An electrochemiluminescence sensor for detecting DNA methylase and its preparation method and application

Abstract

The present invention discloses an electrochemiluminescence sensor for detecting DNA methylase, a preparation method and an application thereof. The sensor is composed of NH2-UIO66@WO 3‑x / Nafion modified substrate electrode, after the palindrome probe is cut, it is covalently bound to the modified electrode, and TdT-mediated branched amplification reaction is carried out on the electrode to capture and bind Ru(phen)3Cl2. The sensor of the present invention is formed by WO 3‑x Quantum dot-encapsulated metal-organic framework materials are used as co-reactants for electrochemiluminescence combined with terminal deoxynucleotidyl transferase-mediated branched polymerization reactions for the quantitative determination of DNA MTase. The procedure is simple, the reaction is rapid, and the sensitivity is high. It shows extraordinary sensitivity and reliability in spiked human serum and has been successfully extended to the screening of potential inhibitors, opening up a new path for detecting DNA MTase activity and screening inhibitors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and in particular relates to an electrochemiluminescence sensor for detecting DNA methylase, and a preparation method and application thereof. Background Art

[0002] DNA methyltransferases (MTases) are ubiquitous in biological organisms and catalyze the methylation reaction, transferring cytosine or adenine bases from specific DNA residues using S-adenosyl-L-methionine (SAM) as a methyl donor. Methyltransferases regulate many major physiological processes, from prokaryotes to eukaryotes. In humans, DNA MTases play important roles in gene expression, genome maintenance, and parental imprinting, and their overexpression may be closely associated with the development and progression of various cancers. In bacteria, DNA MTases regulate bacterial virulence and motility and are key factors in bacterial colonization and disease. Therefore, DNA MTases may serve as effective biomarkers for early clinical diagnosis and as targets for antimicrobial drugs. Recently, numerous methods for detecting DNA MTase activity have been reported, including colorimetric, fluorescent, electrochemical, and photoelectrochemical methods. However, highly sensitive quantitative detection of DNA MTases remains challenging due to the instability of detection probes and the complexity of detection procedures. Therefore, the development of highly sensitive and specific methods for detecting DNA MTase activity and screening inhibitors is of great importance.

[0003] Electrochemiluminescence (ECL) refers to the process in which a luminescent substance forms a high-energy excited state after electrochemical and chemical reactions on an electrode surface, which then relaxes to produce light. Because it fully integrates the characteristics of electrochemistry and chemiluminescence, offering advantages such as high sensitivity, controllable luminescence, and ease of miniaturization, it has become one of the most important analytical methods in the field of biomedical analysis, attracting the attention of numerous researchers and widely used in the detection and diagnosis of various biomarkers. ECL reactions are mainly divided into annihilation-type ECL and coreactant-type ECL. Compared with annihilation-type ECL, coreactant-type ECL systems offer more efficient and stable signals. Due to these superior properties, coreactant-type ECL reactions have gradually become the most mainstream reaction format in commercial ECL analytical instruments and methods. Coreactants play a crucial role in coreactant-type ECL. However, most commercial coreactants in anode-side ECL reactions currently suffer from difficult challenges such as high biotoxicity and strong background signals. Therefore, the search for superior ECL coreactants to replace toxic and volatile alkylamines remains a challenge in the field of electrochemiluminescence diagnostics. At present, semiconductor quantum dots (such as WO 3-xQuantum dots (QDs) have shown great potential in the field of ECL due to their low cost, ease of preparation, stable performance, and tunable size. However, the lack of reactive groups for attaching biomolecules has hindered their widespread application in bioassays. Summary of the Invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides an electrochemiluminescence sensor for detecting DNA methylase. The sensor is based on WO 3-x Quantum dot encapsulated metal organic framework materials (NH2-UIO66@WO 3-x ) as a co-reactant, and coupled with terminal deoxynucleotidyl transferase-mediated branched polymerization reaction for signal amplification to quantitatively detect DNA methylase activity. The electrochemiluminescence sensor has simple operation steps, rapid reaction, high sensitivity, and can realize the quantitative detection of DNA methylase activity.

[0005] Another object of the present invention is to provide a preparation method and application of the electrochemiluminescent sensor.

[0006] Technical solution: In order to achieve the above purpose, the present invention provides an electrochemiluminescent sensor for detecting DNA methylase (MTase), which is composed of NH2-UIO66@WO 3-x The palindrome probe was methylated by Dam MTase and cleaved by DpnI to obtain dsDNA, which was covalently bound to the modified electrode. TdT-mediated branched amplification reaction was carried out on the electrode, capturing and binding Ru(phen)3Cl2 to form an electrochemiluminescent sensor.

[0007] Wherein, the NH2-UIO66@WO 3-x Composed of NH2-UIO66 and WO 3-x Quantum dot preparation, the preparation process includes the following steps:

[0008] (1) Preparation of water-soluble WO by ultrasonic exfoliation 3-x quantum dots;

[0009] (2) 2-aminoterephthalic acid and ZrOCl2·8H2O were dissolved in DMF, CH3COOH was added dropwise under stirring, and the mixture was heated for reaction. After the reaction was completed, the reaction product was centrifuged to obtain a precipitate, which was washed and dried to obtain NH2-UIO66;

[0010] (3) NH2-UIO66 and WO 3-x Quantum dots were coupled in aqueous solution to obtain NH2-UIO66@WO 3-x .

