An electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker

Abstract

The invention discloses an electrochemical analysis method for measuring the activity of T4 polynucleotide kinase based on a three-dimensional DNA walker; the three-dimensional DNA walker provided by the invention is formed by modifying the surface of gold nanoparticles with hairpin DNA, and the hairpin DNA is phosphorylated by T4 polynucleotide kinase and sheared by exonuclease to produce the three-dimensional DNA walker; the three-dimensional DNA walker is driven by endonuclease to walk on the surface of gold nanoparticles, and the electrochemically active fragment Fc-DNA in the ferrocene-labeled DNA probe is sheared and released; the released Fc-DNA is hybridized with the methylene blue-labeled DNA probe on the electrode surface to achieve dual ratio electrochemical response signal amplification; the invention is simple to operate, low in cost, non-radioactive, highly sensitive and highly selective. The ratio electrochemical response signal is proportional to the concentration of T4 polynucleotide kinase, and a high-sensitivity and high-selectivity determination of T4 polynucleotide kinase is achieved.

Description

Technical Field

[0001] The invention relates to the field of biochemical analysis and detection technology, and in particular to an electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker and enzyme-assisted amplification technology. Background Art

[0002] T4 polynucleotide kinase (T4PNK) is isolated from bacteriophage T4, referred to as T4 kinase, with 5' kinase activity, which can catalyze the transfer of ATP's γ-position phosphate to the 5' hydroxyl end of oligonucleotide chains or nucleic acids, and is closely related to normal cell physiological activities such as DNA recombination, replication, and damage repair. Abnormal expression of kinase activity can lead to abnormal phosphorylation of proteins, resulting in a variety of human diseases such as Werner syndrome, Loom's syndrome, Rothmund-Thomson syndrome and other major diseases. In addition, T4PNK is also an important molecular biology tool. The discovery and application of T4PNK has promoted the development of molecular biology to a certain extent. At present, T4PNK has become an indispensable tool enzyme in genetic engineering and bioanalysis research, and is further used in the research of nucleic acid damage repair and enzyme inhibitors. Therefore, the development of T4 polynucleotide kinase detection technology is helpful to provide a powerful tool for biological research and clinical diagnosis.

[0003] As a new analytical detection technology, electrochemical sensors have the characteristics of high sensitivity, strong specificity and good stability, and are widely used in disease diagnosis, food safety and environmental monitoring. The performance of electrochemical sensors is affected by the properties of the sensitive interface, the modified materials and the process. In order to improve the detection performance of electrochemical sensors, people have developed a variety of signal amplification strategies with stable performance and high reliability. Among them, DNA walker signal amplification technology has attracted widespread attention due to its simple structure, easy synthesis, predictability and programmability of sequence. Combined with electrochemical biosensors, DNA walkers are nanoscale molecular devices driven by environmental stimuli, enzyme reactions or strand displacement reactions. They can perform repeated mechanical cyclic motions along DNA tracks composed of part or all of nucleic acids to achieve signal cascade amplification, which provides a promising tool for designing sensor response signal amplification methods. In addition, three-dimensional DNA walkers constructed based on different nanomaterials (such as gold nanoparticles, magnetic beads, etc.) as track carriers can be more stably connected to the track during walking due to their large specific surface area, high loading capacity and high local walking chain concentration, which makes up for the deficiency of monopodial or bipodial DNA walkers that are easily derailed during the reaction, and thus exhibits stronger signal amplification capabilities and better sensing analysis and detection performance.

[0004] Traditional methods for detecting T4PNK activity mainly include radioisotope 32 P labeling, polyacrylamide gel electrophoresis and radioautography. These methods generally have the disadvantages of being discontinuous, cumbersome and time-consuming, requiring special equipment, prone to radioactive contamination, or low selectivity and sensitivity. In recent years, some new methods for detecting T4PNK activity have been developed, such as colorimetric analysis, fluorescence analysis, electrochemical analysis, chemiluminescence and fluorescence imaging analysis technology. Although these methods have high sensitivity, the operation process is cumbersome and the cost is high, and their application is limited to a certain extent.

[0005] Currently, there is a lack of an electrochemical analysis method for measuring T4 polynucleotide kinase activity based on three-dimensional DNA walker and enzyme-assisted amplification technology that can achieve high sensitivity, high selectivity, rapid and quantitative detection of T4PNK activity. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an electrochemical analysis method for determining T4 polynucleotide kinase activity with high sensitivity and good selectivity.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows: In the first aspect, the present invention provides an electrochemical analysis method for determining the activity of T4 polynucleotide kinase based on a three-dimensional DNA walker; in the second aspect, the present invention provides an electrochemical biosensor prepared by the electrochemical analysis method for determining the activity of T4 polynucleotide kinase based on a three-dimensional DNA walker.

