CN114295699B - Photoelectrochemical biosensor for detecting 5-formyl cytosine deoxyribonucleotide and preparation method and application thereof - Google Patents
Photoelectrochemical biosensor for detecting 5-formyl cytosine deoxyribonucleotide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Ti base on accordion shape 3 C 2 The method for detecting 5-formylcytosine deoxyribonucleotide in DNA by photoelectrochemical analysis firstly constructs a flower-shaped Bi containing an electrode 2 S 3 Accordion-like Ti 3 C 2 A polydopamine, a 5-formylcytosine deoxyribonucleotide, a calcined ZIF8. By using Bi 2 S 3 And Ti 3 C 2 The photoelectric property is improved by matching the energy bands, the amino group on the polydopamine and the aldehyde group on the 5-formacyl cytosine deoxyribonucleotide are subjected to specific covalent reaction, C-ZIF-8 is used as an artificial mimic enzyme, and the reaction is carried out in H 2 O 2 The reaction of catalyzing 4-chloro-1-naphthol to be oxidized to generate benzene-4-chlorohexanedione in the presence, and depositing the benzene-4-chlorohexanedione on the surface of the electrode to form a crop insulation film can quench the response of the photoelectrochemical sensor. The detection of 5-formyl cytosine deoxyribonucleotide in DNA is realized, and the detection method is simple and easy to operate and has high detection sensitivity.
Description
Technical Field
The invention relates to the technical field of photoelectrochemical analysis, in particular to a photoelectrochemical biosensor for detecting 5-formoxyl cytosine deoxyribonucleotide and a preparation method and application thereof.
Background
The formylation of DNA is a very important part of the field of epigenetic modification. At the DNA level, 5-formylcytosine deoxyribonucleotides are the predominant methylation-modified form of DNA. Are involved in gene regulation, cell differentiation and certain diseases [2 ]. Although 5-formylcytosine deoxyribonucleotides are present in a variety of organs and cells, they are present in amounts of only 0.02 to 0.002% of cytosine, about one percent of the 5hmC content. Research shows that dynamic regulation of 5-formylcytosine deoxyribonucleotide has obvious influence on gene expression control. Various studies have confirmed the importance of 5-formylcytosine deoxyribonucleotides, but due to the lack of sensitive and highly selective analytical methods, the analysis and assessment of the influence of 5-formylcytosine deoxyribonucleotide content on the subjects to be studied are greatly influenced, and therefore the realization of detection of 5-formylcytosine deoxyribonucleotides and their content is of great significance.
At present, the related detection methods aiming at the 5-formyl cytosine deoxyribonucleotide mainly comprise thin-layer chromatography, gas chromatography, column liquid chromatography, liquid chromatography-mass spectrometry combined method and capillary electrophoresis method. Although the method has great effect on detecting 5-formylcytosine deoxyribonucleotide (5 fC), the method also has the defects of needing precise and complicated instruments, complicated sample pretreatment, needing professional operators, low detection sensitivity, weak specificity and the like. Therefore, it is important to establish a simple, rapid, highly sensitive and highly selective method for the detection of 5-formylcytosine deoxyribonucleotides.
PEC biosensors have been developed based on rapid photoelectric conversion and efficient biomolecule-specific recognition, with the advantages of electrochemical and photochemical analysis. Which excites the electro-optically active material with light to produce photo-generated electrons and holes. And the photo-generated electrons are captured by the electrodes to generate a current. The excitation light source and detection signalThe method is two completely different forms, so that the interference of background signals can be effectively reduced, the sensitivity of analysis and detection is greatly improved, and the method has the advantages of high detection speed, easiness in miniaturization, simplicity in operation, simplicity in instrument, low background signals and the like. The patent with the application number of 201810477154.8 discloses a photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine deoxyribonucleotide and a preparation method thereof, and the detection of 5hmC is realized by utilizing good photoelectric activity and biocompatibility of tungsten sulfide, excellent electron donor property and biocompatibility of polydopamine, specific recognition and combination performance of phos-tag-biotin on phosphate groups and specific reaction of hydroxymethyl and sulfydryl on 5hmC catalyzed by M.HhaI methyltransferase. However, although the patent has better selectivity to 5hmC, the detection range and sensitivity are still to be improved. Therefore, a flower-shaped Bi base material is needed 2 S 3 The photoelectrochemical analysis of the-MXene composite material detects the 5-formacyl cytosine deoxyribonucleotide so as to realize the detection of the 5-formacyl cytosine deoxyribonucleotide with high specificity and high sensitivity.
Disclosure of Invention
In view of the prior art, the present invention aims to provide a photoelectrochemical biosensor for detecting 5-formylcytosine deoxyribonucleotide, and a preparation method and an application thereof. Realizes the detection of the 5-formyl cytosine deoxyribonucleotide with high specificity and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a photoelectrochemical biosensor for detecting 5-formylcytosine deoxyribonucleotide comprises an electrode and flower-shaped Bi sequentially modified on the surface of the electrode 2 S 3 Accordion-like MXene (Ti) 3 C 2 ) Polydopamine, 5-formylcytosine deoxyribonucleotide and calcined ZIF8.
Preferably, the electrode is an ITO electrode.
