CN103940890A - Preparation method and application of DNA-AuNPs (gold nanoparticles) nanometer network structure - Google Patents

Preparation method and application of DNA-AuNPs (gold nanoparticles) nanometer network structure Download PDF

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CN103940890A
CN103940890A CN201410168197.XA CN201410168197A CN103940890A CN 103940890 A CN103940890 A CN 103940890A CN 201410168197 A CN201410168197 A CN 201410168197A CN 103940890 A CN103940890 A CN 103940890A
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CN103940890B (en
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王宗花
赵凯
夏建飞
张菲菲
孙娜
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Qingdao University
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Abstract

The invention discloses a preparation method and application of a DNA-AuNPs (gold nanoparticles) nanometer network structure. The preparation method comprises the steps of preparation of AuNPs, preparation of DNA functionalized AuNPs and layer-by-layer assembly of a DNA-AuNPs nanometer network structure. According to the method, the DNA-AuNPs are adopted as nanometer beacons, and double-stranded DNA is formed by utilizing the complementary characteristic of DNA, thereby forming the nanometer network structure and facilitating adsorption of the RuHeX electroactive material; the complementary characteristic of DNA is utilized to form the DNA-AuNPs network structure which provides a great number of sites for adsorption of the electroactive material, realizes amplification of electrochemical signals and is used for analysis of the activity of protein kinase and inhibitor thereof; the nanometer network structure can be modified on the surface of an electrode favorably due to coordination of zirconium ions, a great number of loading sites are provided for active substances, and the electrochemical signal is greatly enhanced; therefore, the minimum detection limit of a biosensor to the kinase activity is 0.003U/mL, the stability is good and the linear range is wide.

Description

A kind of preparation method and application of DNA-AuNPs nanometer network structure
Technical field
The present invention relates to electrochemical sensor field, relate in particular to a kind of preparation method and application of DNA-AuNPs nanometer network structure.
Background technology
The protein phosphorylation regulating by kinases is a biological metabolic important process, and it is playing important effect aspect signal conduction and management cellular activity.Protein kinase dysregulation can be upset the system of protein phosphorylation, causes the generation of various diseases.Therefore, identification protein kinase activity and inhibitor thereof are not only conducive to bio-metabolic process to be understood clearly, and are conducive to the early detection of disease and treatment in time.
Have or not the existence of phosphate radical according to phosphorylation process, metallic ion Zr 4+become the effective reagent of one of catching enriched phosphorus acidifying.Utilize the strong coordination of metallic ion and phosphate radical, Zr 4+be commonly used to as a kind of phosphorylation site on polypeptide and important coupling agent of beacon of connecting.Because the end of DNA own is with phosphate radical or be easy to modify other group and the part that is often used as beacon.Divide than simple sandwich structure or signal amplifying part, nanometer network structure (or super sandwich structure) shows very high sensitivity and is subject to researcher's favor in the detection of DNA analysis and cell.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of preparation method and application of DNA-Au NPs nanometer network structure, it has the electrochemical signals of amplification, detect the sensitive advantage such as reliable.