[0011] As a preference, the preparation of WO in step (1)3-x The specific process of quantum dots is as follows: 50 mg WS2 is mixed with 50 mL DMF to form a mixed solution; the mixed solution is ultrasonicated at a power of 240 W for 240 minutes to obtain a black solution; the black solution is refluxed at 140°C and 800 rpm for 6 hours; the refluxed solution is centrifuged at 3000 rpm for 10 minutes to obtain a supernatant; the supernatant is heated at 70°C under vacuum conditions for 30 minutes, and the precipitate is washed with ultrapure water; the precipitate is rotary evaporated and dried at a constant temperature of 60°C.

[0012] The specific process for preparing NH2-UIO66 in step (2) is as follows: 23.5 mg of 2-aminoterephthalic acid and 31 mg of ZrOCl2·8H2O were added to 30 mL of DMF. The mixture was continuously sonicated for 10 minutes to fully dissolve it, and 3.6 mL of CH3COOH was added dropwise under stirring. The mixture was then transferred to a reactor and heated at 120°C for 24 hours. After cooling, the solid was collected by centrifugation at 6000 rpm for 10 minutes. The precipitate was washed with ethanol and dried by rotary evaporation at a constant temperature of 60°C.

[0013] Preparation of NH2-UIO66@WO in step (3) 3-x The specific process is as follows: 10 mg of NH2-UIO66 powder was dispersed in 10 mL of H2O (adjusted with HCl) at pH = 4, and ultrasonicated for 10 min to fully disperse it. 10 mL of WO with a concentration of 1 mg / mL was added dropwise under ultrasonication. 3-x The quantum dot aqueous solution was stirred at 1000 rpm for 36 h. After stirring, the mixture was centrifuged at 6000 rpm for 10 min to collect the precipitate, and the precipitate was dried by rotary evaporation at a constant temperature of 60°C.

[0014] The sequence of the palindrome probe is 5'-COOH-TTT TTC GTG GAT CCA CG-P-3', which is a specific sequence that can be self-generated for DNA methylase (MTase) recognition after the palindrome probe is annealed.

[0015] The method for constructing an electrochemiluminescence sensor for detecting DNA methylase (MTase) according to the present invention comprises the following steps:

[0016] (1) Fixing the base material: NH2-UIO66@WO 3-x The mixed solution of PEG / Nafion was drop-coated on the electrode surface and dried overnight;

[0017] (2) Anchoring recognition molecules: applying the methylated and sheared palindrome probe solution to the electrode surface obtained in step (1) and incubating;

[0018] (3) Signal amplification process: TdT, dATP and Poly-T DNA solution are applied to the electrode surface obtained in step (2) and incubated to form double-stranded DNA through branched polymerization reaction;

[0019] (4) Construction of electrochemiluminescent biosensor: The electrode obtained in step (3) is immersed in a ruthenium phenanthroline solution and incubated, so that the ruthenium phenanthroline molecules specifically bind to the double-stranded DNA (dsDNA) groove to construct an electrochemiluminescent biosensor.

[0020] Wherein, the NH2-UIO66@WO in step (1) 3-x The concentration of Nafion in / Nafion is 0.01%-0.1%wt, and the solvent of Nafion solution is ethanol; NH2-UIO66@WO 3-x Mix with Nafion solution in equal volumes.

[0021] Preferably, the NH2-UIO66@WO 3-x The concentration is 0.1-1.0 mg / mL, more preferably 0.5 mg / mL, specifically 0.5 mg / mL NH2-UIO66@WO 3-x The mixture was mixed evenly with 0.05% Nafion in ethanol (v / v=1:1), and 5 μL of the mixed solution was dropped onto the surface of the glassy carbon electrode and dried overnight.

[0022] Wherein, the preparation of the methylated and sheared palindrome probe solution in step (2) is as follows:

[0023] (1) The palindrome probe is annealed at high temperature to obtain a palindrome sequence;

[0024] (2) Methylation reaction: the annealed probe is incubated with Dam MTase and SAM for methylation;

[0025] (3) Shearing enzyme action: The methylated probe is incubated with DpnI for shearing.

[0026] In a further step, 5 μL of the obtained probe solution is drop-coated on the surface of the base electrode obtained in step (2) and incubated at 37° C. for 2 h, so as to be modified onto the electrode surface through -CO-NH- bonds.

[0027] In step (3), dATP, CoCl2, TdT, Poly-T DNA, and TdT buffer are drop-coated on the electrode surface and incubated for branched polymerization reaction. After the reaction is completed, the electrode is cleaned, and Poly-T DNA is drop-coated on the electrode surface again and incubated. The sequence of the Poly-T DNA is TTT TTT TTT TTT TTT TTT.

[0028] Preferably, 5 μL of a solution containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT, 1 μM Poly-T DNA and 1×TdT buffer (wherein 1×TdT buffer ensures that the above 5 μL system contains 100 mM KCl, 30 mM Tris-acera, 0.05% (v / v) Triton X-100, pH 7.5) is drop-coated on the surface of the base electrode obtained in step (3) and incubated at 37°C for 3 h to perform branched polymerization reaction. After the reaction is completed, the electrode is cleaned and 5 μL of 3 μM Poly-T DNA is drop-coated on the surface of the base electrode again and incubated at 37°C for 2 h.