[0008] The present invention discloses an electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker, comprising the following steps: (1) designing a hairpin DNA sequence; the hairpin DNA has a nucleotide sequence shown in SEQ ID No. 1;

[0009] (2) designing a ferrocene-labeled DNA probe sequence; the ferrocene-labeled DNA probe has a nucleotide sequence shown in SEQ ID No. 2;

[0010] (3) Designing a methylene blue labeled DNA probe sequence; the methylene blue labeled DNA probe has a nucleotide sequence shown in SEQ ID No. 3;

[0011] (4) Preparation of a three-dimensional DNA walker: hairpin DNA and ferrocene-labeled DNA are modified on the surface of gold nanoparticles AuNPs by self-assembly, the -OH group at the 5' end of the hairpin DNA is phosphorylated by T4 polynucleotide kinase, and then cut into single-stranded DNA by nuclease exonuclease, which can hybridize with ferrocene-labeled DNA to form a three-dimensional DNA walker. The three-dimensional DNA walker is driven by nuclease endonuclease to walk on the surface of AuNPs, cutting and releasing the electrochemically active Fc-DNA fragment in the ferrocene-labeled DNA;

[0012] (5) constructing an electrochemical biosensor: methylene blue labeled DNA is self-assembled on the surface of a gold electrode through Au-S bonds, and the active sites on the electrode surface are blocked by MCH. The electrochemically active Fc-DNA fragment released in the above step (4) can hybridize with the methylene blue labeled DNA to form a double-stranded structure, thereby realizing dual ratiometric electrochemical response signal amplification and T4 polynucleotide kinase activity determination;

[0013] (6) determining T4 polynucleotide kinase activity;

[0014] (7) Determination of T4 polynucleotide kinase inhibitor;

[0015] (8) Actual sample measurement.

[0016] Furthermore, in step (1), the length of the hairpin DNA is 60 bases, the 5′ end is labeled with a hydroxyl group, and the 3′ end is labeled with a thiol group; in step (2), the length of the ferrocene-labeled DNA probe is 32 bases, the 5′ end is labeled with ferrocene Fc, and the 3′ end is labeled with a thiol group; in step (3), the length of the methylene blue-labeled DNA probe is 49 bases, the 5′ end is labeled with methylene blue MB, and the 3′ end is labeled with a thiol group.

[0017] Furthermore, in step (4), gold nanoparticles AuNPs were prepared by sodium citrate reduction method. 50 mL of 1 mM chloroauric acid solution was heated to reflux and stirred, and 5 mL of 40 mM sodium citrate was added. The color of the solution changed from light yellow to dark red. The solution was refluxed for 30 min and cooled naturally to room temperature under stirring to obtain AuNPs with a diameter of 13 nm.

[0018] Furthermore, in step (4), a three-dimensional DNA walker is prepared: before the self-assembly of the hairpin DNA and the ferrocene-labeled DNA, they are reacted with 15 mM TCEP in the dark for 1 hour, then the hairpin DNA is heated to 95°C for 5 minutes, cooled naturally to room temperature, and the ferrocene-labeled DNA is heated to 95°C for 5 minutes, and then immersed in ice water for sudden cooling; 100 μL of 180 nM hairpin DNA is mixed with 100 μL of 3.6 μM ferrocene-labeled DNA and 100 μL of 9 nM AuNPs solution, and incubated at room temperature for 12 hours; 2 M NaCl solution is added to the mixture six times in succession, 10 μL each time for salt aging treatment; the mixture is incubated at room temperature for 24 hours, centrifuged at 15,000 rpm for 30 minutes to remove the supernatant, and the precipitate is dissolved in DNA hybridization buffer to obtain the hairpin DNA and ferrocene-labeled DNA. Modified AuNPs; take 5 μL of the above two DNA-modified AuNPs solutions, mix with 5 μL T4PNK reaction buffer, the T4PNK reaction buffer contains 1 mM ATP, 2.5 μL 10 units nuclease reaction buffer and different concentrations of T4PNK, dilute to 25 μL, incubate at 37°C for 2 hours for phosphorylation and nuclease exonuclease cleavage reaction to produce a three-dimensional DNA walker; add 5 μL rCutSmart buffer, rCutSmart buffer contains 10 units nuclease, dilute to 50 μL, incubate at 37°C for 2 hours, and the three-dimensional DNA walker walks on the surface of AuNPs. The driving force for the three-dimensional DNA walker to walk on the surface of AuNPs is provided by the nuclease, and the shearing releases the electrochemically active Fc-DNA fragments.

