CN110687171A - Electrochemical biosensor for detecting base excision repair enzyme and preparation method and application thereof - Google Patents
Electrochemical biosensor for detecting base excision repair enzyme and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an electrochemical biosensor for detecting base excision repair enzyme and a preparation method and application thereof. The biosensor comprises a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying an iron-containing nitrogen-rich carbon nanotube and cyclodextrin onto a glassy carbon electrode; the electrochemical biosensor also comprises an MB-hairpin/AuNPs probe, wherein the MB-hairpin/AuNPs probe comprises gold nanoparticles and a thiolated MB-hairpin structure probe modified on AuNP, and a stem region in the MB-hairpin structure probe is designed with 1 to a plurality of target bases of the base excision repair enzyme to be detected. The electrochemical biosensor prepared by the invention has the advantages of high sensitivity, low background signal and the like, and has wide application value in the fields of screening of the base excision repair enzyme inhibitor, analysis of biological samples and the like.
Description
Technical Field
The invention belongs to the technical field of electrochemical detection, and particularly relates to an electrochemical biosensor for detecting base excision repair enzyme, and a preparation method and application thereof.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
The natural enzyme has high activity and good specificity, and plays an important role in organism metabolism and various biochemical reactions. However, natural enzymes face significant challenges including high cost of preparation, purification and storage, susceptibility to denaturation under harsh conditions, and inhibition of catalytic activity in certain complex media (e.g., wastewater). The demand for highly efficient specific enzymes has led chemists to design synthetases with similar structures and analogous functions using novel nanotechnology, and a large number of functional nanomaterials continue to emerge. Noble metals (e.g. Pd and Au), metal oxides (e.g. MnO)2，Fe3O4And CeO2) Carbon-based nanomaterials, metal complexes, porphyrins, and polymers have been extensively studied to mimic the structure and function of natural enzymes. Compared with natural enzymes, the nano material has the advantages of low cost, adjustable catalytic activity, good stability, good robustness under severe environment conditions (such as methanol, ethanol and dimethylformamide), easiness in long-term storage and treatment and the like, and is expected to become a research hotspot of artificial enzymes.
The realization of supramolecular host-guest recognition through non-covalent interactions is a new approach to large functional structure assembly. In general, the host (e.g., β -CD) contains a large volume cavity and has outstanding recognition and encapsulation capabilities for guest molecules. Due to the specificity and bio-orthogonality of the recognition motifs, host-guest interactions have found widespread use in bioassays.
Base Excision Repair (BER) is one of the DNA repair mechanisms, uracil-DNA glycosylase (UDG) plays a key role in maintaining genome integrity. UDG is capable of removing uracil damage from DNA by catalyzing the hydrolysis of the n-glycosidic bond between uracil and deoxyribose. Because DNA glycosylase is crucial to DNA damage repair and is associated with individual and population disease susceptibility, UDG has become a promising biomarker and a potential therapeutic target. Traditional methods for detecting DNA glycosylase include gel electrophoresis, radioactive detection and chromatography, but these methods are generally time-consuming and labor-consuming, involve dangerous radioactive substances and complicated procedures. In addition, several new UDG detection methods have been developed, including colorimetric and fluorescent methods, but they involve the preparation of hairpin probes and functional nucleic acid probes with dye labels. Therefore, there is a need to develop a simple and sensitive method for detecting DNA glycosylase.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an electrochemical biosensor based on subject-object action and simulated enzyme electrocatalytic signal amplification, and a preparation method and application thereof.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the present invention, an electrochemical biosensor for detecting a base excision repair enzyme is provided, which comprises a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying iron-containing nitrogen-rich carbon nanotubes (FeCN) and cyclodextrin (beta-CD) onto a Glassy Carbon Electrode (GCE).
Further, the electrochemical biosensor further comprises an MB-hairpin/AuNPs probe, wherein the MB-hairpin/AuNPs probe comprises gold nanoparticles (AuNPs) and a thiolated MB-hairpin structure (hairpin) probe modified on the AuNPs, a stem region in the MB-hairpin structure probe is designed with a target base of the repair enzyme for base excision to be detected, and the number of the target base can be set according to actual conditions, such as 1, 2, 4, 6 and the like.