The present invention is achieved by the following technical solutions:
A preparation method for DNA-AuNPs nanometer network structure, step is as follows:
1) preparation of Au NPs: 100mL0.01% (w/v) HAuCl 4aqueous solution is heated to reflux, and 2.5mL1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps boiling 15min, solution by colourless become claret gradually till, obtain Au NPs, save backup in 4 DEG C;
2) preparation of DNA functionalization Au NPs: first DNA prevents from forming disulfide bond through TCEP activation 2h; DNA1 or DNA2 are joined in 1mL Au NPs, under room temperature, stir 24h; Fully, after reaction, at room temperature dropwise slowly add 150 μ L1M NaCl, under dark condition, react 24h; Rotating speed centrifuging 10min by completely reacted solution with 12000rpm, is then scattered in 300mM NaCl solution again, obtains DNA1-AuNPs or DNA2-AuNPs, is suspended from 50mM Tris-Hcl damping fluid for subsequent use; The DNA2-AuNPs preparing is processed with 1 μ M cDNA, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', and its nucleotide sequence is as shown in SEQ ID NO.1; The sequence of DNA1 is 5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ', and its nucleotide sequence is as shown in SEQ ID NO.2; The sequence of DNA2 is 5 '-SH-C6-CGTTTAGGATTTGTG-3 ', and its nucleotide sequence is as shown in SEQ ID NO.3;
3) layer assembly of DNA-AuNPs nanometer network structure: before gold electrode is assembled, first gold electrode is used respectively to α-Al of 0.3 and 0.05 μ m 2o 3polishing grinding in paste, and clean 1min in ethanol and deionized water for ultrasonic successively, and at the H of 0.5M 2sO 4in solution, adopt three-electrode system to carry out electrochemical cleaning and activation; It is in 7.4 the phosphate buffer that contains 500 μ M kemptides that the gold electrode of handling well is soaked in to 0.05M pH, and is placed under room temperature and reacts 12h, and then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Seal blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted and contains 20mMMgCl 2in the damping fluid of solution, protein kinase A and 100 μ M ATP, at 37 DEG C of reaction 1h, obtain phosphorylation kemptide modified electrode; The kemptide modified electrode that completes phosphorylation is cleaned up, use respectively subsequently 0.5mM Zr 4+process this electrode with 6 μ L nanometer beacon DNA1-AuNPs, pass through Zr 4+nanometer beacon group is loaded on to the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this TBS, intermediate water rinse and N for modified electrode 2dry up; This electrode continuous after cDNA-DNA2-AuNPs and DNA1-AuNPs process, form DNA-AuNPs nanometer network structure, after layer assembly through TBS process, rinse, obtain DNA-AuNPs nano-electrode; The method is prepared obtained electrode and is tested for phosphorylation.
The present invention also provides a kind of DNA-AuNPs nanometer network structure of preparing according to said method.
The present invention also provides a kind of sensor based on DNA-AuNPs nanometer network structure construction.
The present invention also provides a kind of and detects for the activity of hyclone protein kinase based on DNA-AuNPs nanometer network sensor.
The present invention also provides a kind of and detects for the activity of kinases inhibitor based on DNA-AuNPs nanometer network sensor.
The application of sensor based on DNA-Au NPs nanometer network structure construction in protein kinase activity and inhibitor thereof are analyzed, cyclic voltammetric and electrochemical AC impedance are with 5mM K 3[Fe (CN) 6]/K 4[Fe (CN) 6] be redox probe, and carry out in the electrolyte solution that contains 0.5M KCl, the frequency range of EIS is 100mHz～100kHz; Nano-electrode DNA-Au NPs is immersed in to 50 μ M RuHex reaction 10min, the straight-through N of this process one 2, detection signal in blank TBS damping fluid, scanning voltage :-0.3～+ 0.7V, sweep speed is 100mV/s.
Beneficial effect of the present invention is,
1. the present invention utilizes DNA-AuNPs to do nanometer beacon, utilizes the complementary characteristic of DNA to form double-stranded DNA, and then forms nanometer network structure, because Au NPs and DNA itself are with a large amount of negative charges, is conducive to the absorption of this electroactive material of RuHeX; And utilize the complementary performance of DNA to form the reticulate texture of DNA-Au NPs, for the absorption of electroactive material provides a large amount of sites, thereby realize the amplification of electrochemical signals;
2. the electrochemical signals based on DNA-Au NPs nanometer network structure amplifies for protein kinase activity and inhibitor analysis thereof;
3. by the coordination of zirconium ion, be beneficial to nanometer network structural modification at electrode surface, for active substance provides a large amount of load sites, greatly strengthen electrochemical signals, therefore, this biology sensor reaches 0.03U/mL to the lowest detection limit value of kinase activity, and Simultaneous Stabilization is high, the range of linearity is wide.
