Uracil DNA glycosylase dual-mode detection method based on DNA nanoladder focusing effect and application

Abstract

The application discloses a DNA nanoladder focusing effect-based double-mode detection method for uracil DNA glycosylase and application. A special DNA nanoprism is synthesized first, and then UDG is dropped on the surface of the DNA nanoprism modified electrode, so that the nanoladder (U1, M1 module, M2 module) is triggered to assemble on the electrode surface, and then the in-situ formation of the DNA nanoladder is triggered. Subsequently, the application designs a double-mode detection method: (1) Ru(phen)3 2+ mediated electrochemiluminescence (ECL) detection method; and (2) MnTMPyP mediated electrochemical impedance (EIS) detection method. The UDG activity and the inhibition effect of small molecule inhibitors are monitored through the enhancement of the ECL signal and the EIS signal. So far, there is no double-mode detection method for realizing the UDG activity monitoring by integrating the DNA nanoprism, the UDG shearing effect, the DNA nanoladder and the focusing effect. The application has the advantages of good specificity, high sensitivity, fast detection speed, accurate and reliable results and low cost.

Description

Technical Field

[0001] This invention relates to the fabrication of electrochemical biosensors and the construction of dual-mode detection methods, particularly to a dual-mode detection method and application of uracil DNA glycosylation enzymes based on the DNA nanoladder focusing effect, belonging to the fields of functional biomaterials and biosensing technology. Background Technology

[0002] The genome is composed of DNA bases that form specific base pairs, and their accuracy and stability are crucial for the cell cycle. Uracil DNA glycosylase (UDG) is an important DNA repair enzyme, critical for maintaining genome integrity. UDG removes uracil bases from DNA by initiating base excision repair (BER), thereby generating pyrimidine-free (AP) sites. Abnormal UDG activity is often associated with many diseases, such as high-grade lymphoma, neurodegenerative diseases, and Bloom syndrome, and abnormal UDG levels usually precede lesion progression, thus holding great potential for early monitoring of related diseases. Currently, UDG detection methods include electrochemical, chemiluminescence, fluorescence, and colorimetric methods. Electrochemical methods have attracted considerable attention due to their inherent advantages of simplicity, reliability, and sensitivity. Therefore, designing a more sensitive and effective strategy based on electrochemical detection technology to detect UDG activity has significant practical implications.

[0003] In recent years, the demand for early diagnosis of infectious diseases and malignant tumors has been increasing, thus high-sensitivity and high-selectivity biosensing methods for disease biomarker testing have attracted great attention. Various DNA signal amplification technologies (such as polymerase chain reaction (PCR), ligase chain reaction (LCR), rolling circle amplification (RCA), and hybridization chain reaction (HCR)) have been applied in this field. Among them, HCR-based amplification technology has attracted researchers' interest due to its advantage of enzyme-free isothermal amplification. This method allows the initiator to rapidly trigger an autonomous replication reaction to generate long DNA chains, which can then serve as carriers for loading a large number of signal tags, such as quantum dots, electrochemical redox probes, fluorophores, and electrochemiluminescent probes, to amplify the signal output. This invention utilizes the addressability-guided HCR reaction of DNA to construct a novel DNA nanoladder and uses it for UDG activity monitoring, demonstrating good novelty.