Wherein the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG), and the like; preferably uracil-DNA saccharifying enzyme (UDG).
In a second aspect of the present invention, there is provided a method for preparing the electrochemical biosensor for detecting a base excision repair enzyme, the method comprising:
(1) preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; after drying, dropwise adding the beta-CD solution on FeCN/GCE to obtain a beta-CD/FeCN/GCE electrode;
(2) preparation of MB-hairpin/AuNPs Probe: mixing and incubating gold nanoparticle (AuNP) solution and thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the target product.
In a third aspect of the present invention, there is provided the use of the above electrochemical biosensor for detecting a base excision repair enzyme.
In a fourth aspect of the present invention, there is provided a method for detecting a base excision repair enzyme based on the above electrochemical biosensor, the method comprising: and adding the MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding the beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection.
In a fifth aspect of the present invention, there is provided the use of the electrochemical biosensor and/or the detection method described above in drug screening and enzyme analysis of biological samples related to the enzyme for repairing base excision.
The base excision repair enzyme related drugs include but are not limited to base excision repair enzyme inhibitors and base excision repair enzyme activators;
the biological sample comprises cells of an organism, such as HeLa cells. Tests prove that the biosensor provided by the invention has better analysis capability on real complex biological samples, can be used for quantitative detection on the activity of cell base excision repair enzyme (such as UDG), and has great application potential in the fields of biomedical basic research, clinical diagnosis and the like.
The invention has the beneficial effects that:
1. use of mimetic enzymes: although natural enzymes have good activity and specificity, the wide application of the natural enzymes is limited by high cost and changeability, the iron-nitrogen-rich carbon nanotube (FeCN) used by the invention is prepared by one-step self-assembly of low-cost dicyandiamide and iron (II) chloride tetrahydrate, and then is synthesized by simple heat treatment, the method is simple and convenient, the cost is low, and the iron-nitrogen-rich carbon nanotube (FeCN) simulating peroxidase can electrocatalytic methylene blue with electrocatalytic activity to obviously amplify electrochemical signals.
2. High sensitivity: the catalytic oxidation of MB by FeCN is mediated by the catalytic substrate RSH which is relatively stable in O2 oxidation, but not by external H2O2Greatly simplifying the experimental process, and being capable of sensitively detecting the UDG with the detection limit of 7.413 multiplied by 10-5U mL-1。
3. Low background signal: due to the stem-loop structure of hairpin DNA and the steric effect of AuNPs, supramolecular host-guest reactions occur between MB and β -CD, resulting in very low background signals.
4. A wide range of potential applications: the electrocatalytic amplification biosensor designed by the invention can be used for screening of UDG inhibitors and analysis of biological samples, and has wide potential application in biomedical research.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of an electrochemical biosensor for one-step detection of UDG based on host-guest interaction and FeCN mimic enzyme electrocatalytic signal amplification.
FIG. 2 is a representation of different nanoparticles of the present invention, wherein A is an X-ray diffraction pattern (XRD) of a synthesized iron-containing nitrogen-rich carbon nanotube (FeCN), B is an ultraviolet absorption spectrum of the synthesized nanogold and the MB-modified hairpin probe in combination with the nanogold, C is a Transmission Electron Microscope (TEM) image of the synthesized iron-containing nitrogen-rich carbon nanotube (FeCN), and D is a Transmission Electron Microscope (TEM) image of the gold nanoparticle.
FIG. 3 is a representation of the experimental feasibility analysis of the invention, A being Fe (CN) at 5 mmoles per liter with 0.1 moles per liter KCl6 3-/4-The Electrochemical Impedance Spectroscopy (EIS) of different modified electrodes in (1), wherein a is a bare Glassy Carbon Electrode (GCE), b is FeCN/GCE, c is beta-CD/FeCN/GCE,d is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE; b is an electrochemical Differential Pulse Voltammetry (DPV) curve containing 0.1 mol/L PBS of different modified electrodes, a is MB-hairpin/AuNPs + beta-CD/FeCN/GCE, B is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE, c is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE +10mM cysteine, d is MB-hairpin/AuNPs +1UmL- 1UDG+FeCN/GCE。
FIG. 4 is a graph of experimental condition optimization of the present invention, where A is the optimization of FeCN concentration, B is the optimization of β -CD concentration, C is the incubation time of one-step reaction of UDG with a sensor, and error bars represent the standard deviation of three independent experiments.