Brief description of the drawings
Fig. 1 is the schematic diagram in protein kinase activity detection and inhibitor analysis based on DNA-Au NPs nanometer network structure;
Fig. 2 is Fourier's infrared spectrogram of protein phosphorylation process;
Fig. 3 is electrochemical impedance figure (A) and the cyclic voltammogram (B) of different modifying electrode;
Fig. 4 (A) is the Electronic Speculum figure of Au NPs, is (B) Au NPs (a), the uv absorption spectrogram of Au NPs-DNA (b) and cDNA-DNA2-Au NPs-cDNA (c);
Fig. 5 is the electrochemical response figure of different modifying electrode;
Fig. 6 is temperature (A) and the affect figure of ATP concentration (B) on electrochemical response;
Fig. 7 is the electrochemical response figure of the protein kinase of variable concentrations, and wherein, interior illustration is the linear relationship chart of protein kinase concentration and DPV response;
Fig. 8 is galvanochemistry intensity-a) inhibitor ellagic acid and b) concentration curve of Tyrphostin AG147, and wherein, the protein kinase A that phosphorylation reaction adds is 100U/mL.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
A preparation method for DNA-AuNPs nanometer network structure, step is as follows:
1) preparation of Au NPs: 100mL0.01% (w/v) HAuCl 4aqueous solution is heated to reflux, and 2.5mL1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps boiling 15min, solution by colourless become claret gradually till, obtain Au NPs, save backup in 4 DEG C;
2) preparation of DNA functionalization Au NPs: first DNA prevents from forming disulfide bond through TCEP activation 2h; DNA1 or DNA2 are joined in 1mL Au NPs, under room temperature, stir 24h; Fully, after reaction, at room temperature dropwise slowly add 150 μ L1M NaCl, under dark condition, react 24h; Rotating speed centrifuging 10min by completely reacted solution with 12000rpm, is then scattered in 300mM NaCl solution again, obtains DNA1-AuNPs or DNA2-AuNPs, is suspended from 50mM Tris-Hcl damping fluid for subsequent use; The DNA2-AuNPs preparing is processed with 1 μ M cDNA, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is that the sequence of 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', DNA1 is
5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ', the sequence of DNA2 is 5 '-SH-C6-CGTTTAGGATTTGTG-3 ';
3) layer assembly of DNA-AuNPs nanometer network structure: before gold electrode is assembled, first gold electrode is used respectively to α-Al of 0.3 and 0.05 μ m 2o 3polishing grinding in paste, and clean 1min in ethanol and deionized water for ultrasonic successively, and at the H of 0.5M 2sO 4in solution, adopt three-electrode system to carry out electrochemical cleaning and activation; It is in 7.4 the phosphate buffer that contains 500 μ M kemptides that the gold electrode of handling well is soaked in to 0.05M pH, and is placed under room temperature and reacts 12h, and then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Seal blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted and contains 20mM MgCl 2in the damping fluid of solution, protein kinase A and 100 μ M ATP, at 37 DEG C of reaction 1h, obtain phosphorylation kemptide modified electrode; The kemptide modified electrode that completes phosphorylation is cleaned up, use respectively subsequently 0.5mM Zr 4+process this electrode with 6 μ L nanometer beacon DNA1-AuNPs, pass through Zr 4+nanometer beacon group is loaded on to the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this TBS, intermediate water rinse and N for modified electrode 2dry up; This electrode continuous after cDNA-DNA2-AuNPs and DNA1-AuNPs process, form DNA-AuNPs nanometer network structure, after layer assembly through TBS process, rinse, obtain DNA-AuNPs nano-electrode; The method is prepared obtained electrode and is tested for phosphorylation.
As shown in Figure 1, first by the effect of Au-S key, the kemptide of halfcystine functionalization is directly fixed to the assembled layers of gold electrode surfaces formation one deck densification, mercapto hexane is used to the blank site on enclosed-electrode surface, to eliminate non-specific binding, taking ATP after coreagent protein kinase A catalysis phosphorylation reaction, use respectively Zr 4+the nanometer beacon DNA1-Au NPs having prepared processes this electrode, passes through Zr 4+ground floor nanometer beacon group is loaded on the electrode of kemptide modification of phosphorylation with the coordination of phosphate radical; Subsequently, this electrode after cDNA-DNA2-Au NPs and DNA1-Au NPs processing, forms a DNA-Au NPs nanometer network structure continuously gradually; Because golden nanometer particle and DNA itself are with a large amount of negative charges, be conducive to the absorption of this electroactive material of RuHeX; And utilize the complementary performance of DNA to form the reticulate texture of DNA-Au NPs, for the absorption of electroactive material provides a large amount of sites, be beneficial to the rapid enhancing of electrochemical signals.
The process of protein phosphorylation is determined by Fourier's infrared spectrum, adopts Fourier's infrared spectrum (FTIR) analysis to use QUINX55 spectroscope (Germany), as shown in from Fig. 2, at 995cm -1and 1070cm -1there are two extra peaks at place, infers due to the former because existence of phosphate radical, illustrate that phosphate radical transfers to the electrode of kemptide modification from ATP.