[0029] Preferably, in step (4), the obtained electrode is immersed in a 2 mM Ru(phen) 3 Cl 2 solution and incubated at 37° C. for 2 h, so that the Ru(phen) 3 Cl 2 molecules are specifically bound to the dsDNA groove to construct an electrochemiluminescent biosensor.

[0030] The invention relates to an application of the electrochemiluminescence biosensor for detecting DNA methylase activity in quantitatively detecting DNA methylase activity.

[0031] Wherein, the DNA methylase activity is detected by WO 3-x Quantum dot-encapsulated metal-organic framework material NH2-UIO66@WO 3-x As a coreactant for electrochemiluminescence, terminal deoxynucleotidyl transferase (TdT)-mediated branched polymerization was used for signal amplification of the sensing system.

[0032] Wherein, the electrochemical reactor is made of WO 3-x Quantum dot encapsulated MOFs (NH2-UIO66@WO 3-x ) as a co-reactant and ruthenium phenanthroline as an electrochemiluminescent reagent. The electrochemiluminescent sensor for detecting DNA methylases uses DamMTase as the target molecule for target molecule recognition.

[0033] The electrochemiluminescence biosensor for detecting DNA methylase activity of the present invention is used in screening DNA methylase inhibitors and drug development.

[0034] Exploring ECL co-reactants with better performance to replace toxic and volatile alkylamines has always been a challenge in the field of electrochemiluminescence diagnosis. 3-x Quantum dots (QDs) have shown great potential in the field of ECL due to their low cost, ease of preparation, stable performance, and adjustable size. However, the lack of active groups for linking biomolecules has hindered their widespread application in bioassays. 3-xQuantum dots encapsulated MOFs to prepare NH2-UIO66@WO 3-x Nanocomposite materials, on the one hand, NH2-UIO66 has reactive end groups that can be easily coupled with biomolecules, effectively solving the problem that quantum dots lack active groups for connecting biomolecules. After the MOF is encapsulated by the present invention, the MOF surface has amino groups that can connect biomolecules; on the other hand, NH2-UIO66 can increase WO 3-x The loading of quantum dots further enhances its ECL response. Moreover, the TdT-mediated branched polymerization reaction forms branched dsDNA nanostructures that can specifically bind to Ru(phen)3Cl2, promoting the capture of ECL luminescent agents.

[0035] The present invention prepares metal organic framework materials (MOFs) with large specific surface area, high porosity and active groups, and 3-x Quantum dots encapsulated MOFs to prepare a new composite material NH2-UIO66@WO 3-x , which contains bioactive groups that can improve the coupling ability with biomolecules and increase WO 3-x The loading amount of quantum dots improves its ECL response. Moreover, the branched dsDNA structure produced by the terminal deoxynucleotidyl transferase-mediated branched polymerization reaction can promote the capture of the luminescent agent -Ru(phen)3Cl2 to enhance the ECL signal. Based on these two advantages, an electrochemiluminescence sensor was constructed for quantitative detection of DNA methylase activity. The present invention prepares a metal organic framework material (MOFs) with a large specific surface area, high porosity and active reactive groups, and uses WO 3-x Quantum dots encapsulated MOFs to prepare a new composite material NH2-UIO66@WO 3-x , which not only contains bioactive groups that can improve the coupling ability with biomolecules, but also increases the WO 3-x The loading of quantum dots enhances their ECL response. Furthermore, the terminal deoxynucleotidyl transferase-mediated branched polymerization reaction facilitates the capture of the luminescent agent (Ru(phen)3Cl2), enhancing the ECL signal. Based on this, an electrochemiluminescence sensor for detecting DNA methylase was constructed for quantitative detection of DNA methylase activity. This electrochemiluminescence sensor features simple operation, rapid response, and high sensitivity, enabling quantitative detection of DNA methylase activity.

[0036] The present invention is based on WO 3-x Quantum dot encapsulated MOFs(NH2-UIO66@WO 3-x ) as a coreactant for electrochemiluminescence, and a branched polymerization reaction mediated by terminal deoxynucleotidyl transferase was used to construct a new dual-signal amplification electrochemiluminescence sensor for the quantitative detection of DNA methylase (MTase).3-x As an ECL co-reactant for Ru(phen)3Cl2, compared with the use of WO 3-x Compared with quantum dots as co-reactants, a 7-fold higher ECL intensity was observed. The capture of ECL luminophores was promoted by TdT-mediated template-free branched polymerization, showing a signal amplification capability of about 20 times. The constructed ECL sensor showed a wide linear detection range and a lower detection limit, and can be used for the analysis of actual samples such as human serum spiked samples. The sensor of the present invention is through WO 3-x Quantum dot encapsulated metal organic framework materials (NH2-UIO66@WO 3-x ) as a co-reactant for electrochemiluminescence combined with terminal deoxynucleotidyl transferase-mediated branched polymerization for quantitative determination of DNAMTase. Due to the abundant amino groups on NH2-UIO66, it is not only conducive to biomolecule coupling, but also increases the WO 3-x Quantum dot loading enhances the ECL signal. Using branched double-stranded DNA (dsDNA) generated by a TdT-mediated branched amplification reaction instead of traditional linear dsDNA further facilitates the capture of the ECL luminescent agent, ruthenium o-phenanthroline, and promotes signal amplification. This method demonstrates exceptional sensitivity and reliability in spiked human serum and has been successfully extended to the screening of potential inhibitors, opening a new avenue for detecting DNAMTase activity and screening inhibitors.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0038] The present invention uses WO 3-x Quantum dots encapsulate NH2-UIO66 as a co-reactant, NH2-UIO66@WO 3-x Not only is NH2-UIO66 rich in amino groups beneficial for biomolecule coupling, but it is also beneficial for WO 3-x The loading of quantum dots is increased compared to using only WO 3-x Compared with quantum dots as co-reactants, the ECL intensity was observed to be 7 times higher. The ECL biosensor prepared using this method is simple to operate, has a rapid response, high sensitivity, strong anti-interference and good stability.

