CN103940890B - 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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CN103940890B
CN103940890B CN201410168197.XA CN201410168197A CN103940890B CN 103940890 B CN103940890 B CN 103940890B CN 201410168197 A CN201410168197 A CN 201410168197A CN 103940890 B CN103940890 B CN 103940890B
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CN103940890A (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 of DNA-AuNPs Specific surface area and application
Technical field
The present invention relates to electrochemical sensor field, particularly relate to a kind of preparation method and application of DNA-AuNPs Specific surface area.
Background technology
The protein phosphorylation regulated by kinases is a biological metabolic important process, and it plays important effect in intracellular signaling and management cellular activity.Protein kinase dysregulation can upset the system of protein phosphorylation, causes the generation of various disease.Therefore, identify that protein kinase activity and inhibitor thereof are not only conducive to understanding clearly bio-metabolic process, and be conducive to early detection and the treatment in time of disease.
According to the existence of Phosphorylation events with or without phosphate radical, metallic ion Zr 4+become the effective reagent of the one of catching enriched phosphorus acidifying.Utilize the strong coordination of metallic ion and phosphate radical, Zr 4+be commonly used to the important coupling agent as the phosphorylation site on a kind of connecting peptides and beacon.Because the end of DNA own is with phosphate radical or the part that is easy to modify other group and is often used as beacon.Divide compared to simple sandwich structure or signal amplifying part, Specific surface area (or super sandwich structure) shows very high sensitivity and is subject to the favor of researcher 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 Specific surface area, it has amplifies electrochemical signals, detects the sensitive advantage such as reliably.
The present invention is achieved by the following technical solutions:
A preparation method for DNA-AuNPs Specific surface area, step is as follows:
1) preparation of Au NPs: 100mL0.01% (w/v) HAuCl 4aqueous solution is heated to backflow, and 2.5mL1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps the 15min that seethes with excitement, 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 activates 2h through TCEP and prevents formation disulfide bond; DNA1 or DNA2 is joined in 1mL Au NPs, stirred at ambient temperature 24h; After abundant reaction, at room temperature dropwise slowly add 150 μ L1M NaCl, under dark condition, react 24h; By completely reacted solution with the rotating speed centrifuging 10min of 12000rpm, be then again scattered in 300mM NaCl solution, obtain DNA1-AuNPs or DNA2-AuNPs, be suspended from 50mM Tris-Hcl damping fluid for subsequent use; By the DNA2-AuNPs 1 μM of cDNA process prepared, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', and its nucleotide sequence is as shown in SEQ IDNO.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 Specific surface area: before gold electrode is assembled, first uses the α-Al of 0.3 and 0.05 μm respectively by gold electrode 2o 3polishing grinding in paste, and successively at ethanol and deionized water for ultrasonic cleaning 1min, and at the H of 0.5M 2sO 4three-electrode system is adopted to carry out electrochemical cleaning and activation in solution; The gold electrode handled well is soaked in 0.05M pH be 7.4 containing in the phosphate buffer of 500 μMs of kemptides, and react 12h under being placed in room temperature, then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Close blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted containing 20mMMgCl 2in the damping fluid of solution, protein kinase A and 100 μMs of ATP, at 37 DEG C of reaction 1h, obtain phosphorylated Kemptide modified electrode; The kemptide modified electrode completing phosphorylation is cleaned up, uses 0.5mM Zr respectively subsequently 4+with 6 these electrodes of μ L nanometer beacon DNA1-AuNPs process, pass through Zr 4+nanometer beacon group is loaded on the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this modified electrode TBS, intermediate water rinse and N 2dry up; This electrode, continuous through after cDNA-DNA2-AuNPs and DNA1-AuNPs process, forms DNA-AuNPs Specific surface area, through TBS process, rinse after layer assembly, obtains DNA-AuNPs nano-electrode; The method prepares obtained electrode for phosphorylation assays.
The present invention also provides a kind of DNA-AuNPs Specific surface area prepared according to said method.