FIG. 5 is a graph representing the results of the sensitivity and selectivity experiments of the present invention, A is the electrochemical Differential Pulse Voltammetry (DPV) curve of biosensors incubated with different concentrations of UDG (from a to j: 0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1U per ml), B is the linear relationship between current and logarithm of UDG concentration in the range of 0.0005 to 1U per ml, detection conditions: 0.1 mol/l phosphate buffered solution at pH 7.4 containing 10 mmol/l cysteine, C is a graph characterizing the results of the selectivity experiment, 0.01 mg/ml BSA, 1U/l hAAG, 1U/l FPG, respectively, and the error bars represent the standard deviation of three independent experiments.
FIG. 6 shows the difference in the effect of different concentrations of UGI on the relative activity of UDG in accordance with the present invention. The concentration of UDG was maintained at 1U per ml, and the error bars represent the standard deviation of three independent experiments.
FIG. 7 is a graph of the linear correlation between the current of the invention and the logarithm of the number of HeLa cells from 5 to 10000 cells, with error bars representing the standard deviation of three independent experiments.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
As previously mentioned, conventional DNA glycosylase detection methods are often time consuming, laborious, involve hazardous radioactive materials and complicated procedures. In order to solve the technical problems, the invention provides a method for detecting a base excision repair enzyme based on a host-guest action and a mimic enzyme mediated electrocatalytic amplification biosensor.
In an exemplary embodiment of the present invention, an electrochemical biosensor for detecting a base excision repair enzyme is provided, which includes a β -CD/FeCN/GCE electrode fabricated by modifying an iron-containing nitrogen-rich carbon nanotube (FeCN) and cyclodextrin (β -CD) onto a Glassy Carbon Electrode (GCE).
In another embodiment of the present invention, the electrochemical biosensor further includes an MB-hairpin/AuNPs probe, where the MB-hairpin/AuNPs probe includes gold nanoparticles (AuNPs) and a thiolated MB-hairpin structure (hairpin) probe modified on the AuNPs, a stem region in the MB-hairpin structure probe is designed with 1 to multiple target bases of the base excision repair enzyme to be detected, and the number of the target bases may be set according to actual situations, such as 1, 2, 4, 6, and the like.
In yet another embodiment of the invention, the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), and thymine-DNA glycosylase (TDG).
In another embodiment of the present invention, the stem region of the hairpin structure probe is modified with Methylene Blue (MB) at the 3 'end and thiol at the 5' end, such that the hairpin structure is connected to the gold nanoparticle (AuNP) through a gold-sulfur bond.
In another embodiment of the present invention, when the base excision repair enzyme to be detected is uracil-DNA saccharifying enzyme (UDG), the nucleotide sequence of the hairpin structure can be:
5’-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH2)6-MB-3' (SEQ ID NO. 1). Wherein, italicized letters represent bases that undergo complementary pairing in the stem region of the hairpin structure.
In another embodiment of the present invention, there is provided a method for preparing the electrochemical biosensor for detecting a base excision repair enzyme, the method comprising:
(1) preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; and after drying, dropwise adding the beta-CD solution onto FeCN/GCE to obtain the beta-CD/FeCN/GCE electrode.
(2) Preparation of MB-hairpin/AuNPs Probe: mixing and incubating gold nanoparticle (AuNP) solution and thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the target product.
In another embodiment of the invention, the concentration of the FeCN solution is 0.5-4 mg/mL (preferably 2 mg/mL); the concentration of the beta-CD solution is 0.5-4 mg/mL (preferably 2 mg/mL); experiments prove that when the concentration of the FeCN solution is 2mg/ml and the concentration of the beta-CD solution is 2mg/ml, the electrochemical peak current is strongest, and the detection effect is optimal.