[0019] Further, in step (4), the amount and concentration of the hairpin DNA are 100 μL and 180 nM, respectively, the amount and concentration of the ferrocene-labeled DNA are 100 μL and 3.6 μM, respectively, the amount and concentration of the AuNPs solution are 100 μL and 9 nM, respectively, the incubation temperature and time are 25°C and 12 h, respectively; the concentration and number of salt aging treatments are 2 M NaCl and 6 times, 10 μL each time; the concentration of ATP in the T4PNK reaction buffer is 1 mM, the concentration of T4PNK is 0.001-10 U / mL, and the concentration of the nuclease λ is 0. exo The concentration was 10 units; the amount of T4PNK reaction buffer was 5 μL, the incubation temperature and time were 37°C and 2 h respectively; the concentration of nuclease in rCutSmart buffer was 10 units, the amount of rCutSmart buffer was 5 μL, and the walking temperature and time of the three-dimensional DNA walker were 37°C and 2 h respectively.

[0020] Furthermore, in step (5), methylene blue labeled DNA reacted with 15mM TCEP in the dark for 1 hour, heated to 95°C for 5 minutes, and naturally cooled to room temperature; 10μL of 1μM methylene blue labeled DNA solution was drop-coated on the pretreated gold electrode surface and incubated at 25°C for 2 hours; the modified electrode was immersed in 1mM MCH solution for 1 hour; 10μL of the supernatant after the three-dimensional DNA walker walked in step (4) was drop-coated on the modified gold electrode surface, incubated at 37°C for 2 hours, and rinsed with PBS buffer to obtain a T4PNK electrochemical biosensor.

[0021] Furthermore, in step (5), the amount and concentration of methylene blue labeled DNA were 10 μL and 1 μM, respectively, and the incubation temperature and time were 25°C and 2 h, respectively; the concentration of MCH was 1 mM, and the incubation temperature and time were 25°C and 1 h, respectively; the amount of the supernatant after the three-dimensional DNA walker in step (4) was 10 μL, and the incubation temperature and time were 37°C and 2 h, respectively;

[0022] In step (6), the activity of T4 polynucleotide kinase T4PNK is determined by using an electrochemical workstation with a three-electrode system, using differential pulse voltammetry DPV for quantification, and drawing a standard curve of the relationship between the dual ratiometric response signal DPV peak current and the T4PNK concentration.

[0023] Further, in step (6), an electrochemical workstation is used to measure the electrochemical response signal, and the activity of T4PNK is quantitatively determined according to the size of the dual ratio electrochemical signal; in step (7), the T4 polynucleotide kinase inhibitor is adenosine diphosphate (ADP) and ammonium sulfate (NH 4 ) 2 SO 4 ; In step (8), the actual sample is HeLa cell lysate.

[0024] The three-dimensional DNA walker of the present invention is prepared by an electrochemical analysis method for measuring the activity of T4 polynucleotide kinase based on the three-dimensional DNA walker.

[0025] The present invention discloses an electrochemical biosensor prepared by an electrochemical analysis method for measuring the activity of T4 polynucleotide kinase based on a three-dimensional DNA walker.

[0026] Beneficial effects: The invention has the advantages of simple operation, low cost, non-radioactivity, high sensitivity and good selectivity.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) The present invention provides an electrochemical technique for detecting T4 polynucleotide kinase activity; the present invention utilizes hairpin DNA and ferrocene-labeled DNA to self-assemble on the surface of AuNPs to form a three-dimensional DNA walker to improve the selectivity of T4PNK determination;

[0029] (2) The present invention uses a nuclease to drive a three-dimensional DNA walker to walk on the surface of AuNPs; the present invention uses the electrochemically active Fc-DNA fragments released by the three-dimensional DNA walker walking on the surface of AuNPs to hybridize with the methylene blue labeled DNA modified on the surface of the gold electrode to generate a dual ratiometric electrochemical response signal of Fc and MB, thereby improving the sensitivity of measuring T4PNK activity;

[0030] (3) The electrochemical biosensor prepared by the present invention can be used to detect T4PNK inhibitors adenosine diphosphate and ammonium sulfate; the electrochemical biosensor prepared by the present invention can be used to measure the T4PNK activity in actual sample HeLa cells. The ratiometric electrochemical response signal is proportional to the concentration of T4 polynucleotide kinase, achieving high sensitivity and high selectivity for the determination of T4 polynucleotide kinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 This is a schematic diagram of the preparation technology and detection principle for determining T4 polynucleotide kinase activity using the three-dimensional DNA walker and enzyme-assisted amplification technology of the present invention.