[0039] The present invention uses TdT-mediated branched polymerization reaction to form branched dsDNA nanostructures that can specifically bind to Ru(phen)3Cl2, promote the capture of ECL luminescent agents, and show a signal amplification capability of about 20 times. Compared with other electrochemiluminescence sensors, it has the advantages of high sensitivity and high specificity.

[0040] The present invention uses the co-reactant NH2-UIO66@WO 3-xThe nanocomposite material is modified on the electrode surface, and the DNA fragments are modified on the electrode surface through -CO-NH- bonds. Branched dsDNA nanostructures are formed on the electrode surface through TdT-mediated branched polymerization reaction, and the ECL emitter Ru(phen)3Cl2 is quantitatively captured to construct an electrochemiluminescence sensor for quantitative determination of DNAMTase. This biosensor shortens the coreactant NH2-UIO66@WO 3-x The distance from the luminescent agent Ru(phen)3Cl2 improves the luminescence efficiency of ECL.

[0041] The ECL biosensor prepared in this paper not only exhibits good sensing performance for spiked serum samples but also can be used to screen potential inhibitors. This study provides a promising analytical platform for Dam MTase activity assessment, inhibitor screening, and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 NH2-UIO66@WO of the present invention 3-x Schematic diagram of the design and preparation;

[0043] Figure 2 1 is a cyclic voltammogram during the assembly process of the ECL sensor of the present invention;

[0044] Figure 3 NH2-UIO66@WO of the present invention 3-x Comparison of ECL intensity as ECL co-reactant;

[0045] Figure 4 This is a comparison diagram of the ECL intensity of the TdT-mediated branched polymerization reaction of the present invention;

[0046] Figure 5 is a linear relationship diagram between the ECL intensity of the present invention and the logarithm of the Dam MTase concentration;

[0047] Figure 6 It is a selective investigation diagram of the present invention;

[0048] Figure 7 This is the spike recovery experiment of the ECL sensor of the present invention in human serum;

[0049] Figure 8 This is an ECL intensity graph for screening different inhibitors of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described below with reference to specific examples and accompanying drawings. Materials, reagents, and the like used in the following examples are commercially available unless otherwise specified. Experimental procedures in the examples where specific conditions are not specified were generally performed under conventional conditions or those recommended by the manufacturer.

[0051] The poly-T DNA (5'-TTT TTT TTT TTT TTT TTT TTT-3') and the palindromic probe sequence (5'-COOH-TTT TTC GTG GAT CCACG-P-3) were synthesized by Sangon Biotechnology Co., Ltd.

[0052] M.SsI MTase (catalog number: #M0226V), Dam MTase (catalog number: #M0222S), and TdT (including TdT buffer) (catalog number: #M0315S) were purchased from NEB (Beijing).

[0053] dATP (Product No. B500044-0001) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0054] The abbreviations of the technical terms in the present invention are as follows:

[0055] Oxygen-deficient tungsten oxide quantum dots: WO 3-x Quantum dots; zirconium oxychloride octahydrate: ZrOCl2·8H2O; tungsten disulfide: WS2; N,N-dimethylformamide: DMF; acetic acid: CH3COOH; Dam methylase: Dam MTase; restriction endonuclease: DpnI; S-adenosyl-L-methionine: SAM; double-stranded DNA: dsDNA; terminal deoxynucleotidyl transferase: TdT; hydrochloric acid: HCl; ruthenium o-phenanthroline: Ru(phen)3Cl2; glassy carbon electrode: GCE; metal-organic framework material: MOF; electrochemiluminescence: ECL.

[0056] Example 1

[0057] NH2-UIO66@WO 3-x Preparation, such as Figure 1 As shown:

[0058] (1)WO 3-x Preparation of quantum dots:

[0059] 50 mg of WS2 was mixed with 50 mL of DMF to form a mixed solution; the mixed solution was ultrasonicated at a power of 240 W for 240 minutes to obtain a black solution; the black solution was refluxed at 140°C and 800 rpm for 6 hours to obtain a reflux solution; the reflux solution was centrifuged at 3000 rpm for 10 minutes to obtain a supernatant; the supernatant was heated at 70°C under vacuum for 30 minutes, and the solid precipitated product was washed and precipitated with ultrapure water; the precipitate was rotary evaporated and dried at a constant temperature of 60°C.