The preparation method of the FeCN comprises the following steps: heating dicyandiamide and ferrous salt to 490-510 ℃ in an inert gas atmosphere to self-assemble to form a precursor, heating the precursor to 890-910 ℃ in the inert gas atmosphere, and calcining to obtain the iron-containing nitrogen-rich carbon nanotube FeCN.
Wherein the diameter of the gold nanoparticle (AuNP) is 13nm, and the gold nanoparticle is preferably prepared by a sodium citrate reduction method.
The activating treatment process of the thiolated MB-hairpin structure probe comprises the following steps: the disulfide-bonded oligonucleotide is reduced with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 0.5 to 1.5 hours (preferably 1 hour).
The mixing incubation time is 10-20 hours (preferably 16 hours), namely, the thiolated hairpin probe is connected to AuNPs through a gold-sulfur bond.
In still another embodiment of the present invention, there is provided a use of the above electrochemical biosensor for detecting a base excision repair enzyme.
Wherein the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG).
In another embodiment of the present invention, there is provided a method for detecting a base excision repair enzyme based on the above electrochemical biosensor, the method comprising: and adding the MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding the beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection.
Wherein the incubation treatment condition is 40-120 min (preferably 80min), and the incubation treatment temperature is 30-40 ℃ (preferably 37 ℃).
In another embodiment of the present invention, the electrochemical biosensor and/or the detection method are used for drug screening and enzyme analysis of biological samples related to the enzyme for repairing base excision.
The base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG); further preferably uracil-DNA saccharifying enzyme (UDG).
The base excision repair enzyme related drugs include but are not limited to base excision repair enzyme inhibitors and base excision repair enzyme activators;
the biological sample comprises cells of an organism, such as HeLa cells. Tests prove that the biosensor provided by the invention has better analysis capability on real complex biological samples, can be used for quantitative detection on the activity of cell base excision repair enzyme (such as UDG), and has great application potential in the fields of biomedical basic research, clinical diagnosis and the like.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. In the following examples, the hairpin probes have the sequence from 5 'to 3':
5’-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH2)6-MB-3’。
examples
The principle of the embodiment is as shown in fig. 1:
the principle of an electrochemical biosensor for UDG detection is shown in figure 1. And dripping FeCN solution on the surface of the GCE to obtain FeCN/GCE. After drying at room temperature, the beta-CD solution was dropped onto FeCN/GCE to form beta-CD/FeCN/GCE. The DNA substrate is designed as a hairpin structure with a stem and a loop. The stem contains two complementary strands, including a methylene blue MB-labeled 3 'end and a thiol-labeled 5' end, of which there are six uracil bases. The 5' end sulfhydryl is connected with AuNPs through gold-sulfur bond, MB cannot be recognized by beta-CD/FeCN/GCE due to the stereo effect of AuNPs and DNA of hairpin stem-loop structure, and lower background current is caused. The presence of UDG facilitates the removal of six uracil bases from hairpin DNA, resulting in a linear single-stranded DNA with a MB at the 3' end. MB can assemble onto the electrode through host-guest interaction with β -CD, resulting in a significant enhancement of MB peak current. At the same time, L-cysteine (RSH) having a thiol structure is substituted with O2Conversion to L-cystine (RSSR) and release of H2O2. At H2O2With the help of FeCN, oxidation of MB is catalyzed, generating an amplified electrochemical signal. Due to the high-efficiency electrocatalysis of FeCN mimic enzyme, stable catalytic substrate RSH and supermolecule host-guest strategy, the biosensor has extremely low background and confidenceThe advantage of signal amplification, it can be used for sensitive detection of UDG activity.
The specific process comprises the following steps:
and (3) FeCN synthesis: dicyandiamide (12mmol) was reacted with iron (II) chloride tetrahydrate (FeCl)2·4H2O, 0.84mmol), placing the mixture on a quartz boat, placing in a tube furnace with argon, and heating at 500 ℃ for 2 hours. The temperature was then further raised to 900 ℃ with a slope of 10 ℃/min and held at 900 ℃ for 2 hours with argon gas being passed, to give a black powdery material. After grinding in a mortar, the resulting FeCN powder was stored in a desiccator.