[0004] This invention first synthesizes a special DNA nanoprism containing hairpin DNA, the substrate of UDG. Therefore, after UDG is dropped onto the surface of the DNA nanoprism-modified electrode, uracil can be removed, transforming the hairpin DNA into linear DNA. DNAU1 can be captured by this linear DNA, leading to the assembly of nanoladders (M1 module, M2 module) on the electrode surface, thereby triggering the in-situ formation of DNA nanoladders. Subsequently, this invention designs a dual-modal signal output mode: (1) Ru(phen)3 2+ Its derivatives can be embedded into the grooves of double-stranded helical DNA (dsDNA) with nanoladders, exhibiting good electrochemiluminescence (ECL) properties; (2) In the presence of H2O2, meso-tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP), which has peroxidase-like properties, can promote the oxidation of 3,3'-diaminobenzidine (DAB). The nonconductive insoluble precipitates (IPs) formed in the dsDNA grooves can lead to an enhancement of the electrochemical impedance (EIS) signal. The strong DNA nanoladder network is similar to a stage focusing effect on the electrode surface, which helps the rapid transport of electrons in the electric field. Monitoring UDG activity and the inhibitory effect of its small molecule inhibitors through the enhancement of ECL and EIS signals is currently lacking. A dual-mode detection method integrating DNA nanoprisms, UDG cleavage, DNA nanoladders, and focusing effects to achieve UDG activity monitoring has yet to be found. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a dual-mode detection method and application of uracil DNA glycosylation enzyme based on the DNA nanoladder focusing effect, which has good selectivity, high sensitivity, high stability, accurate and reliable results, and low cost.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a dual-mode detection method and application of uracil DNA glycosylation enzyme based on the DNA nanoladder focusing effect, the specific steps of which are as follows:

[0007] (1) Synthesis of DNA prisms containing UDG cleavage sites

[0008] ① Add L3 (0.1~1.5μM, 5~15μL), S a (0.1~1.5μM, 10~50μL), 10×TAE / Mg 2+(2-10 μL) and tris(2-carboxyethyl)phosphine (TCEP) (10-50 mM, 1-5 μL) are mixed, DEPC water is added to a total volume of 20-100 μL, mixed well, and annealed; ② L3 (0.1-1.5 μM, 5-15 μL), S b (0.1~1.5μM, 10~50μL) and 10×TAE / Mg 2+ Mix (2-10 μL), add DEPC water to a total volume of 20-100 μL, mix well, and anneal; ③ Mix solutions ① and ② in a 1:1 ratio, anneal and assemble, and anneal.

[0009] L3, S a and S b These are the names of the three DNA strands involved in the synthesis of the DNA triangular prism in this invention, wherein S a Containing UDG splicing sites, the three DNA sequences are as follows:

[0010]

[0011] 10×TAE / Mg 2+ Solution composition: 40 mM tris(hydroxymethyl)aminomethane (Tris), 20 mM acetic acid, 2 mM ethylenediaminetetraacetic acid (EDTA), and 12.5 mM Mg. 2+ The pH is 7.4, and the solution was prepared using DEPC water.

[0012] The annealing conditions in the prism synthesis steps were: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 22℃, 30 min; 4℃, 30 min; hold, 4℃.

[0013] (2) Synthesis of M1 and M2

[0014] Given that this invention requires DNA nanoladders to amplify the signal, three special DNA strands (U1, H1, and H2) were designed, with sequences as shown in the table, and the synthesis of M1 and M2 is as follows:

[0015] H1 (final concentration: 4 μM) and H2 (final concentration: 4 μM) were respectively in Tris-HCl-EDTA-Mg 2+ (TAE-Mg 2+ Anneal in buffer solution. The annealing program is as follows: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 25℃, 30 min; 4℃, 30 min.

[0016] Tris-HCl-EDTA-Mg 2+ (TAE-Mg 2+Buffer composition: 40mM Tris-HCl, pH 7.0, 20mM HCl, 2mM EDTA and 12.5mM MgCl2.

[0017] The three special DNA strands (U1, H1, and H2) have the following sequences:

[0018]

[0019] (3) Fabrication of electrochemiluminescence sensor

[0020] Electrode 1 Preparation: Before use, the gold electrode (Au) was polished with Al2O3 powder with diameters of 0.3 μm and 0.05 μm, respectively. It was then ultrasonically cleaned three times with ultrapure water, dried with a syringe, and then immersed in 0.1 M H2SO4 solution. Cyclic voltammetry was performed in the range of -0.3 V to +1.2 V for 5–30 min. Finally, it was cleaned with ultrapure water and dried with a syringe before use.