[0060] (2) Preparation of NH2-UIO66:

[0061] 23.5 mg of 2-aminoterephthalic acid and 31 mg of ZrOCl2·8H2O were added to 30 mL of DMF. The mixture was sonicated continuously for 10 minutes to fully dissolve it. 3.6 mL of CH3COOH was then added dropwise while stirring. The mixture was then transferred to a reactor and heated at 120°C for 24 hours. After cooling, the solid was collected by centrifugation at 6000 rpm for 10 minutes. The precipitate was washed with ethanol and dried by rotary evaporation at 60°C.

[0062] (3)NH2-UIO66@WO 3-x Preparation:

[0063] 10 mg of NH2-UIO66 powder was dispersed in 10 mL of H2O (adjusted with HCl) at pH 4 and ultrasonicated for 10 min to fully disperse it. 10 mL of WO with a concentration of 1 mg / mL was added dropwise under 280 W ultrasonication. 3-x The quantum dot aqueous solution was stirred at 1000 rpm for 36 h. After stirring, the solid was collected by centrifugation at 6000 rpm for 10 min. The precipitate was dried by rotary evaporation at a constant temperature of 60 ° C to obtain NH2-UIO66@WO 3-x Disperse in aqueous solution for later use.

[0064] Example 2

[0065] Preparation of electrochemiluminescent sensor for detecting Dam MTase:

[0066] (1) Identification of target molecules:

[0067] A 1 μM palindrome probe was incubated at 95°C for 5 minutes and cooled to room temperature to obtain a palindrome sequence. The sequence was then incubated with 10 U / mL Dam MTase and 160 μM SAM at 37°C for 2 hours for methylation and incubated with 80 U / mL DpnI at 37°C for 2 hours for shearing to obtain dsDNA. The above concentrations are the final concentrations in the system.

[0068] (2) Electrode pretreatment:

[0069] The working electrode glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powders in sequence, and then ultrasonically cleaned with water, ethanol, and water for 4 min.

[0070] (3) Base material fixation:

[0071] 0.5 mg / mL NH2-UIO66@WO prepared in Example 1 3-xThe mixture was mixed evenly with a 0.05% wt Nafion ethanol solution (v / v=1:1), and 5 μL of the mixed solution was dropwise applied on the surface of the glassy carbon electrode and dried overnight.

[0072] (4) Anchor recognition molecules:

[0073] 5 μL of the dsDNA solution obtained in step (1) was drop-coated on the electrode surface obtained in step (3) and incubated at 37° C. for 2 h to modify the electrode surface through the -CO-NH- bond.

[0074] (5) Signal amplification process:

[0075] Take 5 μL of the system containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT, 1 μM Poly-T DNA and 1×TdT buffer and drop it on the electrode surface obtained in step (4) and incubate it at 37°C for 3 h to perform branched polymerization reaction. After the reaction, wash the electrode with secondary water and drop 5 μL of 3 μM Poly-T DNA again on the surface of the base electrode and incubate it at 37°C for 2 h.

[0076] Among them, the palindrome probe (SEQ ID NO.1) (5'-COOH-TTT TTC GTG GAT CCA CG-P-3')

[0077] Poly-T DNA(SEQ ID NO.2)(5'-TTT TTT TTT TTT TTT TTT TT-3')

[0078] 1× TdT buffer (so that 5 μL of the system contains 100 mM KCl, 30 mM Tris-acera, 0.05% (v / v) Triton X-100, pH 7.5)

[0079] (6) Construction of electrochemiluminescence biosensor:

[0080] The electrode obtained in step (5) was immersed in a 2 mM Ru(phen)3Cl2 solution and incubated at 37°C for 2 h, so that the Ru(phen)3Cl2 molecules were specifically bound to the dsDNA groove to construct an ECL biosensor.

[0081] Example 3

[0082] Cyclic Voltammetry Monitoring the Assembly Process of Electrochemiluminescence Sensors

[0083] In order to explore the successful preparation of the electrochemiluminescence sensor, the signal response of the sensor at each modification stage was recorded by cyclic voltammetry. The glassy carbon electrode (working electrode) obtained in steps (2)-(6) of Example 2 was placed in a 0.01M PBS (pH 7.4) solution containing 2mM K3[Fe(CN)6] and cyclic voltammetry was performed at a rate of 0.1V / s. The results are shown in FIG. Figure 2 As shown. Bare GCE in the presence of 2mM [Fe(CN)6] 3- The CV in 0.1M PBS (pH 7.4) showed a pair of redox peaks with a peak potential difference of less than 85mV, indicating a reversible redox reaction. 3-x After / Nafion was modified to the electrode surface, the peak current value of its CV curve decreased compared to the bare electrode due to the deterioration of electronic conductivity. After the DNA was covalently fixed and the DNA produced by the subsequent TdT-mediated branched polymerization reaction was fixed on the electrode, the peak current further gradually decreased because the DNA blocked the electron transfer of K3Fe(CN)6. After Ru(phen)3Cl2 was embedded in dsDNA, the peak current increased slightly, which may be because Ru(phen)3Cl2 on the electrode mediates the electron transfer between the electrode and K3Fe(CN)6 in the solution. This example illustrates the use of electrochemical methods to monitor the sensor assembly process, indicating that the corresponding materials and DNA were successfully modified to the electrode surface during the sensor preparation process.