Synthesis of gold nanoparticles (AuNPs) (diameter 13 nm) and MB-hairpin/AuNP probes: for the synthesis of gold nanoparticles, first, all glassware used in the preparation process needs to be in aqua regia (3 parts concentrated hydrochloric acid, 1 part concentrated HNO)3) Thoroughly cleaning, washing with secondary water, and drying for later use (attention is required: aqua regia is extremely dangerous and should be handled with caution, requiring gloves and goggles for handling). First 0.01% HAuCl4The solution 100 ml was boiled with vigorous stirring and then 2.5 ml of 1% sodium citrate solution was added rapidly. The mixed solution was kept boiling for 30 minutes and then cooled to room temperature. When AuNPs were formed, the solution turned deep red, then stirred and cooled to room temperature. The AuNPs obtained were stored in brown glass bottles at 4 ℃ for further use.
The MB-hairpin probe was diluted to 10. mu. mol per liter of buffer (1.5 mmol per liter MgCl)210 mmol per liter pH 8.0Tris-HCl) and then incubated at 95 ℃ for 5 minutes, slowly cooled to room temperature over 30 minutes to completely fold into a hairpin structure. The thiolated MB-hairpin probe (10. mu. mol per liter) was then activated with 1 mmol per liter of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 1h to open the disulfide bond. AuNPs were mixed with MB-hairpin probe for 16h, and the resulting solution was centrifuged at 12000rpm for 20min to remove excess oligonucleotide. The wine red MB-hairpin/AuNP precipitate was washed with 0.1 mol/L PBS and dispersed with 0.1 mol/L PBS. The resulting probe solution was stored in a refrigerator for use. The MB-hairpin @ AuNPs are identified by an ultraviolet spectrophotometry.
Preparing an electrochemical biosensor: the electrochemical sensor is constructed on the GCE electrode. The electrodes were treated with 1.0, 0.3 and 0.05 micron alpha-Al prior to modification2O3The GCE electrodes were powder polished and then sonicated with pure water and ethanol for 3 minutes, respectively. 10 μ l of FeCN solution (2 mg per ml, solvent is ultrapure water) was added dropwise onto the GCE surface to obtain FeCN/GCE. After drying at room temperature, 10. mu.l of a beta-CD solution (2 mmol/l, solvent is ultrapure water) was dropped on FeCN/GCE to obtain beta-CD/FeCN/GCE.
Electrochemical detection of UDG and inhibitor assays: 5 microliters of MB-hairpin/AuNP probe was added to a 10 microliter reaction system containing different concentrations of UDG and 1 microliter of a 10 XUDG reaction buffer. beta-CD/FeCN/GCE was incubated with 10. mu.l of MB-hairpin/AuNP probe solutions containing different concentrations of the target UDG at 37 ℃ for 80 minutes, the electrodes were thoroughly washed and nitrogen dried and stored at 4 ℃. Furthermore, the UDG inhibition experiment was studied using UGI as a model inhibitor. Except that the inhibitor UGI with different concentrations contains 1U mL-1Similar approach was used for UDG inhibition assays, except for the pre-mixing of reaction buffer for UDG
Preparation of cell extract: HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin in an incubator containing 5% carbon dioxide at 37 ℃. When the hela cells grew to the exponential growth phase, they were collected by trypsinization, washed twice with cold PBS (pH 7.4, Gibco, usa) and the resulting solution was centrifuged at 1000 rpm for 5 minutes. These cells were suspended in 100. mu.l lysis buffer, incubated on ice for 30 min, and centrifuged at 12000g for 20min at 4 ℃. The supernatant was transferred to a fresh tube and stored at-80 ℃. Results of the experiment
1. Characterization of materials
This example investigated the crystal structure of a FeCN catalyst using an X-ray diffraction pattern (XRD) (fig. 2A). The peak at 26.2 ℃ represents the characteristic diffraction of the carbon (002) plane, consistent with the structural characteristics of carbon nanotubes. The peak in the range of 40 to 50 ℃ may be directed to Fe3Diffraction of C Structure (JCPDF No.89-2867) indicated thermal polycondensation and carbonizationFeCN is then formed. Uv-vis absorption spectroscopy was used to characterize the formation of MB-hairpin/AuNPs conjugates (fig. 2B). The adsorption peak for 10nm AuNPs was 518nm (FIG. 2B, black curve). After hairpin probe modification of AuNPs, the AuNPs increased in diameter due to coupling of hairpin DNA to AuNPs, resulting in a slight shift of the surface plasmon resonance peak from 518 to 522nm (fig. 2B, red curve). Notably, a new absorption peak appeared at 260nm, which is a characteristic absorption peak of the double bond of the DNA base, indicating the formation of the MB-hairpin/AuNPs conjugate. Fig. 2C shows a Transmission Electron Microscope (TEM) image of the synthesized FeCN doped with small amount of iron nanoparticles. In addition, the morphology of AuNPs was also studied using transmission electron microscopy. Uniform shape with an average diameter of 13nm (FIG. 2D).