[0021] Electrode 2 preparation: Drop 2.5–5 μL of the synthesized DNA triangular prism solution onto the surface of Electrode 1 and place it in a refrigerator overnight.

[0022] Electrode 3 preparation: After washing with DEPC water, 2.5-5 μL of 1×UDG reaction buffer and different concentrations of UDG were dropped onto the surface of the triangular prism-modified electrode and incubated at 37°C for 1-3 h. The electrode was then slowly rinsed with DEPC water. The successfully prepared electrode was labeled Electrode 2 and prepared for later use.

[0023] Electrode 4 preparation:

[0024] For the ECL detection method: U1 (2.5–5 μL, 100–200 nM) was dropped onto Electrode 3 and incubated at 37 °C for 1–2 h. To form a DNA nanoladder, a mixture containing M1, M2 (1–2 μM) and Ru(phen)3Cl2·H2O (1–5 μM) was incubated with the electrode for 8–16 h, followed by electrochemiluminescence assay in phosphate buffer solution (PB, pH 7.0, 0.1 M, containing 10 mM TPrA).

[0025] For the EIS detection method: U1 (2.5–5 μL, 100–200 nM) was dropped onto Electrode 3 and incubated at 37 °C for 1–2 h. To form a DNA nanoladder, a mixture containing M1, M2 (1 μM), and MnTMPyP (3 μM) was incubated with the electrode for 16–20 h. Subsequently, to obtain precipitation on the electrode surface, the modified electrode was immersed in a precipitation solution containing 5 mM MDAb and 1 mM H2O2 and incubated at room temperature for 25–50 min. After washing with PB buffer, the resulting electrode was subjected to precipitation in a solution containing 5 mM [Fe(CN)6]. 3- / 4- EIS measurements were performed in 0.1M PB (pH 7.0) and 0.1M KCl.

[0026] During the preparation of the Electrode 3 electrode, the concentration of UDG was varied to investigate its effect on electrochemical impedance and electrochemiluminescence signal. The electrochemiluminescence parameters used were as follows: A BPCL T15 measurement and analysis system was used, with a high voltage of 800 V, a scan rate of 100 mV / s, and a scan voltage of 0–1.35 V. The electrochemical impedance parameters used were as follows: AC frequency range of 10... -2 ~10 5 Hz, with an initial voltage of 0.245V and an amplitude of 5mV;

[0027] Invention Principle: This invention first assembles DNA triangular prisms on the electrode surface, then uses UDG to remove uracil, turning hairpin DNA into straight-chain DNA, which can capture DNA U1, thereby triggering the in-situ formation of DNA nanoladders (involving M1 and M2). Subsequently, we designed a dual-modal signal output mode: (1) Ru(phen)3 2+ Its derivatives can be embedded into the grooves of double-stranded helical DNA (dsDNA) with nanoladders, exhibiting good electrochemiluminescence (ECL) properties; (2) In the presence of H2O2, it has peroxidase-like properties. MnTMPyP can promote the oxidation of diaminobenzidine (DAB), and the non-conductive insoluble precipitates (IPs) formed in the dsDNA grooves can lead to enhanced electrochemical impedance (EIS) signals. The powerful DNA nanoladder network is similar to a stage focusing effect on the electrode surface, which helps the rapid transport of electrons in the electric field. Obviously, within a certain concentration range, the higher the UDG concentration, the more obvious the current response. Experimental results show that the magnitude of the current is linearly related to the concentration of UDG within a certain range, realizing the detection of UDG. Its advantages are:

[0028] (1) High sensitivity. Experiments showed that the linear correlation equation between the sensor's electrochemiluminescence response and the logarithm of UDG concentration was y = 2718lgC. UDG +13976, R2 =0.9982, linear range is 0.00001~0.1U / mL, detection limit is 2.7×10 - 6 U / mL; the linear correlation equation between the sensor's electrochemical impedance response and the logarithmic value of UDG concentration is y = 1911lgC. UDG +9674, R 2 =0.9970, linear range is 0.00001~0.1U / mL, detection limit is 3.1×10 -6 U / mL. This demonstrates that the sensor can achieve highly sensitive detection of UDG.

