Construction and application of an efficient DNA walker for rapid and sensitive detection of DNA methylation
By constructing an efficient DNA walker, using bimetal MOF material to combine with thiol-modified probes, the problem of unsatisfactory DNA methylation walking efficiency was solved in traditional DNA walkers, and rapid and sensitive DNA methylation detection was achieved, which significantly improved the accuracy and efficiency of the detection.
Patent Information
- Application Number
- CN202210620803.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-06-02
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2042-06-02
AI Technical Summary
Traditional DNA walkers have poor walking efficiency in detecting DNA methylation, resulting in poor detection results and making it difficult to achieve fast and sensitive detection.
By constructing an efficient DNA walker, the bimetallic MOF material Au@NiCo-MOF is combined with the thiol-modified fuel probe FP and fixed probe IP to form a functionalized FP/Au@NiCo-MOF, which is used for electrochemical detection with DNA of different methylation degrees.
The walking efficiency of DNA walkers is improved, the operating time is reduced, and the DNA methylation can be detected quickly and sensitively, which significantly enhances the catalytic current and improves the accuracy and efficiency of detection.
Smart Images
Figure CN115011667B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of electrochemical biosensors, and in particular to a construction method and application of an efficient DNA walker for rapid and sensitive detection of DNA methylation. Background Art
[0002] DNA methylation refers to the epigenetic modification in which DNA methyltransferase catalyzes the 5-terminal carbon atom of the cytosine molecule on the CpG island using S-adenosylmethionine as a methyl donor, introducing a methyl group through a covalent bond. Abnormal DNA methylation is closely related to the occurrence and development of a variety of diseases, including the formation and growth of tumor cells, and the control of the active expression of tumor suppressor genes. Therefore, DNA methylation can be used as an important marker for clinical detection, and the effective detection of DNA methylation is of great significance for the early diagnosis of diseases and the study of genetic mechanisms.
[0003] As a classic DNA molecular machine, DNA walker has the advantages of efficient self-assembly nanostructure, cargo loading and unloading capabilities and bio-mimicry phenomena. It has attracted widespread attention from scientists as a signal amplification method. However, the walking efficiency of traditional DNA walkers is not ideal. One of the reasons is that the walking chain is easy to fall off the track and wander around in the sample, which cannot effectively react and amplify the signal, further limiting the application effect of the walker in disease marker detection. In addition, the ordered pore structure of MOF materials makes its metal active sites have the same microenvironment, which is conducive to improving the selectivity of catalytic reactions, thus having important value in electrochemical catalysis. Studies have shown that compared with single metal MOFs, bimetallic or multimetallic MOFs exhibit advantages such as higher conductivity, enhanced catalytic activity and adjustable electrochemical activity. At the same time, MOF as a carrier to carry DNA chains can effectively increase the effective concentration of DNA chains, thereby enhancing the walking efficiency of DNA walkers. Summary of the invention
[0004] The purpose of the present invention is to provide a method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation, which can improve the walking efficiency of the DNA walker, reduce the operation time, and achieve rapid and sensitive detection of DNA methylation.
[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides a method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation, comprising the following steps:
[0007] S1: First, NiCo-MOF was synthesized by solvothermal method, and then Au nanoparticles were coated on the surface of NiCo-MOF to obtain Au@NiCo-MOF.
[0008] S2: Mix the thiol-modified fuel probe FP and Au@NiCo-MOF to prepare functionalized FP / Au@NiCo-MOF;
[0009] S3: Pre-treat the surface of the gold electrode AuE, fix the thiol-modified fixed probe IP to the surface of the gold electrode AuE, and block it with mercaptohexanol MCH to obtain MCH / IP / AuE, which serves as the track of the DNA walker;
[0010] S4: DNA with different methylation levels is dropped onto the MCH / IP / AuE interface to start the DNA walker;
[0011] S5: The sensing interface after the reaction is used as a working electrode to form a three-electrode system with an Ag / AgCl reference electrode and a platinum wire counter electrode, and placed in a phosphate buffer solution and a nitrogen atmosphere for electrochemical detection.