The present invention also provides a kind of sensor built based on DNA-AuNPs Specific surface area.
The present invention also provides a kind of based on the Activity determination of DNA-AuNPs nanometer network sensor for protein kinase in hyclone.
The present invention also provides a kind of based on the Activity determination of DNA-AuNPs nanometer network sensor for kinases inhibitor.
Based on the application of sensor in protein kinase activity and inhibitor thereof are analyzed that DNA-Au NPs Specific surface area builds, 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 containing in the electrolyte solution of 0.5M KCl, the frequency range of EIS is 100mHz ~ 100kHz; Nano-electrode DNA-Au NPs is immersed in 50 μMs of RuHex and reacts 10min, this process one leads directly to N 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 Specific surface area, because Au NPs and DNA itself is with a large amount of negative charge, be conducive to the absorption of this electroactive material of RuHeX; And utilizing the performance of the complementation of DNA to form the reticulate texture of DNA-Au NPs, the absorption for electroactive material provides a large amount of sites, thus realizes the amplification of electrochemical signals;
2. the electrochemical signals based on DNA-Au NPs Specific surface area amplifies for protein kinase activity and inhibitor analysis thereof;
3. by the coordination of zirconium ion, be beneficial to Specific surface area and be modified at electrode surface, for active substance provides a large amount of load sites, greatly enhance electrochemical signals, therefore, the lowest detection limit value of this biology sensor to kinase activity reaches 0.03U/mL, and Simultaneous Stabilization is high, the range of linearity is wide.
Accompanying drawing explanation
Fig. 1 is at the schematic diagram that protein kinase activity detects and inhibitor is analyzed based on DNA-Au NPs Specific surface area;
Fig. 2 is the FTIR spectrum figure of protein phosphorylation process;
Fig. 3 is electrochemical impedance figure (A) and the cyclic voltammogram (B) of different modifying electrode;
The Electronic Speculum figure that Fig. 4 (A) is Au NPs, (B) is Au NPs (a), Au NPs-DNA (b) and the uv absorption spectrogram of cDNA-DNA2-Au NPs-cDNA (c);
Fig. 5 is the electrochemical response figure of different modifying electrode;
Fig. 6 is that temperature (A) and ATP concentration (B) are to the effect diagram of 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 electrochemical strengths-a) concentration curve of inhibitor ellagic acid and b) Tyrphostin AG147, 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 Specific surface area, step is as follows:
1) preparation of Au NPs: 100mL0.01% (w/v) HAuCl 4aqueous solution is heated to backflow, and 2.5mL1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps the 15min that seethes with excitement, 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 activates 2h through TCEP and prevents formation disulfide bond; DNA1 or DNA2 is joined in 1mL Au NPs, stirred at ambient temperature 24h; After abundant reaction, at room temperature dropwise slowly add 150 μ L1M NaCl, under dark condition, react 24h; By completely reacted solution with the rotating speed centrifuging 10min of 12000rpm, be then again scattered in 300mM NaCl solution, obtain DNA1-AuNPs or DNA2-AuNPs, be suspended from 50mM Tris-Hcl damping fluid for subsequent use; By the DNA2-AuNPs 1 μM of cDNA process prepared, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', the sequence of DNA1 is
The sequence of 5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ', DNA2 is 5 '-SH-C6-CGTTTAGGATTTGTG-3 ';
3) layer assembly of DNA-AuNPs Specific surface area: before gold electrode is assembled, first uses the α-Al of 0.3 and 0.05 μm respectively by gold electrode 2o 3polishing grinding in paste, and successively at ethanol and deionized water for ultrasonic cleaning 1min, and at the H of 0.5M 2sO 4three-electrode system is adopted to carry out electrochemical cleaning and activation in solution; The gold electrode handled well is soaked in 0.05M pH be 7.4 containing in the phosphate buffer of 500 μMs of kemptides, and react 12h under being placed in room temperature, then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Close blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted containing 20mMMgCl 2in the damping fluid of solution, protein kinase A and 100 μMs of ATP, at 37 DEG C of reaction 1h, obtain phosphorylated Kemptide modified electrode; The kemptide modified electrode completing phosphorylation is cleaned up, uses 0.5mM Zr respectively subsequently 4+with 6 these electrodes of μ L nanometer beacon DNA1-AuNPs process, pass through Zr 4+nanometer beacon group is loaded on the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this modified electrode TBS, intermediate water rinse and N 2dry up; This electrode, continuous through after cDNA-DNA2-AuNPs and DNA1-AuNPs process, forms DNA-AuNPs Specific surface area, through TBS process, rinse after layer assembly, obtains DNA-AuNPs nano-electrode; The method prepares obtained electrode for phosphorylation assays.