2. Experimental verification of principle
To demonstrate the feasibility of this solution, this example characterizes the modified electrode by Electrochemical Impedance Spectroscopy (EIS) at 5 millimoles per liter [ Fe (CN)6]3-/4-Electrochemical behavior at different stages in the solution. When FeCN is coated on GCE, due to good conductivity of FeCN, electron transfer resistance (R) is compared to bare Glassy Carbon Electrode (GCE) (fig. 3A, curve a)et) Decreasing to 22 Ω (fig. 3A, curve b). Electron transfer resistance (R) when Cyclodextrin (. beta. -CD) is assembled onto FeCN/GCEet) Further down to 50 omega (fig. 3A, curve c). After assembling MB-hairpin/AuNPs probes reactive with 1U per ml UDG on a beta-CD/FeCN/GCE surface (FIG. 3A, Curve d), RetIncreased to 560. omega. because the negatively charged phosphate backbone of the DNA electrostatically repels the negatively charged redox probe [ Fe (CN)6]3-/4-The electron transfer is reduced.
The feasibility of biosensors with different modified electrodes was explored using Differential Pulse Voltammetry (DPV). As in fig. 3B, a large MB peak current was observed when the target UDG was present (fig. 3B, curve B), but in the absence of UDG no electrochemical signal was detected (fig. 3B, curve a). Notably, the amplification of the electrochemical signal (FIG. 3B, curve B) initially resulted from the addition of no H2O2On the premise of (1), the electrocatalytic reduction of MB by FeCN. DPV of MB when cysteine is added to the electrolyteThe peak current was further increased (FIG. 3B, curve c), which can be explained by the presence of a thiol-structured L-cysteine bound to O2Oxidative release of H2O2And the catalytic oxidation of the electron medium MB by FeCN is promoted, and an amplified electrochemical signal is generated. In contrast, in the absence of β -CD, MB cannot accumulate on the electrode via host-guest interactions because of the absence of β -CD, and the electrochemical signal is also very low in the presence of UDG (fig. 3B, curve d).