[0029] (2) High specificity. Other common related enzymes do not interfere with this detection system. This is because this invention is based on UDG removing uracil, ultimately promoting the in-situ formation of DNA nanoladders. The amount of UDG affects Ru(phen)3. 2+ With the insertion of MnTMPyP, other enzymes cannot remove uracil, thus having no interference with this detection system.

[0030] (3) The results are accurate. The recovery rates are all between 90% and 110%.

[0031] (4) Inhibitors. This biosensor was used to detect the insertion of Ru(phen)3. 2+ By observing the electrochemiluminescence response and the electrochemical impedance response to the insertion of MnTMPyP, the detection of UDG inhibitors can be achieved, and the correlation between the sensor's electrochemiluminescence and electrochemical impedance responses and UDG inhibitors can be derived.

[0032] (5) Preparation and detection methods require less reagent, have a fast detection speed, and are low in cost.

[0033] In summary, the dual-mode detection method and application of uracil DNA glycosylation enzyme based on the DNA nanoladder focusing effect has the advantages of high sensitivity, good selectivity, simple operation, rapid analysis and ease of use. It can realize the detection of low concentrations of UDG and the screening of small molecule inhibitors, and has good application prospects. Attached Figure Description

[0034] Figure 1 This is a feasibility experiment diagram of the dual-mode detection method of the sensor of the present invention;

[0035] Figure 2 The present invention describes the electrochemiluminescence response and electrochemical impedance response of the sensor in response to the presence or absence of UDG.

[0036] Figure 3 This is a logarithmic calibration curve of the electrochemiluminescence and electrochemical impedance response of the sensor of the present invention to different concentrations of UDG with respect to UDG concentration.

[0037] Figure 4 To demonstrate the inhibitory effect of different concentrations of UGI on UDG activity under dual-mode detection;

[0038] Figure 5 This is an experimental diagram showing the selectivity of the sensor of this invention under dual-mode detection. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0040] Example 1: Sensor Fabrication

[0041] (1) prism synthesis

[0042] ① Add L3 (1.18 μM, 10 μL), S a (1.21μM, 29.2μL), 10×TAE / Mg 2+ (5.9 μL) and TCEP (30 mM, 3 μL) were mixed, and DEPC water was added to a total volume of 59 μL. The mixture was then annealed. ② L3 (1.18 μM, 10 μL), S b (1.1μM, 32.2μL) and 10×TAE / Mg 2+ (5.9μL) Mix, add DEPC water to a total volume of 59μL, mix well, and anneal; ③ Mix solutions ① and ② in a 1:1 ratio, anneal and assemble, and anneal.

[0043] 10×TAE / Mg 2+ The solution consists of: 40 mM tris(hydroxymethyl)aminomethane (Tris), 20 mM acetic acid, 2 mM ethylenediaminetetraacetic acid (EDTA), and 12.5 mM Mg. 2+ The pH is 7.4, and the solution was prepared using DEPC water.

[0044] The annealing conditions in the prism synthesis steps were: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 22℃, 30 min; 4℃, 30 min; hold, 4℃.

[0045] (2) Synthesis of M1 and M2

[0046] Given that this invention requires DNA nanoladders to amplify the signal, three special DNA strands (U1, H1, and H2) were designed. The synthesis of M1 and M2 is as follows:

[0047] H1 (final concentration: 4 μM) and H2 (final concentration: 4 μM) were respectively in Tris-HCl-EDTA-Mg 2+ (TAE-Mg 2+Anneal in buffer solution. The annealing program is as follows: 95℃, 5 min; 65℃, 30 min; 50℃, 30 min; 37℃, 30 min; 25℃, 30 min; 4℃, 30 min.