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 that gold electrode surfaces forms one deck densification, mercapto hexane is used to the blank site on enclosed-electrode surface, to eliminate non-specific binding, after taking ATP as coreagent protein kinase A catalytic phosphorylation reaction, use Zr respectively 4+this electrode of nanometer beacon DNA1-Au NPs process prepared, passes through Zr 4+on the electrode kemptide that ground floor nanometer beacon group is loaded on phosphorylation modified with the coordination of phosphate radical; Subsequently, this electrode, continuously across after cDNA-DNA2-AuNPs and DNA1-Au NPs process, forms a DNA-Au NPs Specific surface area gradually; Because golden nanometer particle and DNA itself are with a large amount of negative charge, be conducive to the absorption of this electroactive material of RuHeX; And utilizing the performance of the complementation of DNA to form the reticulate texture of DNA-Au NPs, the absorption for electroactive material provides a large amount of sites, is beneficial to the rapid enhancing of electrochemical signals.
The process of protein phosphorylation is determined by FTIR spectrum, adopts FTIR 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 that reason is caused by the existence of phosphate radical, illustrates that phosphate radical is transferred 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-doing signal probe adopts cyclic voltammetry to use CHI802B electrochemical analyser (Shanghai, occasion China) to characterize the schematic diagram of electrode assembling process, and as can be seen from the figure, naked gold electrode shows a pair perfect redox peak; When electrode face finish one deck kemptide film, CV electric current declines, and two peak-to-peak width become large, this is because the inertia of kemptide itself hinders the transmission of electronics at electrode surface; The blank site of electrode surface is after the Seal treatment and generation phosphorylation reaction of mercapto hexane, peak current obviously declines, the difference of the spike potential at two peaks is larger, and this illustrates that the process of phosphorylation has introduced electronegative phosphate radical, thus produces Coulomb repulsion to signal probe.Zr is passed through at ground floor DNA-Au NPs 4+when modifying the electrode surface of phosphorylation, the change of electric current and spike potential difference is increasing, and this, mainly because Au NPs and DNA itself is electronegative, hinders Fe (CN) 6 4-/3-near electrode surface; Along with layer assembly is constantly carried out, electric current constantly declines increasing with spike potential difference, illustrates that this network structure is successfully formed at electrode surface.The measurement of electrochemical impedance spectroscopy (EIS) uses 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 of electrode surface electro transfer, along with kemptide, sealer are modified at the generation of electrode surface and Phosphorylation events, compared with naked gold electrode, the diameter of half circle constantly increases, and describes being successfully completed of these processes.Along with the layer assembly of DNA-Au NPs, half circle in AC impedance curve also constantly becomes large, and also further illustrate the impedance of electro transfer in continuous increase, Specific surface area is formed 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, UV detect is in Ultravioblet spectrophotometer UV3900 (Japan, Hitachi) on to carry out, as shown in Figure 4, result shows that the diameter of the golden nanometer particle synthesized is approximately 15 ~ 18nm.Single Au NPs has a characteristic absorption peak at 520nm place, when DNA by Au-S key be bonded in golden nanometer particle on the surface time, characteristic absorption peak transfers to 525nm from 520nm; And when forming complementary strand, Absorption Characteristics peak continues to transfer to 528nm, thus demonstrates the formation of cDNA-DNA2-Au NPs.