[0084] Example 4

[0085] Determination of NH2-UIO66@WO 3-x Signal amplification capability of electrochemiluminescence sensors when used as ECL co-reactant:

[0086] (1) Identification of target molecules:

[0087] A 1 μM palindrome probe was annealed at high temperature to obtain a palindrome sequence, which was then incubated with 10 U / mL Dam MTase and 160 μM SAM at 37°C for 2 h for methylation and incubated with 80 U / mL DpnI at 37°C for 2 h for shearing to obtain dsDNA. The above concentrations are the final concentrations in the system.

[0088] (2) Electrode pretreatment:

[0089] The working electrode glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powders in sequence, and then ultrasonically cleaned with water, ethanol, and water for 4 min.

[0090] (3) Base material fixation:

[0091] 0.5 mg / mL NH2-UIO66@WO prepared in Example 13-x Mix evenly with 0.05% Nafion (v / v=1:1), take 5 μL of the mixed solution and drop it on the surface of the glassy carbon electrode and dry overnight.

[0092] For comparison, 0.5 mg / mL WO 3-x The quantum dots (prepared in Example 1) were mixed evenly with a 0.05% Nafion ethanol solution (v / v=1:1). 5 μL of the mixed solution was drop-coated on the surface of the glassy carbon electrode and dried overnight.

[0093] (4) Anchor recognition molecules:

[0094] 5 μL of the dsDNA solution obtained in step (1) was drop-coated on the electrode surface obtained in step (3) and incubated at 37° C. for 2 h.

[0095] (5) Signal amplification process:

[0096] Take 5 μL of the system containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT, 1 μM Poly-T DNA and 1×TdT buffer and drop it on the electrode surface obtained in step (4) and incubate it at 37°C for 3 h to perform branched polymerization reaction. After the reaction is completed, clean the electrode and drop 5 μL of 3 μM Poly-T DNA again on the surface of the base electrode and incubate it at 37°C for 2 h.

[0097] (6) Construction of electrochemiluminescence biosensor:

[0098] The electrode obtained in step (5) was immersed in a 2 mM Ru(phen)3Cl2 solution and incubated at 37°C for 2 h, so that the Ru(phen)3Cl2 molecules were specifically bound to the dsDNA groove to construct an ECL biosensor.

[0099] (7) Test the ECL luminescence signal of the sensor:

[0100] The sensor was tested in 0.1M PBS (pH 7.4) solution with a CV test in the potential range of 0-1.2V and a rate of 0.1V / s. The ECL luminescence signal was recorded synchronously with a photomultiplier voltage (PMT) of 800V. Figure 3 As shown, compared with the single WO 3-x Compared with quantum dots, NH2-UIO66@WO 3-x The nanocomposite showed a 7-fold signal enhancement.

[0101] Example 5

[0102] Determining the Signal Amplification Capacity of TdT-Mediated Branched Polymerization Reactions in Electrochemiluminescence Sensors:

[0103] (1) Identification of target molecules:

[0104] A 1 μM palindrome probe was annealed at high temperature to obtain a palindrome sequence, which was then incubated with 10 U / mL Dam MTase and 160 μM SAM at 37°C for 2 h for methylation and incubated with 80 U / mL DpnI at 37°C for 2 h for shearing to obtain dsDNA. The above concentrations are the final concentrations in the system.

[0105] (2) Electrode pretreatment:

[0106] The working electrode glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powders in sequence, and then ultrasonically cleaned with water, ethanol, and water for 4 min.

[0107] (3) Base material fixation:

[0108] 0.5 mg / mL NH2-UIO66@WO prepared in Example 1 3-x Mix evenly with 0.05% Nafion ethanol solution (v / v=1:1), take 5 μL of the mixed solution and drop it on the surface of the glassy carbon electrode and dry overnight.

[0109] (4) Anchor recognition molecules:

[0110] 5 μL of the dsDNA solution obtained in step (1) was drop-coated on the electrode surface obtained in step (3) and incubated at 37° C. for 2 h.

[0111] (5) Signal amplification process:

[0112] Take 5 μL of the system containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT, 1 μM Poly-T DNA and 1×TdT buffer and drop it on the electrode surface obtained in step (4) and incubate it at 37°C for 3 h to perform branched polymerization reaction. After the reaction is completed, clean the electrode and drop 5 μL of 3 μM Poly-T DNA again on the surface of the base electrode and incubate it at 37°C for 2 h.

[0113] As a comparison 1, the electrode obtained in step (4) was not subjected to any treatment;

[0114] As a comparison 2, 5 μL of the system containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT and 1× TdT buffer in step (5) was drop-coated on the electrode surface obtained in step (4) and incubated at 37°C for 3 h to perform linear polymerization reaction. After the reaction, the electrode was cleaned and 5 μL of 3 μM Poly-T DNA was drop-coated on the base electrode surface again and incubated at 37°C for 2 h.

[0115] (6) Construction of electrochemiluminescence biosensor:

[0116] The electrode obtained in step (5) was immersed in a 2 mM Ru(phen)3Cl2 solution and incubated at 37°C for 2 h, so that the Ru(phen)3Cl2 molecules were specifically bound to the dsDNA groove to construct an ECL biosensor.

