparation 3o 4nano particle and TiO 2@Fe 3o 4composite nanoparticle infrared spectrogram.
Fig. 2 is the impedance diagram of each step electrode modification process electrode.
In figure:
A is the impedance diagram of naked gold electrode.
B is the impedance diagram of the gold electrode of probe S1 modification.
C is probe S1 and S3-TiO 2@Fe 3o 4the impedance diagram of the gold electrode of modifying after effect.
D is the impedance diagram of the gold electrode of probe S1 and S2 and the modification of phosphorylation S3 hybridization.
E is for being probe S1 and S2 and S3-TiO 2@Fe 3o 4the impedance diagram of the gold electrode of modifying after effect.
Fig. 3 (A) is based on TiO 2@Fe 3o 4assemble the cyclic voltammogram of electrochemica biological sensor to the catalysis of variable concentrations hydrogen peroxide with the tree-shaped polymkeric substance that DNA forms.
Fig. 3 (B) is the typical curve of sensor to the catalysis of variable concentrations hydrogen peroxide for this reason.
Fig. 4 is the cyclic voltammogram of different modifying electrode pair hydrogen peroxide catalysis.
In Fig. 4 (A):
A is the cyclic voltammogram of ordinary construction sensor.
B is based on TiO 2@Fe 3o 4the cyclic voltammogram of the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA.
In Fig. 4 (B):
A is and S3-TiO 2@Fe 3o 4arbitrarily the DNA of pairing is used for the cyclic voltammogram of the sensor of assembling.
B is and S3-TiO 2@Fe 3o 4the DNA that has two base mispairings is for the cyclic voltammogram of the sensor assembled.
C is and S3-TiO 2@Fe 3o 4the DNA that has a complete complementary pairing is for the cyclic voltammogram of the sensor assembled.
Fig. 5 (A) is based on TiO 2@Fe 3o 4assemble with the tree-shaped polymkeric substance that DNA forms the cyclic voltammogram that electrochemica biological sensor detects variable concentrations target dna molecule.
Fig. 5 (B) is based on TiO 2@Fe 3o 4assemble with the tree-shaped polymkeric substance that DNA forms the chronoamperogram that electrochemica biological sensor detects variable concentrations target dna molecule.
Fig. 5 (C) is based on TiO 2@Fe 3o 4assemble the typical curve of the change of electrochemica biological sensor to the detection of variable concentrations target dna molecule with the tree-shaped polymkeric substance that DNA forms.
Fig. 6 is based on TiO 2@Fe 3o 4the electrochemica biological sensor of the tree-shaped polymkeric substance assembling forming with DNA prepare schematic diagram.
Embodiment
Describe the present invention with reference to the accompanying drawings below, it is a kind of preferred embodiment in numerous embodiments of the present invention.
Embodiment mono-:
TiO 2@Fe 3o 4the preparation of nano composite material: utilize hydrothermal synthesis method, first synthesize the Fe of size uniform 3o 4nano particle, then by the Fe being successfully prepared 3o 4nano particle utilizes hydrothermal synthesis method at the coated one deck TiO in its surface 2coating, this compound substance is syncaryon (Fe effectively 3o 4) living things catalysis performance and shell (TiO 2) biologic specificity performance.Therefore the compound substance of preparation is had to good using value for the detection of biology sensor.
Based on TiO 2@Fe 3o 4as follows with the tree-shaped polymer electrochemical biology sensor preparation of DNA formation and the step of application:
A, the DNA sequence dna (sulfhydrylation: S1, target dna: S2, one end hydroxylation: S3) of buying is dissolved in respectively in Tris-HCl (pH7.4) buffer solution, and saves backup down at low temperatures.
B, by gold electrode first successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO 3: H 2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
C, the buffer solution that contains probe S1 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 6-10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1.Meanwhile, in the buffer solution that contains probe S3, add T4PNK, ATP and cultivate this mixed solution 3-5h, obtain the S3 of phosphorylation; Add again the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 1-3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
D, the gold electrode that S1 is modified are put into and are contained S2 and DNA-TiO 2@Fe 3o 4in the buffer solution of biological composite, cultivate 3-5h, utilize the base complementrity pairing effect between DNA, based on TiO 2@Fe 3o 4the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA is successfully prepared.
E, due to the concentration difference of S2, the DNA-TiO of assembling 2@Fe 3o 4the amount of biological composite can be different, along with the increase of S2 concentration, DNA-TiO 2@Fe 3o 4the amount of biological composite also can increase thereupon, and therefore this sensor can quantitatively detect variable concentrations target dna S2.
Embodiment bis-:
Based on TiO 2@Fe 3o 4as follows with the tree-shaped polymer electrochemical biology sensor preparation of DNA formation and the step of application:
A, the DNA sequence dna (sulfhydrylation: S1, target dna: S2, one end hydroxylation: S3) of buying is dissolved in respectively in 0.05MTris-HCl (pH7.4) buffer solution, and saves backup at 4 ℃.
B, by gold electrode first successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO 3: H 2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
C, the buffer solution that contains probe S1 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 6-10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1.Meanwhile, in the buffer solution that contains probe S3, add T4PNK, ATP and cultivate this mixed solution 3-5h, obtain the S3 of phosphorylation; Add again the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 1-3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
D, the gold electrode that S1 is modified are put into and are contained S2 and DNA-TiO 2@Fe 3o 4in the buffer solution of biological composite, cultivate 3-5h, utilize the base complementrity pairing effect between DNA, based on TiO 2@Fe 3o 4the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA is successfully prepared.