[0033] Figure 2 The Fc and MB dual signal response curve diagram for measuring T4 polynucleotide kinase activity using the present invention, the abscissa is the peak potential, the unit is V, and the ordinate is the DPV response peak current, the unit is μA.

[0034] Figure 3 This is a working curve diagram for measuring the activity of T4 polynucleotide kinase using the technology of the present invention. The horizontal axis is the concentration of T4PNK, the unit is U / mL, and the vertical axis is the peak current ratio of the Fc and MB dual signals, without unit. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples. It should be understood that these examples are explanations and examples of the present invention and are not intended to limit the scope of the present invention in any form.

[0036] The present invention discloses an electrochemical analysis method for determining T4 polynucleotide kinase activity based on a three-dimensional DNA walker, comprising the following steps: (1) designing a hairpin DNA sequence; the hairpin DNA has a nucleotide sequence shown in SEQ ID No. 1; the hairpin DNA is 60 bases long, with a hydroxyl group labeled at the 5′ end and a thiol group labeled at the 3′ end;

[0037] (2) Designing a ferrocene-labeled DNA probe sequence; the ferrocene-labeled DNA probe has a nucleotide sequence shown in SEQ ID No. 2; the ferrocene-labeled DNA probe is 32 bases in length, with a ferrocene Fc labeled at the 5′ end and a thiol labeled at the 3′ end;

[0038] (3) Designing a methylene blue labeled DNA probe sequence; the methylene blue labeled DNA probe has a nucleotide sequence shown in SEQ ID No. 3; the methylene blue labeled DNA probe is 49 bases in length, with methylene blue MB labeled at the 5′ end and thiol labeled at the 3′ end.

[0039] (4) Preparation of three-dimensional DNA walker: hairpin DNA and ferrocene-labeled DNA were modified on the surface of gold nanoparticles AuNPs by self-assembly. The -OH group at the 5' end of the hairpin DNA was phosphorylated by T4 polynucleotide kinase and then cleaved into single-stranded DNA by nuclease exonuclease. The single-stranded DNA could hybridize with ferrocene-labeled DNA to become a three-dimensional DNA walker. The three-dimensional DNA walker was driven by nuclease endonuclease to walk on the surface of AuNPs, shearing and releasing the electrochemically active Fc-DNA fragments in the ferrocene-labeled DNA. Gold nanoparticles AuNPs were prepared by sodium citrate reduction method. 50 mL of 1 mM chloroauric acid solution was heated to reflux and stirred. 5 mL of 40 mM sodium citrate was added. The color of the solution changed from light yellow to dark red. The solution was refluxed for 30 min and cooled to room temperature naturally with stirring to obtain AuNPs with a diameter of 13 nm.

[0040] Preparation of three-dimensional DNA walker: Before self-assembly of hairpin DNA and ferrocene-labeled DNA, react with 15mM TCEP in the dark for 1h, then heat the hairpin DNA to 95℃ for 5min, cool naturally to room temperature, heat the ferrocene-labeled DNA to 95℃ for 5min, and quench in ice water; take 100μL 180nM hairpin DNA, mix with 100μL 3.6μM ferrocene-labeled DNA and 100μL 9nM AuNPs solution, and incubate at room temperature for 12h; add 2M NaCl solution to the mixture 6 times in succession, 10μL each time for salt aging treatment; incubate the mixture at room temperature for 24h, centrifuge at 15000rpm for 30min to remove the supernatant, dissolve the precipitate in DNA hybridization buffer, and obtain hairpin DNA and ferrocene-labeled DNA modified AuNPs; take 5μL of the above two DNA modified AuNPs The AuNPs solution was mixed with 5 μL T4PNK reaction buffer, wherein the T4PNK reaction buffer contained 1 mM ATP, 2.5 μL 10 units exonuclease reaction buffer and different concentrations of T4PNK, diluted to 25 μL, and incubated at 37°C for 2 hours for phosphorylation and exonuclease cleavage reaction to produce a three-dimensional DNA walker; 5 μL rCutSmart buffer was added, wherein the rCutSmart buffer contained 10 units endonuclease, diluted to 50 μL, and incubated at 37°C for 2 hours, and the three-dimensional DNA walker walked on the AuNPs surface. The driving force for the three-dimensional DNA walker to walk on the AuNPs surface was provided by the endonuclease, and the shearing released the electrochemically active Fc-DNA fragments.