The electrode of kemptide and the modification of unphosphorylated kemptide that Fig. 5 illustrates phosphorylation is at the electrochemical behavior after Specific surface area and electroactive material process, both all have peak current, but the former peak current intensity is more much larger than the latter, this illustrates that kemptide modified electrode introduces phosphate radical in Phosphorylation events, passes through Zr 4+coordination, be beneficial to Specific surface area and be modified at electrode surface, for active substance provides a large amount of load sites, greatly enhance electrochemical signals.Interior illustration is a series of contrast test, shows at Zr 4+in non-existent situation, electrochemical signals does not significantly strengthen, and illustrates in the assembling process of this sensor, Zr 4+serve vital effect.Compared with a in Fig. 2, the electrochemical response of the gold electrode only having kemptide and mercapto hexane to modify just seems very weak, and this is mainly caused by the non-specific adsorption effect that electrode pair DNA-Au NPs is few.Above result shows, this Specific surface area can be used as the sensor that the good carrier of electroactive material prepares 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 activity of enzyme reduce even inactivation.Therefore, we optimize temperature required in phosphorylation reaction process.As shown in Figure 6,37 DEG C time, the response signal of electrode is the strongest, and the activity of protein kinase is the highest, and therefore, 37 DEG C is the Optimal Temperature of phosphorylation reaction.Because ATP is used to provide phosphate radical in phosphorylation reaction process, therefore the concentration of ATP is also the key factor affecting 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 μMs, electrode response reaches maximal value, and along with the increase of ATP concentration not in change.Therefore, when protein kinase A concentration is 25U/mL, the optimal concentration of ATP is 40 μMs.
The present invention also provides a kind of DNA-AuNPs Specific surface area prepared according to said method.
The present invention also provides a kind of sensor built based on DNA-AuNPs Specific surface area.
The present invention also provides a kind of based on the Activity determination of DNA-AuNPs nanometer network sensor for protein kinase in hyclone.
The present invention also provides a kind of based on the Activity determination of DNA-AuNPs nanometer network sensor for kinases inhibitor.
Based on the application of sensor in protein kinase activity and inhibitor thereof are analyzed that nano-electrode DNA-Au NPs builds, 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 containing in the electrolyte solution of 0.5M KCl, the frequency range of EIS is 100mHz ~ 100kHz; Nano-electrode DNA-Au NPs is immersed in 50 μMs of RuHex and reacts 10min, this process one leads directly to N 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, by the electrochemical response of the protein kinase of variable concentrations come the kinase whose active situation of evaluating protein wherein differential pulse Voltammetric detection (DPV) 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 is also confirmed further, and the certain density PKA of the electrode detection prepared with same Zhi Xinxian (25U/mL) assesses.Learn from analysis result, the deviation detected between different sensors is 4.96%.Above result shows, this sensor can be successfully applied to Sensitive Determination protein kinase activity.
Inhibitor is analyzed
The biology sensor prepared is used for equally the research of small-molecule substance kinases inhibitor, wherein differential pulse Voltammetric detection (DPV) 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 μM, the signal of DPV response also constantly weakens, and when its concentration is increased to 10 μMs, response signal is the most weak to no longer changing.Through calculating, the IC of this inhibitor 50=4.01 μMs; 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 the selectivity assessing this sensor, along with the increase of Tyrphostin AG1478 concentration, DPV response does not change, and which illustrates the successful screening that this sensor can realize inhibitor.
The application of actual sample
This sensor is used for the activity analysis of protein kinase A in hyclone, first the TBS damping fluid of 0.1ml peptide cow's serum with 1ml50 μM of pH=7.4 is diluted, 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 detected is respectively 2.17% and 1.60%, is all less than 5%.Result shows that this sensor is successfully applied in the Activity determination of protein kinase in hyclone as a kind of reliable method.