3. Optimization of experimental conditions
In order to achieve the best detection performance, experimental conditions such as the concentration of FeCN and β -CD and the incubation time of UDG with the biosensor were optimized (fig. 4). As shown in fig. 4A, the electrochemical peak current increases as the concentration of FeCN increases from 0.5 to 2mg per ml, and when the concentration is greater than 2mg, the current value decreases because a high concentration of FeCN may cause FeCN to fall off the electrode. Therefore, 2mg per ml of FeCN was used in subsequent experiments. The effect of β -CD concentration on assay performance was further investigated (fig. 4B). The peak current increases as the concentration of β -CD increases from 0.5 to 2 mmoles per litre, and when the concentration exceeds 2 mmoles per litre, the current value decreases due to electron transfer inhibition caused by saturated host-guest interactions and β -CD non-conduction. Thus, 2 mmoles per liter of β -CD were used for subsequent studies. In addition, the incubation time for the biosensor to detect UDG in one step was optimized (fig. 4C). With the change of the incubation time from 40min to 80min, the current value rapidly increased with the increase of the incubation time and tended to be stable above 80min, so that 80min was selected as the optimal incubation time in the subsequent studies
4. Sensitivity and selectivity assays
To evaluate the sensitivity of this protocol for detecting UDG, the effect of different concentrations of UDG on DPV current was measured in 0.1 mol per liter PBS (pH 7.4) containing 10 mmol per liter of cysteine under optimal experimental conditions (fig. 5A). The Differential Pulse Voltammetry (DPV) signal increased with increasing UDG concentration from 0.0005 to 1U per ml, and a good linear relationship was obtained between the change in DPV current and the logarithm of the UDG concentration (0.0005 to 1U per ml). The corresponding equation is 1.329+0.2836log10C, correlation coefficient 0.9767 (fig. 5B), where I is the current intensity and C is the UDG concentration (U per ml). The detection limit of the biosensor was calculated to be 7.413 × 10 based on 3 times the standard deviation of the blank response-5U per ml is 27 times higher than reported UDG determination method, 270 times higher than color method, and 107 times higher than electrochemical method. The high sensitivity of the present solution can be attributed to three factors: (1) due to the stem-loop structure of hairpin dnas and the steric effect of AuNPs, supramolecular host-guest reactions between MB and β -CD occur leading to extremely low background; (2) the simulated peroxidase FeCN has good electrocatalytic activity and stability in a wider pH and temperature range; (3) l-cysteine by O2Conversion to L-cystine (RSSR) with release of H2O2And the catalytic oxidation of MB by FeCN is promoted, and an amplified electrochemical signal is generated.
In order to evaluate the specificity of detecting UDG in this embodiment, Bovine Serum Albumin (BSA), human alkyl adenine DNA saccharifying enzyme (hAAG) and formamidopyrimidine DNA saccharifying enzyme (FPG) were used as negative controls to evaluate the specificity of detection. BSA cannot recognize and remove uracil from DNA substrates. hAAG cleaves alkylated adenine to generate uracil-free sites, and FPG cleaves 4, 6-diamino-5-carboxamidopyrimidine, 8-hydroxyguanine, 2, 6-diamino-4-hydroxy-5-carboxamidopyrimidine but does not cleave the DNA substrate used in this study. As shown in fig. 5C, the target UDG detected a high electrochemical signal, while the current responses detected by 0.01 mg per ml BSA, 1U per ml hAAG, 1U per ml FPG were negligible. Experimental results show that the technical scheme has better specificity on UDG.
5. Reproducibility and stability experiments of electrochemical biosensors
The reproducibility of the electrochemical biosensor was investigated by 6 consecutive experiments in the presence of 1U per ml of UDG. The Relative Standard Deviation (RSD) was 1.6%, indicating that the electrochemical biosensor has good reproducibility. In addition, after the test, after the electrode is polished and cleaned, under the same condition, the current signal changes by 5.8% compared with the previous response, which indicates that the electrochemical biosensor has good stability and regeneration capability.
6. UDG inhibitor assay
To verify the feasibility of the proposed electrochemical biosensor for UDG inhibition experiments, Uracil Glycosylase Inhibitor (UGI) was used as model inhibitor. UGI can bind UDG in a 1:1 molar stoichiometric ratio to form a tight, physiologically irreversible complex. Relative Activity of UDG (RA) according to 
Calculation, No represents the peak current without UDG, Nt represents the peak current in the presence of 1U per ml of UDG, and Ni represents the current value in the presence of 1U per ml of UDG and UGI simultaneously. As shown in fig. 6, the relative activity of UDG decreased monotonically with increasing UGI concentration. The experimental result shows that the electrochemical biosensor can be used for screening the UDG inhibitor.