[0041] The amount and concentration of hairpin DNA were 100 μL and 180 nM, the amount and concentration of ferrocene-labeled DNA were 100 μL and 3.6 μM, the amount and concentration of AuNPs solution were 100 μL and 9 nM, the incubation temperature and time were 25 °C and 12 h, respectively; the concentration and number of salt aging treatments were 2 M NaCl and 6 times, 10 μL each time; the concentration of ATP in T4PNK reaction buffer was 1 mM, the concentration of T4PNK was 0.001-10 U / mL, and the concentration of exonuclease λ was 0. exo The concentration was 10 units; the amount of T4PNK reaction buffer was 5 μL, the incubation temperature and time were 37°C and 2 h respectively; the concentration of nuclease in rCutSmart buffer was 10 units, the amount of rCutSmart buffer was 5 μL, and the walking temperature and time of the three-dimensional DNA walker were 37°C and 2 h respectively.

[0042] (5) constructing an electrochemical biosensor: methylene blue labeled DNA is self-assembled on the surface of a gold electrode through Au-S bonds, and the active sites on the electrode surface are blocked by MCH. The electrochemically active Fc-DNA fragment released in the above step (4) can hybridize with the methylene blue labeled DNA to form a double-stranded structure, thereby realizing dual ratiometric electrochemical response signal amplification and T4 polynucleotide kinase activity determination;

[0043] The methylene blue labeled DNA was reacted with 15mM TCEP in the dark for 1h, heated to 95°C for 5min, and naturally cooled to room temperature; 10μL of 1μM methylene blue labeled DNA solution was drop-coated on the pretreated gold electrode surface and incubated at 25°C for 2h; the modified electrode was immersed in 1mM M MCH solution for 1h; 10μL of the supernatant after the three-dimensional DNA walker walked in step (4) was drop-coated on the modified gold electrode surface, incubated at 37°C for 2h, and rinsed with PBS buffer to prepare the T4PNK electrochemical biosensor.

[0044] The amount and concentration of methylene blue labeled DNA were 10 μL and 1 μM, respectively, and the incubation temperature and time were 25°C and 2 h, respectively; the concentration of MCH was 1 mM, and the incubation temperature and time were 25°C and 1 h, respectively; the amount of the supernatant after the three-dimensional DNA walker in step (4) was 10 μL, and the incubation temperature and time were 37°C and 2 h, respectively;

[0045] (6) Determination of T4 polynucleotide kinase activity; Determination of T4 polynucleotide kinase T4PNK activity: Use an electrochemical workstation with a three-electrode system for testing, use differential pulse voltammetry DPV for quantification, and draw a standard curve of the relationship between the peak current of the dual ratio response signal DPV and the T4PNK concentration. Use an electrochemical workstation to measure the electrochemical response signal, and quantitatively determine the activity of T4PNK based on the size of the dual ratio electrochemical signal.

[0046] (7) Determine T4 polynucleotide kinase inhibitor; the T4 polynucleotide kinase inhibitor is adenosine diphosphate (ADP) and ammonium sulfate (NH 4 ) 2 SO 4 ;

[0047] (8) Actual sample determination: The actual sample is HeLa cell lysate.

[0048] The three-dimensional DNA walker of the present invention is prepared by an electrochemical analysis method for measuring the activity of T4 polynucleotide kinase based on the three-dimensional DNA walker.

[0049] The present invention discloses an electrochemical biosensor prepared by an electrochemical analysis method for measuring the activity of T4 polynucleotide kinase based on a three-dimensional DNA walker.

[0050] Example 1

[0051] The principle of the method for determining T4PNK activity based on three-dimensional DNA walker and enzyme-assisted amplification technology of the present invention is as follows Figure 1 As shown. First, the hairpin DNA and ferrocene-labeled DNA are fixed on the surface of AuNPs through the 3'-terminal thiol group to form a gold-sulfur bond. The -OH group at the end of the hairpin DNA is catalytically phosphorylated by T4PNK, and then cut into single-stranded DNA by nuclease exonuclease. The single-stranded DNA can hybridize with ferrocene-labeled DNA to become a three-dimensional DNA walker. The three-dimensional DNA walker is driven by endonuclease to walk on the surface of AuNPs, and the electrochemically active Fc-DNA fragments in the ferrocene-labeled DNA are released by shearing. Methylene blue-labeled DNA is self-assembled on the surface of the gold electrode through Au-S bonds, and the active sites on the electrode surface are blocked by MCH. The Fc-DNA fragments released by the three-dimensional DNA walker walking on the surface of AuNPs hybridize with the methylene blue-labeled DNA to form a double-stranded structure, generating amplification of the dual ratiometric electrochemical response signals of Fc and MB. The ratiometric electrochemical signal generated is proportional to the concentration of T4PNK, realizing the quantitative detection of T4PNK.