[0117] (7) Test the ECL luminescence signal of the sensor:

[0118] The sensor was tested in 0.1M PBS (pH 7.4) solution with a CV test in the potential range of 0-1.2V and a rate of 0.1V / s. The ECL luminescence signal was recorded synchronously with a photomultiplier voltage (PMT) of 800V. Figure 4 As shown, TdT-mediated branched polymerization resulted in a 20-fold enhanced ECL response compared to no polymerization and a 3.2-fold signal enhancement compared to conventional linear polymerization.

[0119] Example 6

[0120] Linear relationship between the electrochemiluminescence sensor ECL intensity and Dam MTase concentration for measuring Dam MTase

[0121] (1) Identification of target molecules:

[0122] A palindrome sequence was obtained after high-temperature annealing of a 1 μM palindrome probe. The palindrome sequence was then incubated with various concentrations of Dam MTase and 160 μM SAM at 37°C for 2 h for methylation. The dsDNA was then sheared with 80 U / mL DpnI at 37°C for 2 h. The above concentrations are the final concentrations in the system.

[0123] The concentration of Dam MTase was 1×10 -3 U / mL, 1×10 -2 U / mL, 0.1U / mL, 1U / mL, 10U / mL, 100U / mL, and three parallel experimental groups were set for each concentration.

[0124] (2) Electrode pretreatment:

[0125] The working electrode glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powders in sequence, and then ultrasonically cleaned with water, ethanol, and water for 4 min.

[0126] (3) Base material fixation:

[0127] 0.5 mg / mL NH2-UIO66@WO prepared in Example 1 3-x Mix evenly with 0.05% Nafion ethanol solution (v / v=1:1), take 5 μL of the mixed solution and drop it on the surface of the glassy carbon electrode and dry overnight.

[0128] (4) Anchor recognition molecules:

[0129] 5 μL of dsDNA solution of various concentrations in step (1) was drop-coated on the electrode surface obtained in step (3) and incubated at 37° C. for 2 h to modify the electrode surface through -CO-NH- bonds.

[0130] (5) Signal amplification process:

[0131] Take 5 μL of the system containing 50 mM dATP, 2.5 mM CoCl2, 2 U TdT, 1 μM Poly-T DNA and 1×TdT buffer and drop it on the electrode surface obtained in step (4) and incubate it at 37°C for 3 h to perform branched polymerization reaction. After the reaction is completed, clean the electrode and drop 5 μL of 3 μM Poly-T DNA again on the surface of the base electrode and incubate it at 37°C for 2 h.

[0132] (6) Construction of electrochemiluminescence biosensor:

[0133] The electrode obtained in step (5) was immersed in a 2 mM Ru(phen)3Cl2 solution and incubated at 37°C for 2 h, so that the Ru(phen)3Cl2 molecules were specifically bound to the dsDNA groove to construct an ECL biosensor.

[0134] (7) Test the ECL luminescence signal of the sensor:

[0135] The sensor was tested in 0.1M PBS (pH 7.4) solution by CV with a potential range of 0-1.2V and a rate of 0.1V / s. The ECL luminescence signal was recorded simultaneously with a photomultiplier voltage (PMT) of 800V. The linear relationship between the ECL signal intensity and the logarithmic value of the Dam MTase concentration was analyzed. The results are shown in Fig. Figure 5 With the increase of Dam MTase concentration, the ECL signal intensity gradually increased. -3 The lowest detection limit of the sensor is 2.4×10 -4 U / mL. This example illustrates that the electrochemiluminescence sensor of the present invention can quantitatively detect DNA methylase activity with good detection effect, wide detection range and low detection limit.

[0136] Example 6

[0137] Analysis of target detection specificity of electrochemiluminescence sensors for Dam MTase assays

[0138] To investigate whether the sensor of the present invention has a non-specific response to the structural analogs of the target analyte, the detection process of the electrochemiluminescence sensor system is the same as that of Example 4, except that in step (1), M.SsI MTase is selected as the interfering protein, and a single component of Dam MTase or DpnI is selected as the interference control. The results are shown in FIG. Figure 6 As shown, the ECL responses of Dam MTase, DpnI, and M.SssI+DpnI were negligible compared with those of Dam MTase+DpnI, indicating that the biosensor was highly selective for DamMTase+DpnI analysis.

[0139] Example 7

[0140] Electrochemiluminescence sensor for quantitative detection of Dam MTase in real samples

[0141] In order to investigate the feasibility of the electrochemiluminescence sensor of the present invention for quantitative detection of Dam MTase in human serum, the detection process of the electrochemiluminescence sensor system was the same as that of Example 4, except that the preparation method of the DamMTase solution to be tested used in step (1) was as follows: different concentrations of DamMTase were dissolved in a 10% volume fraction human serum solution (human serum was diluted 10 times by diluting it with PBS at pH = 7.4) until the final concentration of DamMTase was 1×10 -3 U / mL, 1×10 -2 U / mL, 0.1U / mL, 1U / mL, 10U / mL, 100U / mL. like Figure 7 The results show that the recovery rate of the electrochemiluminescence sensor system of the present invention for detecting Dam MTase in human serum is between 96.45% and 103.41%, and the relative standard deviation between different control groups is less than 6.99%, indicating that this method can accurately detect Dam MTse activity in human serum.