Cyclic voltammetric and electrochemical impedance are often used to characterize the process that electrode surface fills layer by layer, and as shown in Figure 3, (B) figure is with Fe (CN) 6 4-/3-do signal probe and adopt cyclic voltammetry to use CHI802B electrochemical analyser (Shanghai, occasion China) to characterize the schematic diagram of electrode assembling process, as can be seen from the figure, naked gold electrode shows a pair of perfect redox peak; In the time of electrode face finish one deck kemptide film, CV electric current declines, and two peak-to-peak width become large, and this is because the inertia of kemptide itself has hindered the transmission of electronics at electrode surface; After the sealing processing and generation phosphorylation reaction of the blank site of electrode surface through mercapto hexane, peak current obviously declines, the difference of the spike potential at two peaks is larger, and the process of this explanation phosphorylation has been introduced electronegative phosphate radical, thereby signal probe is produced to Coulomb repulsion.Pass through Zr at ground floor DNA-Au NPs 4+while modifying the electrode surface of phosphorylation, the poor variation of electric current and spike potential is increasing, and this is mainly because Au NPs and DNA itself are electronegative, hinders Fe (CN) 6 4-/3-near electrode surface; Along with layer assembly is constantly carried out, the continuous decline of electric current and spike potential are poor increasing, illustrate that this network structure successfully forms at electrode surface.The measurement of electrochemical impedance spectroscopy (EIS) is used SP-150 electro-chemical systems (Bio-Logic, France), result is as shown in the impedance curve of (A) figure, the diameter of half circle is equivalent to the impedance that electrode surface electronics shifts, along with kemptide, sealer are modified at the generation of electrode surface and phosphorylation process, compared with naked gold electrode, the diameter of half circle constantly increases, and being successfully completed of these processes has been described.Along with the layer assembly of DNA-Au NPs, it is large that half circle in AC impedance curve also constantly becomes, and also further illustrates the impedance of electronics transfer in continuous increase, and nanometer network structure forms gradually.This is consistent with the result that cyclic voltammetry characterizes.
Synthetic Au NPs uses transmission electron microscope and ultra-violet absorption spectrum to characterize, wherein sem image (TEM) records through FDAC H-800, it is in Ultravioblet spectrophotometer UV3900 (Japan that ultraviolet detects, Hitachi) on carry out, as shown in Figure 4, result shows that the diameter of synthetic golden nanometer particle is approximately 15～18nm.Single Au NPs has a characteristic absorption peak at 520nm place, and in the time that DNA is bonded on golden nanometer particle surface by Au-S key, characteristic absorption peak is transferred to 525nm from 520nm; And in the time forming complementary strand, Absorption Characteristics peak continues to transfer to 528nm, thereby has proved the formation of cDNA-DNA2-Au NPs.
Fig. 5 has shown that the kemptide of phosphorylation and the electrode of unphosphorylated kemptide modification are at the electrochemical behavior through after nanometer network structure and electroactive material processing, both all have peak current, but the former peak current intensity is more much larger than the latter, this explanation kemptide modified electrode has been introduced phosphate radical in phosphorylation process, passes through Zr 4+coordination, be beneficial to nanometer network structural modification at electrode surface, for active substance provides a large amount of load sites, greatly strengthened electrochemical signals.Interior illustration is a series of contrast test, has shown at Zr 4+in non-existent situation, electrochemical signals does not significantly strengthen, and Zr is described in the assembling process of this sensor 4+play vital effect.Compare with a in Fig. 2, only have the electrochemical response of the gold electrode of kemptide and mercapto hexane modification just to seem very weak, this is mainly to be caused by the few non-specific adsorption effect of electrode pair DNA-Au NPs.Above result shows, this nanometer network structure can be used as the sensor that carrier that electroactive material is good is prepared a sensitive simple general-purpose, for the activity analysis of protein kinase.