Table 1 is used for the Activity determination of protein kinase A in hyclone based on DNA-AuNPs nanometer network sensor
Wherein, protein kinase A (PKA, catalytic subunit is from cor bovinum), six ammino rutheniums (RuHex), ellagic acid (Ellagicacid), 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 '), synthesis and the purifying of DNA1 (5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ') and DNA2 (5 '-SH-C6-CGTTTAGGATTTGTG-3 ') complete 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 amendment or distortion that creative work can make still within protection scope of the present invention.

Claims (5)

1. a preparation method for DNA-AuNPs Specific surface area, is characterized in that, comprises the steps:
1) preparation of Au NPs: 100mL 0.01% (w/v) HAuCl 4aqueous solution is heated to backflow, and 2.5mL 1% (w/v) sodium citrate is rapidly to entering in three-neck flask, and whole reaction keeps the 15min that seethes with excitement, 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 activates 2h through TCEP and prevents formation disulfide bond; DNA1 or DNA2 is joined in 1mL Au NPs, stirred at ambient temperature 24h; After abundant reaction, at room temperature dropwise slowly add 150 μ L 1M NaCl, under dark condition, react 24h; By completely reacted solution with the rotating speed centrifuging 10min of 12000rpm, be then again scattered in 300mM NaCl solution, obtain DNA1-AuNPs or DNA2-AuNPs, be suspended from 50mM Tris-Hcl damping fluid for subsequent use; By the DNA2-AuNPs 1 μM of cDNA process prepared, obtain cDNA-DNA2-Au NPs, wherein the sequence of cDNA is 5 '-CAGACTACTACAAGCTTTCACAAATCCTAAACG-3 ', the sequence of DNA1 be the sequence of 5 '-P-GCTTGTAGTAGTCTG-C6-SH-3 ', DNA2 is 5 '-SH-C6-CGTTTAGGATTTGTG-3 ';
3) layer assembly of DNA-AuNPs Specific surface area: before gold electrode is assembled, first uses the α-Al of 0.3 μm and 0.05 μm respectively by gold electrode 2o 3polishing grinding in paste, and successively at ethanol and deionized water for ultrasonic cleaning 1min, and at the H of 0.5M 2sO 4three-electrode system is adopted to carry out electrochemical cleaning and activation in solution; The gold electrode handled well is soaked in 0.05M pH be 7.4 containing in the phosphate buffer of 500 μMs of kemptides, and react 12h under being placed in room temperature, then gold electrode rinses through a large amount of phosphate buffers and intermediate water, dry, obtains kemptide modified electrode; Close blank site with 1mM mercapto hexane, then kemptide modified electrode is inserted containing 20mMMgCl 2in the damping fluid of solution, protein kinase A and 100 μMs of ATP, at 37 DEG C of reaction 1h, obtain phosphorylated Kemptide modified electrode; The kemptide modified electrode completing phosphorylation is cleaned up, uses 0.5mM Zr respectively subsequently 4+with 6 these electrodes of μ L nanometer beacon DNA1-AuNPs process, pass through Zr 4+nanometer beacon group is loaded on the kemptide modified electrode of phosphorylation with the coordination of phosphate radical; Finally, this modified electrode TBS, intermediate water rinse and N 2dry up; This electrode, continuous through after cDNA-DNA2-AuNPs and DNA1-AuNPs process, forms DNA-AuNPs Specific surface area, through TBS process, rinse after layer assembly, obtains DNA-AuNPs nano-electrode.
2. a kind of DNA-AuNPs Specific surface area of preparing of the method for claim 1.
3. based on a kind of sensor that DNA-AuNPs Specific surface area according to claim 2 builds.
4. based on the application of sensor according to claim 3 Activity determination of protein kinase or kinases inhibitor in hyclone.
5. the application of sensor as claimed in claim 4, 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 containing in the electrolyte solution of 0.5M KCl, the frequency range of EIS is 100mHz ~ 100kHz; Nano-electrode DNA-Au NPs is immersed in 50 μMs of RuHex and reacts 10min, this process one leads directly to N 2, detection signal in blank TBS damping fluid, scanning voltage :-0.3 ~+0.7V, sweep speed is 100mV/s.
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