[0012] Preferably, the step S1 specifically comprises: dissolving 2,5-dihydroxyterephthalic acid (DHTA), an inorganic salt of nickel, an inorganic salt of cobalt and polyvinylpyrrolidone (PVP) in dimethylformamide (DMF), and then placing the mixed solution in a reactor and reacting at 120° C. for 24 hours; after cooling to room temperature, washing and drying to obtain a NiCo-MOF sample; and quenching the mixture with chloroauric acid (HAuCl 4 ) as the precursor, sodium borohydride (NaBH 4 ) as a reducing agent and polyvinyl pyrrolidone (PVP) as a surfactant to prepare nano-gold colloidal particles, which were cross-linked with NiCo-MOF to obtain Au@NiCo-MOF.
[0013] Preferably, the inorganic salt of nickel is NiCl 2 or NiNO 3 The inorganic salt of cobalt is CoCl 2 or CoNO 3 .
[0014] Preferably, the step S2 specifically comprises: reacting the thiol-modified fuel probe FP with tri(2-carboxyethyl)phosphine for 60 minutes to open the disulfide bond, and then reacting with Au@NiCo-MOF for 12 hours to obtain FP / Au@NiCo-MOF; wherein the sequence of the fuel probe FP is 5′-TCACAACGTCTACACATGGCGTTGTGATAGAGTATCCATGTGTAGACTGAATCGA-(CH 2 ) 6 -SH-3′′ (SEQ ID NO. 1).
[0015] Preferably, the step S3 specifically comprises: reacting the thiol-modified fixed probe IP with tri(2-carboxyethyl)phosphine for 60 minutes to open the disulfide bond, then dripping it onto the treated surface of the gold electrode AuE, incubating it for 12 hours under light-proof conditions, and then dripping mercaptohexanol MCH onto the sensing interface for 2 hours after rinsing to obtain MCH / / IP / AuE, wherein the sequence of the thiol-modified fixed probe IP is 5′-SH-(CH 2 ) 6 -ACTACTTCTCGATTCAATACTCTATCACAACGCCATGTGTAGACGTTGTGATAGAGTAT-3' (SEQ ID NO. 2).
[0016] Preferably, step S4 specifically comprises: simultaneously dropping DNA samples with different methylation degrees and FP / Au@NiCo-MOF onto the MCH / IP / AuE surface, and reacting at room temperature for 20 minutes, wherein the sequence of the DNA is 5′-mCGTTGTGATAGAGTATTGAATmCGAGAAGTAGT-3′ (SEQ ID NO. 3).
[0017] In a second aspect, the present invention also provides a DNA walker obtained by the above-mentioned construction method.
[0018] In a third aspect, the present invention also provides the use of the above-mentioned DNA walker in detecting DNA methylation.
[0019] Compared with the prior art, the present invention constructs an electrochemical sensor for rapid and sensitive detection of methylated DNA by constructing an efficient DNA walker and preparing a bimetallic MOF material with high catalytic performance. The fully methylated DNA target is used as a walking chain to start the DNA walker, and the bimetallic MOF carries the fuel chain to drive the methylated DNA to undergo a chain transfer reaction between tracks to achieve walking, which effectively enhances the walking efficiency. At the same time, the bimetallic MOF can catalyze the reduction of hydrogen peroxide to obtain a significantly enhanced catalytic current, thereby achieving rapid and sensitive detection of methylated DNA. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a schematic diagram of the principle of an efficient DNA walker for rapid and sensitive detection of DNA methylation;
[0021] Figure 2 Characterization diagrams of bimetallic MOF: AB, SEM images of NiCo-MOF; C, TEM image of NiCo-MOF; D, particle size distribution of NiCo-MOF; GH, element mapping; I, XPS spectrum; J, SEM image of Au@NiCo-MOF; K, TEM image of Au@NiCo-MOF;
[0022] Figure 3 It is a characterization diagram of polyacrylamide gel electrophoresis;
[0023] Figure 4 is the electrochemical response diagram of the sensing interface to DNA with different methylation degrees;
[0024] Figure 5 It is the response signal of the sensor to different concentrations of methylated DNA and its linear relationship diagram, where the concentrations from a to i are: 0.5fM, 1fM, 10fM, 50fM, 500fM, 5pM, 50pM, 500pM, 5nM. DETAILED DESCRIPTION
[0025] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0026] Figure 1 Shown is a schematic diagram of the principle of an efficient DNA walker for rapid and sensitive detection of DNA methylation.