In enzymic catalytic reaction, temperature is one of key factor affecting enzymatic activity.Too low temperature or too high temperature can make the even inactivation of activity decreased of enzyme.Therefore, we have optimized temperature required in phosphorylation reaction process.As shown in Figure 6, in the time of 37 DEG C, the response signal of electrode is the strongest, and the activity of protein kinase is the highest, therefore, and 37 DEG C of Optimal Temperature that are phosphorylation reaction.Because ATP is used to provide phosphate radical in phosphorylation reaction process, therefore the concentration of ATP is also a key factor that affects phosphorylation reaction.Along with the continuous increase of ATP concentration, the response signal of modified electrode also constantly strengthens, and when ATP concentration arrives 40 μ M, electrode response reaches maximal value, and is not changing along with the increase of ATP concentration.Therefore,, in the time that protein kinase A concentration is 25U/mL, the optimal concentration of ATP is 40 μ M.
The present invention also provides a kind of DNA-AuNPs nanometer network structure of preparing according to said method.
The present invention also provides a kind of sensor based on DNA-AuNPs nanometer network structure construction.
The present invention also provides a kind of and detects for the activity of hyclone protein kinase based on DNA-AuNPs nanometer network sensor.
The present invention also provides a kind of and detects for the activity of kinases inhibitor based on DNA-AuNPs nanometer network sensor.
The application of the sensor building based on nano-electrode DNA-Au NPs in protein kinase activity and inhibitor thereof are analyzed, cyclic voltammetric and electrochemical AC impedance are with 5mM K 3[Fe (CN) 6]/K 4[Fe (CN) 6] be redox probe, and carry out in the electrolyte solution that contains 0.5M KCl, the frequency range of EIS is 100mHz～100kHz; Nano-electrode DNA-Au NPs is immersed in to 50 μ M RuHex reaction 10min, the straight-through N of this process one 2, detection signal in blank TBS damping fluid, scanning voltage :-0.3～+ 0.7V, sweep speed is 100mV/s.
Protein kinase activity detects
At optimal conditions, the electrochemical response of the protein kinase by variable concentrations come the kinase whose active situation of evaluating protein wherein differential pulse volt-ampere detect (DPV) and use CHI802B electrochemical analyser (Shanghai, occasion China), as shown in Figure 7, along with the raising of protein kinase concentration, the DPV response of this sensor is more and more stronger, arrives a saturation point (concentration is 100U/mL) always; Interior illustration is the DPV response of sensor and the linear relationship curve of PKA concentration, and the range of linearity is 0.1～40U/mL, and detection is limited to: 0.03U/mL (S/N=3).The reappearance of this sensor also further confirmed, the certain density PKA of electrode detection (25U/mL) preparing with same Zhi Xinxian assesses.Learn from analysis result, the deviation detecting between different sensors is 4.96%.Above result shows, this sensor can be successfully applied to Sensitive Determination protein kinase activity.
Inhibitor is analyzed
By the biology sensor preparing equally for the research of small-molecule substance kinases inhibitor, wherein differential pulse volt-ampere detects (DPV) and uses CHI802B electrochemical analyser (Shanghai, occasion China), as shown in a line in Fig. 8, along with inhibitor ellagic acid concentration constantly increases from 0-15 μ M, the signal of DPV response also constantly weakens, and in the time that its concentration is increased to 10 μ M, response signal is the most weak to no longer changing.Through calculating, the IC of this inhibitor 50=4.01 μ M; As shown in b line in Fig. 8, TYR inhibitors of kinases Tyrphostin AG1478 but not the inhibitor of protein kinase is also used to assess the selectivity of this sensor, along with the increase of Tyrphostin AG1478 concentration, DPV response does not change, and this has illustrated that this sensor can realize the successful screening to inhibitor.
The application of actual sample
Activity analysis by this sensor for hyclone protein kinase A, first the TBS damping fluid dilution with 1ml50 μ M pH=7.4 by 0.1ml peptide cow's serum, get the TBS damping fluid of 45 μ L with hyclone, add 5 μ L protein kinase A, make the ultimate density of protein kinase A be respectively 25U/mL and 45U/mL, detect its activity, result is as shown in table 1, the relative deviation detecting is respectively 2.17% and 1.60%, is all less than 5%.Result shows that this sensor is successfully applied in the activity detection of protein kinase in hyclone as a kind of reliable method.