[0052] Example 2

[0053] DNA oligonucleotide sequence design

[0054] The DNA oligonucleotide sequence designed by the present invention was synthesized by Takara Biotechnology (Dalian) Co., Ltd., purified and tested by HPLC, and freeze-dried. The oligonucleotide sequence designed by the present invention is as follows:

[0055] The hairpin DNA has the nucleotide sequence shown in SEQ ID No. 1, hairpin DNA: 5′-HO-CTCGCCTAACTCCTCAGCTCATCGACTCGTGTTAGGCGAGTTTTTTT TTTTTTTTTTTTT-SH-3′;

[0056] The ferrocene-labeled DNA probe has the nucleotide sequence shown in SEQ ID No. 2; ferrocene-labeled DNA probe: 5′-Fc-CGCATCACGAGTCGATGAGCTGAGGATGGTTG-SH-3′;

[0057] The methylene blue labeled DNA probe has the nucleotide sequence shown in SEQ ID No.3; the methylene blue labeled DNA probe:

[0058] 5′-MB-CGCATCACGAGATCCAGACAGTCCACAGCTCATCGACTCGTGATGC GTT-SH-3′;

[0059] The oligonucleotides were dissolved in ultrapure sterilized water and stored at -18°C until use.

[0060] Example 3

[0061] Preparation of three-dimensional DNA walker

[0062] The hairpin DNA and ferrocene-labeled DNA were reacted with 15 mM TCEP in the dark for 1 hour, and then the hairpin DNA was heated to 95°C for 5 minutes and cooled naturally to room temperature. The ferrocene-labeled DNA was heated to 95°C for 5 minutes and then immersed in ice water for quenching. 100 μL of 180 nM hairpin DNA was mixed with 100 μL of 3.6 μM ferrocene-labeled DNA and 100 μL of 9 nM AuNPs solution and incubated at room temperature for 12 hours. 2 M NaCl solution was added to the mixture for 6 consecutive times, 10 μL each time for salt aging treatment. The mixture was incubated at room temperature for 24 hours, centrifuged at 15,000 rpm for 30 minutes to remove the supernatant, and the precipitate was dissolved in DNA hybridization buffer to obtain the hairpin. DNA and ferrocene labeled DNA-modified AuNPs; 5 μL of the above two DNA-modified AuNPs solutions were mixed with 5 μL T4PNK reaction buffer (containing 1 mM ATP), 2.5 μL 10 units exonuclease reaction buffer and different concentrations of T4PNK, diluted to 25 μL, and incubated at 37°C for 2 h for phosphorylation and exonuclease cleavage reactions to produce a three-dimensional DNA walker; 5 μL rCutSmart buffer (containing 10 units endonuclease) was added, diluted to 50 μL, and incubated at 37°C for 2 h. The three-dimensional DNA walker walked on the surface of AuNPs, and the electrochemically active Fc-DNA fragments were released by shearing.

[0063] Example 4

[0064] Preparation of electrochemical biosensors

[0065] The gold electrode (3 mm in diameter) was first immersed in the piranha solution for 10 min, rinsed with ultrapure water, and then soaked in Al2O3 with particle sizes of 0.3 μm and 0.05 μm. 2 O 3 The powder was polished and ultrasonically cleaned with ultrapure water, anhydrous ethanol and ultrapure water in sequence; 2 SO 4 The electrode was activated by cyclic voltammetry scanning for 30 cycles in the solution, with a potential range of -0.2 to 1.6 V and a scan rate of 0.1 V / s. 2 Blow dry and set aside;

[0066] Methylene blue labeled DNA was reacted with 15mM TCEP in the dark for 1h, heated to 95℃ for 5min, and naturally cooled to room temperature; 10μL of 1μM methylene blue labeled DNA solution was drop-coated on the pretreated gold electrode surface and incubated at 25℃ for 2h; the modified electrode was immersed in 1mM M MCH solution for 1h; 10μL of the supernatant after the three-dimensional DNA walker walked on the AuNPs surface was drop-coated on the modified gold electrode surface, incubated at 37℃ for 2h, and rinsed with PBS buffer to prepare the T4PNK electrochemical biosensor.