[0027] First, the bimetallic MOF was prepared by solvothermal method: DHTA, NiCl 2 and CoCl 2 The NiCo-MOF sample was obtained by reacting at 120°C.
[0028] HAuCl 4 As precursor, NaBH 4 Nano-gold colloidal particles were prepared by using 2-ni-pyridine as reducing agent and PVP as surfactant, and then cross-linked with bimetallic MOF to obtain Au@NiCo-MOF.
[0029] Then, the thiol-modified fuel probe FP (1 μmol / L) was reacted with tri(2-carboxyethyl)phosphine for 60 min to open the disulfide bond, and then reacted with Au@NiCo-MOF (1 mg / mL) for 12 h to obtain functionalized FP / Au@NiCo-MOF; wherein the sequence of the fuel probe FP is 5′-TCACAACGTCTACACATGGCGTTGTGATAGAGTATCCATGTGTAGACTGAATCGA-(CH 2 ) 6 -SH-3′ (SEQ ID NO. 1).
[0030] The gold electrode was surface pretreated, and the thiol-modified fixed probe IP (1 μmol / L) was reacted with tri(2-carboxyethyl)phosphine for 60 minutes to open the disulfide bond, and then added to the treated gold electrode AuE surface, incubated for 12 hours under light-proof conditions, and then rinsing and then mercaptohexanol MCH (1 μmol / L) was added to the sensing interface for 2 hours to block, thereby obtaining MCH / / IP / AuE as the track of the DNA walker; wherein the sequence of the thiol-modified fixed probe IP is 5′-SH-(CH 2 ) 6 -ACTACTTCTCGATTCAATACTCTATCACAACGCCATGTGTAGACGTTGTGATAGAGTAT-3' (SEQ ID NO. 2).
[0031] The target DNA with different methylation degrees was used as a walking chain and was simultaneously added to the MCH / IP / AuE interface with FP / Au@NiCo-MOF. The reaction was carried out at room temperature for 20 minutes to start the DNA walker. The sequence of the DNA was 5′-mCGTTGTGATAGAGTATTGAATmCGAGAAGTAGT-3′ (SEQ ID NO. 3).
[0032] The sensing interface after the reaction was used as a working electrode to form a three-electrode system with a Ag / AgCl reference electrode and a platinum wire counter electrode, and was placed in a phosphate buffer solution and a nitrogen atmosphere for electrochemical detection.
[0033] The target DNA opens the hairpin structure of IP with the help of toehold (red and green fragments), exposing a single-stranded fragment at the 5′ end of IP. The fuel chain FP uses the single-stranded fragment as a toehold to perform a chain displacement reaction, displacing part of the target DNA from the IP-target DNA double strand. At this time, the target DNA maintains hybridization with the red fragment of IP and has not yet left the walking track. It further hybridizes with the adjacent IP through a chain transfer reaction. The bimetallic MOF carries the fuel chain FP to continue to undergo a chain displacement reaction, prompting the target DNA to continuously undergo a chain transfer reaction, that is, to achieve walking. Au@NiCo-MOF can not only improve the walking efficiency of the DNA walker, but also has good catalytic performance for hydrogen peroxide, and can provide significantly enhanced electrochemical signals for sensors for rapid and sensitive detection of DNA methylation.