[0142] Example 8

[0143] Screening of Dam MTase Inhibitors Using Electrochemiluminescence Sensors

[0144] MTase targeted inhibitors can prevent its methylation process. Gentamycin, penicillin, and 5-fluorouracil have been approved by the U.S. Food and Drug Administration (FDA) and are widely used in clinical applications. Therefore, these three therapeutic agents were selected as model MTase inhibitors for research. The detection process of the electrochemiluminescence sensor system is the same as that of Example 4, except that 1 μM gentamicin, penicillin, and 5-fluorouracil were added to the Dam MTase solution used in step (1) as inhibitors to evaluate the feasibility of the DNA MTase inhibition test. Figure 8As shown in the figure, the ECL signal decreased to varying degrees after the addition of 1 μM gentamicin, penicillin, and 5-fluorouracil, respectively, indicating that the three inhibitors have different inhibitory effects on Dam MTase activity. The ECL signal decreased most significantly after the addition of 1 μM 5-fluorouracil, indicating that 5-fluorouracil has the highest inhibitory efficiency on DNA MTase activity. These results demonstrate that the proposed electrochemiluminescence biosensor can be used for the screening and evaluation of DNA MTase inhibitors.

Claims

1. An electrochemiluminescence sensor for detecting DNA methylase (MTase), characterized in that: By NH2-UIO66@WO 3-x The palindrome probe was methylated by Dam MTase and cleaved by DpnI to obtain dsDNA, which was covalently bound to the modified electrode. TdT-mediated branched amplification reaction was performed on the electrode to capture and bind Ru(phen)3Cl2 to form an electrochemiluminescent sensor. The NH2-UIO66@WO 3-x Composed of NH2-UIO66 and WO 3-x Quantum dot preparation, the preparation process includes the following steps: (1) Preparation of water-soluble WO by ultrasonic exfoliation 3-x quantum dots; (2) 2-aminoterephthalic acid and ZrOCl2·8H2O were dissolved in DMF, CH3COOH was added dropwise under stirring, and the mixture was heated to react. After the reaction was completed, the reactant was centrifuged to obtain a precipitate, which was washed and dried to obtain NH2-UIO66; (3) Combine NH2-UIO66 with WO 3-x Quantum dots were coupled in aqueous solution to obtain NH2-UIO66@WO 3-x ; The sequence of the palindrome probe is 5'-COOH-TTT TTC GTG GAT CCA CG-P-3'.

2. A method for constructing an electrochemiluminescence sensor for detecting DNA methylase (MTase) according to claim 1, characterized in that: The steps include: (1) Fixing the substrate material: NH2-UIO66@WO 3-x The mixed solution of PEG / Nafion was drop-coated on the electrode surface and dried overnight; (2) Anchoring recognition molecules: applying the methylated and sheared palindrome probe solution to the electrode surface obtained in step (1) and incubating; (3) Signal amplification process: TdT, dATP and Poly-T DNA solution are applied to the electrode surface obtained in step (2) and incubated to form double-stranded DNA through branched polymerization reaction; (4) Construction of electrochemiluminescent biosensor: The electrode obtained in step (3) is immersed in a ruthenium 1,2-phenanthroline solution and incubated, so that the ruthenium 1,2-phenanthroline molecules specifically bind to the double-stranded DNA (dsDNA) groove to construct an electrochemiluminescent biosensor.

3. The construction method according to claim 2, characterized in that NH2-UIO66@WO in step (1) 3-x The concentration of Nafion in / Nafion is 0.01%-0.1% (wt), and the solvent of Nafion solution is ethanol; NH2-UIO66@WO 3-x Mix with Nafion solution in equal volumes.

4. The construction method according to claim 2, characterized in that The preparation of the methylated and sheared palindrome probe solution in step (2) is as follows: (1) The palindrome probe is annealed at high temperature to obtain a palindrome sequence; (2) Methylation reaction: The annealed probe is incubated with Dam MTase and SAM for methylation; (3) Shearing enzyme action: The methylated probe is incubated with DpnI for shearing.

5. The construction method according to claim 2, characterized in that In step (3), dATP, CoCl2, TdT, Poly-T DNA, and TdT buffer are drop-coated on the electrode surface and incubated for branched polymerization reaction. After the reaction is completed, the electrode is cleaned and Poly-T DNA is drop-coated on the electrode surface again and incubated; the sequence of the Poly-T DNA is TTT TTT TTT TTT TTT TTT TT.

6. Use of the electrochemiluminescence sensor for detecting DNA methylase (MTase) according to claim 1 in quantitatively detecting DNA methylase activity.

7. The use according to claim 6, characterized in that In the detection of DNA methylase activity, WO 3-x Quantum dot-encapsulated metal-organic framework material NH2-UIO66@WO 3-x As a co-reactant for electrochemiluminescence, terminal deoxynucleotidyl transferase (TdT)-mediated branched polymerization reaction is used for signal amplification of the sensing system.

8. Use of the electrochemiluminescence sensor for detecting DNA methylase (MTase) according to claim 1 in screening DNA methylase inhibitors and drug development.

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