[0048] Tris-HCl-EDTA-Mg 2+ (TAE-Mg 2+ Buffer composition: 40mM Tris-HCl, pH 7.0, 20mM HCl, 2mM EDTA and 12.5mM MgCl2.

[0049] (3) Fabrication of electrochemiluminescence sensor

[0050] Preparation of Electrode 1: Before use, the gold electrode (Au) was polished with Al2O3 powder with diameters of 0.3 μm and 0.05 μm, respectively. It was then ultrasonically cleaned three times with ultrapure water, dried with a syringe, and then immersed in 0.1 M H2SO4 solution. Cyclic voltammetry was performed for 5 min in the range of -0.3 V to +1.2 V. Finally, it was cleaned with ultrapure water and dried with a syringe for later use.

[0051] Preparation of Electrode 2: 5 μL of the synthesized DNA triangular prism solution was dropped onto the surface of Electrode 1 and placed in a refrigerator overnight.

[0052] Preparation of Electrode 3: After washing with DEPC water, 5 μL of 1×UDG reaction buffer and different concentrations of UDG were dropped onto the surface of the triangular prism-modified electrode and incubated at 37°C for 1 h. The electrode was then slowly rinsed with DEPC water. The electrode was successfully prepared and labeled as Electrode 2 for later use.

[0053] (4) Electrochemiluminescence (ECL) and electrochemical impedance (EIS) testing

[0054] For the ECL detection method: U1 (5 μL, 100 nM) was dropped onto Electrode 3 and incubated at 37 °C for 2 h. To form a DNA nanoladder, a mixture containing M1, M2 (1 μM) and Ru(phen)3Cl2·H2O (5 μM) was incubated with the electrode for 16 h, followed by electrochemiluminescence assay in phosphate buffer (PB, pH 7.0, 0.1 M, containing 10 mM TPrA).

[0055] For the EIS detection method: U1 (5 μL, 100 nM) was dropped onto Electrode 3 and incubated at 37 °C for 2 h. To form a DNA nanoladder, a mixture containing M1, M2 (1 μM), and MnTMPyP (3 μM) was incubated with the electrode for 16 h. Subsequently, to obtain precipitation on the electrode surface, the modified electrode was immersed in a precipitation solution containing 5 mM MDAb and 1 mM H2O2 and incubated at room temperature for 25 min. After washing with PB buffer, the resulting electrode was subjected to precipitation in a solution containing 5 mM [Fe(CN)6]. 3- / 4- EIS tests were performed in 0.1M PB (pH 7.0) and 0.1M KCl.

[0056] The electrochemical impedance and electrochemiluminescence response of the four prepared electrodes were measured, see [reference needed]. Figure 1 It can be seen that the prepared Electrode 4 electrode exhibits a significantly stronger electrochemical response compared to the other three electrodes, indicating that the sensor has been successfully prepared.

[0057] Example 2: Electrochemiluminescence and electrochemical impedance Response with and without UDG

[0058] An electrochemical biosensor based on DNA nanoladder signal enhancement was constructed using uracil DNA glycosylation enzymes. Our biosensor was prepared based on Example 1. See [link to example]. Figure 2 In the presence of UDG, the sensor showed virtually no electrochemiluminescence and electrochemical impedance response in PB (0.1M, pH 7.0), while in the presence of UDG, there was a significant electrochemiluminescence and electrochemical impedance response, proving that the sensor can be used for UDG activity detection.