7. Biological sample testing
In order to evaluate the capability of the biosensor proposed in the present technical solution to analyze a real complex biological sample, this example uses HeLa cells as a model for detecting UDG activity of cells. As shown in FIG. 7, the peak current is linearly related to the logarithm of the HeLa cell number, ranging from 5 to 10000 cells, and the linear equation is that I is 0.7468log10N-0.4502 with correlation coefficient 0.9931, wherein I is the current intensity and N is the number of HeLa cells. The limit of detection was determined to be 5 cells based on 3 standard deviations of the blank response. These results clearly indicate that the electrochemical biosensor can be used for quantitative detection of cell UDG activity, and has great application potential in clinical diagnosis.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> electrochemical biosensor for detecting base excision repair enzyme, and preparation method and application thereof
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>32
<212>DNA
<213> Artificial sequence
<400>1
uuugucugug aaggaggtag atcacagaca aa 32
Claims (10)
1. The electrochemical biosensor for detecting the base excision repair enzyme is characterized by comprising a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying an iron-containing nitrogen-rich carbon nanotube and cyclodextrin onto a glassy carbon electrode.
2. The electrochemical biosensor of claim 1, further comprising an MB-hairpin/AuNPs probe comprising gold nanoparticles and a thiolated MB-hairpin probe modified on the AuNP, wherein the stem region of the MB-hairpin probe is designed with 1 to multiple target bases of the base excision repair enzyme to be detected.
3. The electrochemical biosensor of claim 1, wherein the base excision repair enzymes are DNA glycosylases including alkyl adenine DNA glycosylases, formamidopyrimidine DNA glycosylases, uracil-DNA glycosylases, and thymine-DNA glycosylases.
4. The electrochemical biosensor as set forth in claim 2, wherein the stem region of the MB-hairpin probe is modified at the 3 '-end with methylene blue and at the 5' -end with a thiol group;
preferably, when the base excision repair enzyme to be detected is uracil-DNA glucoamylase, the nucleotide sequence of the hairpin structure is shown as SEQ ID NO. 1.
5. The method for preparing an electrochemical biosensor for detecting a base excision repair enzyme according to any one of claims 1 to 4, comprising:
preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; after drying, dropwise adding the beta-CD solution on FeCN/GCE to obtain a beta-CD/FeCN/GCE electrode;
preparation of MB-hairpin/AuNPs Probe: mixing and incubating the gold nanoparticle solution and the thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the gold nanoparticle probe.
6. The method according to claim 5, wherein the FeCN solution has a concentration of 0.5 to 4mg/mL (preferably 2 mg/mL); the concentration of the beta-CD solution is 0.5-4 mg/mL (preferably 2 mg/mL).
7. The method according to claim 5, wherein the gold nanoparticles have a diameter of 13nm and are prepared by a sodium citrate reduction method; or the like, or, alternatively,
the activating treatment process of the thiolated MB-hairpin structure probe comprises the following steps: reducing the disulfide bond-bonded oligonucleotide with tris (2-carboxyethyl) phosphine hydrochloride for 0.5 to 1.5 hours (preferably 1 hour); or the like, or, alternatively,
the mixing incubation time is 10 to 20 hours (preferably 16 hours).
8. Use of the electrochemical biosensor according to any one of claims 1 to 4 for detecting a base excision repair enzyme.
9. A method for detecting a base excision repair enzyme based on the electrochemical biosensor as set forth in any one of claims 1 to 4, comprising: adding an MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding a beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection;
preferably, the incubation condition is 40 to 120min (more preferably 80min), and the incubation temperature is 30 to 40 ℃ (more preferably 37 ℃).
10. Use of the electrochemical biosensor according to any one of claims 1 to 4 and/or the detection method according to claim 9 for screening drugs related to base excision repair enzymes, enzyme analysis of biological samples;
preferably, the base excision repair enzyme is a DNA glycosylase comprising alkyl adenine DNA glycosylase, formamidopyrimidine DNA glycosylase, uracil-DNA glycosylase and thymine-DNA glycosylase;
preferably, the base excision repair enzyme related drug comprises a base excision repair enzyme inhibitor and a base excision repair enzyme activator;
preferably, the biological sample comprises cells of an organism (HeLa cells).
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Inventor after: Zhang Chunyang Inventor after: Cui Lin Inventor after: Zhao Minhui Inventor before: Zhang Chunyang Inventor before: Cui Lin Inventor before: Zhao Minhui |