E, due to the concentration difference of S2, the DNA-TiO of assembling 2@Fe 3o 4the amount of biological composite can be different, along with the increase of S2 concentration, DNA-TiO 2@Fe 3o 4the amount of biological composite also can increase thereupon, and therefore this sensor can quantitatively detect variable concentrations target dna S2.
Based on TiO 2@Fe 3o 4as follows with the tree-shaped polymer electrochemical biology sensor preparation of DNA formation and the step of application:
A, the DNA sequence dna (sulfhydrylation: S1, target dna: S2, one end hydroxylation: S3) of buying is dissolved in respectively in 0.05MTris-HCl (pH7.4) buffer solution, and saves backup at 4 ℃.
B, by gold electrode first successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO 3: H 2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
C, the buffer solution that contains probe S1 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1.Meanwhile, in the buffer solution that contains probe S3, add T4PNK, ATP and cultivate this mixed solution 5h, obtain the S3 of phosphorylation; Add again the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
D, the gold electrode that S1 is modified are put into and are contained S2 and DNA-TiO 2@Fe 3o 4in the buffer solution of biological composite, cultivate 5h, utilize the base complementrity pairing effect between DNA, based on TiO 2@Fe 3o 4the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA is successfully prepared.
E, due to the concentration difference of S2, the DNA-TiO of assembling 2@Fe 3o 4the amount of biological composite can be different, along with the increase of S2 concentration, DNA-TiO 2@Fe 3o 4the amount of biological composite also can increase thereupon, and therefore this sensor can quantitatively detect variable concentrations target dna S2.
By reference to the accompanying drawings the present invention is exemplarily described above; obviously specific implementation of the present invention is not subject to the restrictions described above; as long as the various improvement that adopted method design of the present invention and technical scheme to carry out; or directly apply to other occasion without improvement, all within protection scope of the present invention.

Claims (10)

1. one kind based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, comprises the steps:
(1) DNA sequence dna: sulfhydrylation: S1, target dna: S2, one end hydroxylation: S3, be dissolved in buffer solution respectively, and save backup;
(2) buffer solution that contains probe S1 is dripped and is coated onto on gold electrode, cultivate, the gold electrode of modifying by Au-S covalent bond effect self assembly S1;
(3) with step (2) simultaneously, in the buffer solution that contains probe S3, add T4PNK, ATP and cultivate this mixed solution,
Obtain the S3 of phosphorylation;
(4) add the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate, and obtains DNA-TiO 2@Fe 3o 4biological composite;
(5) gold electrode of S1 being modified is put into and is contained S2 and DNA-TiO 2@Fe 3o 4in the buffer solution of biological composite, cultivate, utilize the base complementrity pairing effect between DNA, based on TiO 2@Fe 3o 4the tree-shaped polymkeric substance assembling electrochemica biological sensor forming with DNA is successfully prepared.
2. as claimed in claim 1 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that TiO described in step (4) 2@Fe 3o 4the preparation of nano composite material comprises the steps:
(4-1) utilize hydrothermal synthesis method, synthesize the Fe of size uniform 3o 4nano particle;
(4-2) by the Fe being successfully prepared 3o 4nano particle utilizes hydrothermal synthesis method at the coated one deck TiO in its surface 2coating;
(4-3) compound substance syncaryon Fe 3o 4living things catalysis performance and shell TiO 2biologic specificity.
3. as claimed in claim 1 or 2 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, in step (1), DNA sequence dna is dissolved in respectively in 0.05M Tris-HCl (pH7.4) buffer solution, and saves backup at 4 ℃.
As described in any one in claim 1-3 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, described in step (2), gold electrode carries out polishing and cleaning before in step (2).
5. as claimed in claim 4 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, described polishing and cleaning comprise the steps: by gold electrode first successively with 0.3 and the aluminium powder of 0.5mm carry out polishing; Put into successively HNO 3: H 2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
As described in any one in claim 1-5 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, cultivates 6-10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1 in step (2); And/or step (3) adds T4PNK, ATP and cultivates this mixed solution 3-5h in the buffer solution that contains probe S3, obtains the S3 of phosphorylation; And/or step (4) adds the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 1-3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
7. as claimed in claim 6 based on TiO 2@Fe 3o 4the preparation method of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, cultivates h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1 in step (2); Step (3) adds T4PNK, ATP and cultivates this mixed solution 5h in the buffer solution that contains probe S3, obtains the S3 of phosphorylation; Step (4) adds the TiO preparing 2@Fe 3o 4compound substance, continues to cultivate 3h, obtains DNA-TiO 2@Fe 3o 4biological composite.
8. one kind based on TiO 2@Fe 3o 4tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, adopts the preparation method described in claim 1-7 to prepare.
One kind as claimed in claim 8 based on TiO 2@Fe 3o 4the purposes of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that, for the detection to target dna molecule.
10. as claimed in claim 9 based on TiO 2@Fe 3o 4the purposes of tree-shaped polymer electrochemical biology sensor with DNA forms, is characterized in that the concentration difference of S2, the DNA-TiO of assembling 2@Fe 3o 4the amount difference of biological composite, realizes variable concentrations target dna S2 and quantitatively detects.
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