Table 1 detects for the activity of hyclone protein kinase A based on DNA-AuNPs nanometer network sensor
Wherein, protein kinase A (PKA, catalytic subunit is from cor bovinum), six ammino rutheniums (RuHex), ellagic acid (Ellagic acid), TYR inhibitor (Tyrphostin AG1478) is all bought from Sigma company (U.S.), kemptide (the Kemptide of cysteine modified, CLRRASLG) buy from gill biochemistry (Shanghai), ATP Disodium Salt (ATP) is provided by ancient cooking vessel state biology (Beijing), mercapto hexane (1-Hexanethiol) derives from hundred Ming Wei companies (Beijing), HAuCl43H2O (48%w/w) buys from Solution on Chemical Reagents in Shanghai company, cDNA (5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 '), synthetic and the purifying of DNA1 (5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ') and DNA2 (5 '-SH-C6-CGTTTAGGATTTGTG-3 ') completes preparation by the raw work in Shanghai, other analytical reagent is all bought in Beijing chemical company.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendments that creative work can make or distortion still in protection scope of the present invention.

Claims (6)

1. a preparation method for DNA-AuNPs nanometer network structure, is characterized in that, comprises the steps:
1) preparation of Au NPs: 100mL0.01% (w/v) HAuCl 4aqueous solution is heated to reflux, and 2.5mL1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps boiling 15min, solution by colourless become claret gradually till, obtain Au NPs, save backup in 4 DEG C;
2) preparation of DNA functionalization Au NPs: first DNA prevents from forming disulfide bond through TCEP activation 2h; DNA1 or DNA2 are joined in 1mL Au NPs, under room temperature, stir 24h; Fully, after reaction, at room temperature dropwise slowly add 150 μ L1M NaCl, under dark condition, react 24h; Rotating speed centrifuging 10min by completely reacted solution with 12000rpm, is then scattered in 300mM NaCl solution again, obtains DNA1-AuNPs or DNA2-AuNPs, is suspended from 50mM Tris-Hcl damping fluid for subsequent use; The DNA2-AuNPs preparing is processed with 1 μ McDNA, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is that the sequence of 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', DNA1 is 5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ', and the sequence of DNA2 is 5 '-SH-C6-CGTTTAGGATTTGTG-3 ';
3) layer assembly of DNA-AuNPs nanometer network structure: before gold electrode is assembled, first gold electrode is used respectively to α-Al of 0.3 μ m and 0.05 μ m 2o 3polishing grinding in paste, and clean 1min in ethanol and deionized water for ultrasonic successively, and at the H of 0.5M 2sO 4in solution, adopt three-electrode system to carry out electrochemical cleaning and activation; It is in 7.4 the phosphate buffer that contains 500 μ M kemptides that the gold electrode of handling well is soaked in to 0.05M pH, and is placed under room temperature and reacts 12h, and then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Seal blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted and contains 20mMMgCl 2in the damping fluid of solution, protein kinase A and 100 μ M ATP, at 37 DEG C of reaction 1h, obtain phosphorylation kemptide modified electrode; The kemptide modified electrode that completes phosphorylation is cleaned up, use respectively subsequently 0.5mM Zr 4+process this electrode with 6 μ L nanometer beacon DNA1-AuNPs, pass through Zr 4+nanometer beacon group is loaded on to the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this TBS, intermediate water rinse and N for modified electrode 2dry up; This electrode continuous after cDNA-DNA2-AuNPs and DNA1-AuNPs process, form DNA-AuNPs nanometer network structure, after layer assembly through TBS process, rinse, obtain DNA-AuNPs nano-electrode.
2. a kind of DNA-AuNPs nanometer network structure that prepared by the method for claim 1.
3. a kind of sensor based on DNA-AuNPs nanometer network structure construction claimed in claim 2.
4. the application that the activity based on sensor claimed in claim 3 protein kinase in hyclone detects.
5. the application detecting in the activity of kinases inhibitor based on sensor claimed in claim 3.
6. the application of the sensor as described in claim 4 or 5, is characterized in that, cyclic voltammetric and electrochemical AC impedance are with 5mM K 3[Fe (CN) 6]/K 4[Fe (CN) 6] be redox probe, and carry out in the electrolyte solution that contains 0.5M KCl, the frequency range of EIS is 100mHz～100kHz; Nano-electrode DNA-Au NPs is immersed in to 50 μ M RuHex reaction 10min, the straight-through N of this process one 2, detection signal in blank TBS damping fluid, scanning voltage :-0.3～+ 0.7V, sweep speed is 100mV/s.
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