[0034] The synthesis process of bimetallic MOF includes the following steps:
[0035] 0.06 g DHTA, 0.6 g NiCl 2 6H 2 O, 0.3 g CoCl 2 6H2 O and 1g PVP were dissolved in 10mL dimethylformamide (DMF) and placed in a reactor for reaction at 120°C for 24 hours. After cooling to room temperature, the samples were washed and dried to obtain NiCo-MOF. Figure 2 As shown in Figures 2A, 2B, and 2C, NiCo-MOF is spherical and well dispersed on the silicon wafer substrate. The size of NiCo-MOF was then analyzed using Nano Measurer 1.2 ( Figure 2 D), it was found that the NiCo-MOF particles were concentrated in the range of 250nm to 550nm, with an average diameter of about 400nm. Figure 2 EH is the result of elemental mapping analysis, and it can be seen that C, O, Ni, and Co elements are evenly distributed in the spherical structure of NiCo-MOF. XPS spectrum ( Figure 2 I) The elemental composition of NiCo-MOF was further demonstrated.
[0036] Add 0.03 g PVP to 3 mL 1 mM HAuCl 4 The aqueous solution was stirred for 1 hour, and then 100 μL of freshly prepared 100 mM NaBH 4 , a brown-grey nano-gold colloidal solution (average particle size 6nm) was obtained. It was stirred with 500μL, 2.5mg / mL NiCo-MOF for 4 hours, and then centrifuged and washed to obtain Au@NiCo-MOF. Through SEM images ( Figure 2 J) It can be seen that the spherical structure and size of MOF did not change significantly after the reaction. Figure 2 K can observe that gold nanoparticles are wrapped on the surface of spherical NiCo-MOF.
[0037] Characterization of the walking principle of the walker: The walking principle of the walker is verified by polyacrylamide electrophoresis experiment. Figure 3 As shown, the target chain methylated DNA (band 1) has a higher electrophoretic migration rate, while the track chain IP (band 2) and the fuel probe FP (band 3) show relatively low mobility due to their increased molecular weight. After annealing and hybridization of IP and FP, double-stranded IP / FP is obtained, and its electrophoretic migration rate is further reduced (band 4). When the target chain does not exist, FP and IP chains are directly mixed and reacted (band 5), and the migration rate of the product is equivalent to that of IP and FP, indicating that FP cannot directly hybridize with IP, and the brightening of the band color is due to the close number of bases between FP and IP. When methylated DNA is introduced (band 6), a new bright band can be observed, and its migration rate is equivalent to that of band 4, indicating that methylated DNA triggers the operation of the walker.
[0038] Feasibility test of the sensor: The signal response of MCH / / IP / AuE to different solutions was recorded by differential pulse voltammetry and current-time technique to verify the feasibility of detecting methylated DNA. Figure 4 As shown in A, the MCH / IP / AuE interface does not show an obvious peak current signal (curve a), indicating that the interface is sensitive to H 2 O 2 There is no obvious catalytic activity. In the absence of the target, incubating MCH / IP / AuE with FP / Au@NiCo-MOF can obtain a smaller peak current (curve b), which is caused by nonspecific hybridization between the two hairpin probes. After the introduction of unmethylated DNA, the current signal did not increase significantly (curve c), which is because the bisulfite pretreatment caused two mismatched bases in the unmethylated DNA. The head or middle hemimethylated DNA further increased the current signal, but the current signal of the middle hemimethylated DNA (curve e) was greater than that of the head hemimethylated DNA (curve d), which is because the middle mismatch has a greater impact on the hybridization efficiency and the stability of double-stranded DNA. The fully methylated target DNA enables the sensor to obtain a significantly enhanced current signal (curve f). In addition, according to the current-time curve ( Figure 4 B) Analysis showed that no obvious current response was detected at the MCH / IP / AuE interface (curve a). The current signal was smaller in the absence of the target (curve b), and the current signal of fully methylated DNA was stronger than that of unmethylated, head hemimethylated, and middle hemimethylated DNA (curve f vs. c, d, and e). These experimental phenomena were consistent with the CV response, indicating that the sensor can be used to detect methylated DNA.