[0059] Example 3: UDG Activity Detection

[0060] An electrochemical biosensor for uracil DNA glycosylation enzyme was constructed based on DNA nanoladder signal enhancement. The sensor preparation steps were the same as in Specific Example 1. During the Electrode3 construction process, the concentration of UDG was changed sequentially to: 0, 0.00001, 0.00002, 0.00005, 0.0001, 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 U / mL. The electrochemical impedance and electrochemiluminescence response of the sensor in PB (0.1 M, pH 7.0) were recorded. Based on the experimental results, a series of electrochemical response curves corresponding to different concentrations of UDG were obtained, and a quantitative relationship between the electrochemiluminescence and electrochemical impedance responses and the UDG concentration was established. Based on the quantitative relationship between the two, the concentration of UDG in the sample to be tested was determined. The experimental results are as follows. Figure 3As shown, the electrochemical response of the sensor becomes more pronounced with increasing UDG concentration, and the linear correlation equations are y = 2718lgC. UDG +13976, R 2 =0.9982; y = 1911lgC UDG +9674, R 2 =0.9970, linear range was 0.00001~0.1U / mL, detection limit was 2.7×10 -6 U / mL, 3.1×10 -6 The U / mL indicates that the sensor can achieve highly sensitive detection of UDG activity.

[0061] Example 4: Detection of UGI, the UDG inhibitor

[0062] An electrochemical biosensor based on DNA nanoladder signal enhancement was constructed using uracil DNA glycosylation enzyme. The sensor preparation steps were the same as in Specific Example 1. During the Electrode 3 construction process, the concentration of UDG was 0.1 U / mL, and different concentrations of the inhibitor UGI were added sequentially: 0, 0.001, 0.002, 0.003, 0.005, 0.008, 0.01, 0.02, 0.03, 0.05, 0.08, 0.1, 0.2, 0.3, 0.5, and 0.8 U / mL. This UGI concentration was then used to prepare the sensor. The electrochemiluminescence and electrochemical impedance responses of the sensor in PB (0.1 M, pH 7.0) were recorded. The experimental results (e.g.) indicate... Figure 5 As the concentration of the inhibitor UGI increases, the corresponding electrochemiluminescence and electrochemical impedance responses become weaker, indicating that UGI has a stronger inhibitory effect on UDG activity. The half-inhibitory concentrations are 0.037 U / mL and 0.035 U / mL, respectively.

[0063] Example 5: Selective Detection

[0064] In the selectivity experiment, the concentration of UDG and other enzymes was 0.1 U / mL. The abbreviations of the other enzymes used are as follows: protein kinase (PKA), terminal transferase (TdT), histone acetyltransferase (HAT), glucose oxidase (GOx), and horseradish peroxidase (HRP). Following the sensor preparation steps in Example 1 above, during the Electrode 3 construction process, other enzymes of the same concentration were used instead of UDG to prepare the sensor. The results are as follows... Figure 5 As shown, compared with UDG, the sensor has a very small electrochemical response to other enzymes, which is close to the blank signal, indicating that the sensor has good selectivity for the detection of UDG.

[0065] Of course, the above description is not intended to limit the present invention, nor is the present invention limited to the examples given above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a dual-mode detection sensor for uracil DNA glycosylation enzyme based on the DNA nanoladder focusing effect, characterized in that, First, a special DNA nanoprism was synthesized containing hairpin DNA, the substrate of UDG. After UDG was dropped onto the surface of the DNA nanoprism-modified electrode, uracil was removed, turning the hairpin DNA into straight-chain DNA. A DNA nanoladder suitable for UDG activity detection was designed. Since DNA U1 can be captured by straight-chain DNA, it leads to the assembly of nanoladder modules M1 and M2 on the electrode surface, thereby triggering the in-situ formation of the DNA nanoladder. Subsequently, the signal is output in a dual-modal mode: (1) Ru(phen)3 2+ Its derivatives can be embedded into the grooves of double-stranded helical DNA (dsDNA) with nanosteps, exhibiting good electrochemiluminescence (ECL) properties; (2) In the presence of H2O2, MnTMPyP, which has peroxidase-like properties, can promote the oxidation of diaminobenzidine (DAB), and the non-conductive insoluble precipitates (IPs) formed in the dsDNA grooves can lead to an enhancement of the electrochemical impedance (EIS) signal. The L3, S of the triangular prism a and S b The DNA sequence is as follows: The DNA sequences of the DNA nanoladders U1, H1, and H2 are as follows:

Images