[0067] Example 5

[0068] Determination of T4PNK activity

[0069] The test was carried out using an electrochemical workstation with a three-electrode system, with a gold electrode as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The activity of T4PNK was determined by differential pulse voltammetry, using 100 mM PBS (pH 7.4) as the buffer, a potential range of -0.5 to 0.6 V, a potential increment of 50 mV, and a pulse period of 20 ms. The differential pulse curve Fc and MB dual signal response peak current and T4PNK concentration curve and working curve are shown in the figure. Figure 2 and Figure 3 As shown in Figure 2, the peak current and T4PNK concentration showed a good linear relationship in the range of 0.001–10 U / mL, with a correlation coefficient of R 2 is 0.9913, and the linear equation is I Fc / I MB =0.15lgC+0.49, and the detection limit is 0.0003U / mL. Compared with other sensors, the sensor proposed in the present invention has a wider linear range and a lower detection limit. The electrochemical sensor constructed by using a three-dimensional DNA walker and enzyme-assisted amplification technology can realize quantitative and sensitive determination of T4PNK activity.

[0070] Example 6

[0071] Determination of T4PNK activity inhibitors

[0072] Different concentrations of inhibitors adenosine diphosphate (ADP) and ammonium sulfate (NH 4 ) 2 SO 4 Mixed with T4PNK buffer, the experimental method was the same. As the inhibitor concentration increased, the relative activity of T4PNK decreased significantly. When the concentrations of the two inhibitors were 1.54mM and 9.56mM, respectively, the relative activity of T4PNK decreased by 50%, indicating that this method can be used for the detection and analysis of T4PNK inhibitors.

[0073] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. An electrochemical analysis method for determining T4 polynucleotide kinase activity based on a three-dimensional DNA walker, characterized in that The method comprises the following steps: (1) designing a hairpin DNA sequence; the hairpin DNA has a nucleotide sequence shown in SEQ ID No. 1; the hairpin DNA is 60 bases long, the 5′ end is labeled with a hydroxyl group, and the 3′ end is labeled with a thiol group; (2) Designing a ferrocene-labeled DNA probe sequence; the ferrocene-labeled DNA probe has a nucleotide sequence shown in SEQ ID No. 2; the ferrocene-labeled DNA probe is 32 bases in length, with a ferrocene Fc labeled at the 5′ end and a thiol labeled at the 3′ end; (3) Designing a methylene blue labeled DNA probe sequence; the methylene blue labeled DNA probe has a nucleotide sequence as shown in SEQ ID No. 3; the methylene blue labeled DNA probe is 49 bases in length, with methylene blue MB labeled at the 5′ end and thiol labeled at the 3′ end; (4) Preparation of a three-dimensional DNA walker: The hairpin DNA designed in step (1) and the ferrocene-labeled DNA designed in step (2) are self-assembled and modified on the surface of gold nanoparticles AuNPs through Au-S bonds, the -OH group at the 5' end of the hairpin DNA is phosphorylated by T4 polynucleotide kinase, and then cleaved into single-stranded DNA by nuclease exonuclease, and the single-stranded DNA can hybridize with the ferrocene-labeled DNA to form a three-dimensional DNA walker, and the three-dimensional DNA walker is driven by nuclease endonuclease to walk on the surface of AuNPs, and the electrochemically active Fc-DNA fragment in the ferrocene-labeled DNA is cleaved and released; (5) constructing an electrochemical biosensor: the methylene blue labeled DNA designed in step (3) is self-assembled on the surface of the gold electrode AuNPs through Au-S bonds, and the active sites on the electrode surface are blocked by MCH. The electrochemically active Fc-DNA fragment released in the above step (4) can hybridize with the methylene blue labeled DNA to form a double-stranded structure, thereby realizing dual ratiometric electrochemical response signal amplification and T4 polynucleotide kinase activity determination; (6) determining T4 polynucleotide kinase activity; (7) Determination of T4 polynucleotide kinase inhibitor; (8) Actual sample measurement.

2. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (4), gold nanoparticles AuNPs were prepared by sodium citrate reduction method. 50 mL of 1 mM chloroauric acid solution was heated to reflux and stirred, and 5 mL of 40 mM sodium citrate was added. The color of the solution changed from light yellow to dark red. The solution was refluxed for 30 min and cooled naturally to room temperature under stirring to obtain AuNPs with a diameter of 13 nm.

3. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (4), a three-dimensional DNA walker is prepared: before the self-assembly of the hairpin DNA and the ferrocene-labeled DNA, the hairpin DNA is reacted with 15 mM TCEP in the dark for 1 hour, then the hairpin DNA is heated to 95°C for 5 minutes and cooled naturally to room temperature, the ferrocene-labeled DNA is heated to 95°C for 5 minutes and then quenched in ice water; 100 μL of 180 nM hairpin DNA is mixed with 100 μL of 3.6 μM ferrocene-labeled DNA and 100 μL of 9 nM AuNPs solution, and incubated at room temperature for 12 hours; 2 M NaCl solution is added to the mixture six times in succession, 10 μL each time for salt aging treatment; The mixed solution was incubated at room temperature for 24 hours, centrifuged at 15000 rpm for 30 minutes to remove the supernatant, and the precipitate was dissolved in DNA hybridization buffer to obtain hairpin DNA and ferrocene-labeled DNA-modified AuNPs; 5 μL of the above two DNA-modified AuNPs solutions were mixed with 5 μL T4 PNK reaction buffer, the T4 PNK reaction buffer contained 1 mM ATP, 2.5 μL 10 units exonuclease reaction buffer and different concentrations of T4 PNK, diluted to 25 μL, and incubated at 37°C for 2 hours for phosphorylation and exonuclease cleavage reaction to produce a three-dimensional DNA walker; 5 μL rCutSmart buffer was added, the rCutSmart buffer contained 10 units endonuclease, diluted to 50 μL, and incubated at 37°C for 2 hours, and the three-dimensional DNA walker walked on the surface of AuNPs. The driving force for the three-dimensional DNA walker to walk on the surface of AuNPs was provided by the endonuclease, and the shearing released the electrochemically active Fc-DNA fragment.

4. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (4), the amount and concentration of the hairpin DNA were 100 μL and 180 nM, the amount and concentration of the ferrocene-labeled DNA were 100 μL and 3.6 μM, the amount and concentration of the AuNPs solution were 100 μL and 9 nM, the incubation temperature and time were 25°C and 12 h, respectively; the concentration and number of salt aging treatments were 2 M NaCl and 6 times, 10 μL each time; the concentration of ATP in the T4 PNK reaction buffer was 1 mM, the concentration of T4 PNK was 0.001-10 U / mL, and the concentration of exonuclease λ was 0. exo The concentration was 10 units; the amount of T4 PNK reaction buffer was 5 μL, the incubation temperature and time were 37°C and 2 h, respectively; the concentration of nuclease in rCutSmart buffer was 10 units, the amount of rCutSmart buffer was 5 μL, and the walking temperature and time of the three-dimensional DNA walker were 37°C and 2 h, respectively.

5. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (5), methylene blue labeled DNA reacts with 15mM TCEP in the dark for 1 hour, heated to 95°C for 5 minutes, and naturally cooled to room temperature; 10μL of 1μM methylene blue labeled DNA solution is drop-coated on the pretreated gold electrode surface and incubated at 25°C for 2 hours; the modified electrode is immersed in 1mM MCH solution for 1 hour; 10μL of the supernatant after the three-dimensional DNA walker walks in step (4) is drop-coated on the modified gold electrode surface, incubated at 37°C for 2 hours, and rinsed with PBS buffer to obtain a T4PNK electrochemical biosensor.

6. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (5), the amount and concentration of methylene blue labeled DNA were 10 μL and 1 μM, respectively, and the incubation temperature and time were 25°C and 2 h, respectively; the concentration of MCH was 1 mM, and the incubation temperature and time were 25°C and 1 h, respectively; the amount of the supernatant after the three-dimensional DNA walker in step (4) was 10 μL, and the incubation temperature and time were 37°C and 2 h, respectively; In step (6), the activity of T4 polynucleotide kinase T4 PNK is measured by using an electrochemical workstation with a three-electrode system, using differential pulse voltammetry DPV for quantification, and drawing a standard curve of the relationship between the dual ratiometric response signal DPV peak current and the T4 PNK concentration.

7. The electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker according to claim 1, characterized in that: In step (6), an electrochemical workstation is used to measure the electrochemical response signal, and the activity of T4 PNK is quantitatively determined according to the size of the dual ratio electrochemical signal; in step (7), the T4 polynucleotide kinase inhibitor is adenosine diphosphate ADP and ammonium sulfate (NH4)2SO4; in step (8), the actual sample is HeLa cell lysate.

8. The three-dimensional DNA walker obtained by the electrochemical analysis method for measuring T4 polynucleotide kinase activity based on the three-dimensional DNA walker according to any one of claims 1 to 7.

9. The electrochemical biosensor prepared by the electrochemical analysis method for measuring T4 polynucleotide kinase activity based on a three-dimensional DNA walker as claimed in any one of claims 1 to 7.

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