[0039] The detection performance analysis of the sensor: The response signal of the sensor interface to different concentrations of methylated DNA was recorded using the current-time curve. Figure 5 A can observe H 2 O 2 The current signal increases with the increase of methylated DNA concentration. Figure 5 B is the calibration curve for quantitative detection of methylated DNA. It can be seen that the current signal and the logarithmic value of the methylated DNA concentration show a good linear relationship. The linear range is 0.5fM to 5nM, and its linear equation is i=0.06372lgc+1.01586(R 2 =0.9922), and the detection limit was 200aM according to the 3σ rule. In addition, 5pmol / L methylated DNA was measured six times using this method, and the relative standard deviation was 2.9%, indicating that the sensor has good reproducibility. Sequence Listing <110> Jiangsu Normal University <120> Construction and application of an efficient DNA walker for rapid and sensitive detection of DNA methylation <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 55 <212> DNA <213> Artificial Sequence <400> 1 tcacaacgtc tacacatggc gttgtgatag agtatccatg tgtagactga atcga 55 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <400> 2 actacttctc gattcaatac tctatcacaa cgccatgtgt agacgttgtg atagagtat 59 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <400> 3 mcgttgtgat agagtattga atmcgagaag tagt 34
Claims
1. A method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation, characterized in that: The following steps are involved: S1: First, NiCo-MOF is synthesized by a solvothermal method, and gold nanoparticles are further coated on the surface of NiCo-MOF to obtain Au@NiCo-MOF; the step S1 specifically comprises: dissolving 2, 5-dihydroxyterephthalic acid, an inorganic salt of nickel, an inorganic salt of cobalt and polyvinylpyrrolidone in dimethylformamide, and then placing the mixed solution in a reactor, and reacting at 120 °C for 24 hours; after cooling to room temperature, fully washing and drying to obtain a NiCo-MOF sample; using chloroauric acid as a precursor, sodium borohydride as a reducing agent, and polyvinylpyrrolidone as a surfactant to prepare nano-gold colloidal particles, and cross-linking with NiCo-MOF to obtain Au@NiCo-MOF; the inorganic salt of nickel is NiCl2 or NiNO3, and the inorganic salt of cobalt is CoCl2 or CoNO3; S2: The thiol-modified fuel probe FP was mixed with Au@NiCo-MOF to prepare functionalized FP / Au@NiCo-MOF; wherein the sequence of the fuel probe FP is 5ʹ-TCACAACGTCTACACATGGCGTTGTGATAGAGTATCCATGTGTAGACTGAATCGA-(CH2)6-SH-3ʹ; S3: Surface pretreatment of the gold electrode AuE was performed, and the thiol-modified fixed probe IP was fixed to the surface of the gold electrode AuE, and then blocked with mercaptohexanol MCH to obtain MCH / IP / AuE as the track of the DNA walker; wherein the sequence of the thiol-modified fixed probe IP is 5ʹ-SH-(CH2)6-ACTACTTCTCGATTCAATACTCTATCACAACGCCATGTGTAGACGTTGTGATAGAGTAT-3ʹ; S4: DNA with different methylation levels is dropped onto the MCH / IP / AuE interface to start the DNA walker.
2. The method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation according to claim 1, characterized in that: The step S2 specifically includes: reacting the thiol-modified fuel probe FP with tri(2-carboxyethyl)phosphine for 60 minutes to open the disulfide bond, and then reacting with Au@NiCo-MOF for 12 hours to obtain FP / Au@NiCo-MOF.
3. The method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation according to claim 1, characterized in that: The step S3 specifically includes: reacting the thiol-modified fixed probe IP with tri(2-carboxyethyl)phosphine for 60 minutes to open the disulfide bond, then dropping it onto the treated gold electrode surface, incubating it for 12 hours under light-proof conditions, and then dropping mercaptohexanol onto the sensing interface for sealing for 2 hours after rinsing, thereby obtaining MCH / / IP / AuE.
4. The method for constructing an efficient DNA walker for rapid and sensitive detection of DNA methylation according to claim 1, characterized in that: The step S4 specifically includes: simultaneously dropping DNA samples with different methylation degrees and FP / Au@NiCo-MOF onto the MCH / IP / AuE surface, and reacting at room temperature for 20 minutes, wherein the sequence of the DNA is 5ʹ-mCGTTGTGATAGAGTATTGAATmCGAGAAGTAGT-3ʹ.
5. A DNA walker obtained by the construction method according to any one of claims 1 to 4.