Description of terms: MXene in the application is Ti 3 C 2 。
In a second aspect of the present invention, there is provided a method for preparing the photoelectrochemical biosensor, comprising the steps of:
(1) Will contain flower-shaped Bi 2 S 3 The dispersion liquid is dripped on a pretreated electrode and dried to obtain Bi 2 S 3 An electrode;
(2) Will contain accordion-shaped Ti 3 C 2 Dropwise adding the dispersion of (2) to Bi obtained in step (1) 2 S 3 Drying on the electrode to obtain Ti 3 C 2 /Bi 2 S 3 An electrode;
(3) Dropwise adding the dispersion liquid containing polydopamine to the Ti obtained in the step (2) 3 C 2 /Bi 2 S 3 Drying on the electrode to obtain PDA/Ti 3 C 2 /Bi 2 S 3 An electrode;
(4) Dropwise adding 5-formyl cytosine deoxyribonucleotide to the PDA/Ti obtained in the step (3) 3 C 2 /Bi 2 S 3 Reaction on an electrode to give 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 An electrode;
(5) Dropwise adding the dispersion containing calcined ZIF8 (C-ZIF 8) to the 5fdCTP/PDA/Ti obtained in step (4) by utilizing the specific binding between phosphate and zinc 3 C 2 /Bi 2 S 3 On an electrode, after the reaction is finished, the mixture is placed in a nano enzyme reaction solution to be stirred to obtain C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The electrode is a photoelectrochemical biosensor for detecting the 5-formacyl cytosine deoxyribonucleotide.
Preferably, in step (1), the pretreated electrode is prepared by respectively using acetone and 1-4M NaOH aqueous alcohol solution (V) Anhydrous ethanol :V Secondary water 1-1) and secondary water for 15-40min respectively, and drying in the air.
Preferably, in step (1), the flower-like Bi is contained 2 S 3 The dispersion of (A) is prepared by mixing flower-like Bi 2 S 3 Dispersing the nano material in deionized water; the drying is irradiation of an infrared lamp;
more preferably, bi is flower-shaped 2 S 3 The nano material is prepared by the following method:
adding Bi (NO) 3 ) 3 ·5H 2 Dissolving O and thiourea in ethylene glycol, carrying out hydrothermal reaction, collecting precipitate, centrifugally washing with water and ethanol, drying, and collecting solid to obtain flower-shaped Bi 2 S 3 A nano-material.
Preferably, in the step (2), the solution contains accordion-shaped Ti 3 C 2 The dispersion of (A) is prepared by mixing accordion-shaped Ti 3 C 2 Dispersing in deionized water to obtain the product; the drying is irradiation of an infrared lamp;
more preferably, the accordion-like Ti 3 C 2 The preparation method comprises the following steps:
mixing Ti 3 AlC 2 Mixing the aqueous solution and HF solution, stirring at 60 deg.C for 24 hr to obtain a mixture containing accordion-shaped Ti 3 C 2 Centrifuging to collect precipitate, centrifuging with water and ethanol, drying, and collecting solid to obtain accordion-shaped Ti 3 C 2 。
Preferably, in the step (3), the dispersion liquid containing polydopamine is obtained by dispersing polydopamine nano-material in deionized water; the drying is irradiation of an infrared lamp;
more preferably, the polydopamine nano-material is prepared by the following method:
mixing dopamine and secondary water, adding a sodium hydroxide solution, stirring at 50 ℃ for 5 hours to form a polydopamine dispersion, centrifuging, collecting precipitates, washing with water by centrifugation, drying, and collecting solids to obtain the polydopamine nano-material.
Preferably, in the step (4), the reaction is carried out for 0.5 to 3 hours under the humid condition at 37 ℃, and then the washing is carried out for 1 to 5 times.
Preferably, in the step (5), the calcined ZIF 8-containing dispersion is obtained by dispersing a calcined ZIF8 nanomaterial in deionized water; the reaction is carried out for 0.5 to 5 hours under the humid condition of 37 ℃, and then the washing is carried out for 1 to 5 times;
more preferably, the calcined ZIF8 nanomaterial is prepared by the following method:
1) Will be provided withZn(NO 3 ) 2 ·6H 2 Dissolving O and dimethyl imidazole in deionized water, stirring for 1 hour, collecting precipitate, centrifugally washing with water and ethanol, drying, and collecting solid to obtain a ZIF8 nano material;
2) Calcining the ZIF8 nano material obtained in the step 1) in a tubular furnace for 2 hours to obtain a calcined ZIF8 nano material, namely C-ZIF8.
Preferably, the nanoenzyme reaction solution is prepared by adding H to NaAc-HAc solution 2 O 2 Mixing the solution and 4-chloro-1-naphthol solution.
In a third aspect of the invention, there is provided the use of the photoelectrochemical biosensor in the detection of 5-formylcytosine deoxyribonucleotides.
In a fourth aspect of the present invention, there is provided a method for detecting 5-formylcytosine deoxyribonucleotide using the photoelectrochemical biosensor, comprising the steps of:
the photoelectrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a Pt wire is used as an auxiliary electrode to form a three-electrode system for photoelectrochemical signal detection, a detection solution is a Tris buffer solution (pH is 5.5-8.5) containing 5-500mM ascorbic acid, the relation between the current and the concentration of 5-hydroxymethylcytosine nucleotide is established, and the content of 5-hydroxymethylcytosine nucleotide is detected.
Preferably, the detection method is a current-time method, and the applied potential is-0.5-0.5V;
preferably, the detection range is 0.001-200nM, with a detection limit of 0.33pM.
The invention has the beneficial effects that:
(1) The invention utilizes calcined ZIF8 to Bi 2 S 3 -Ti 3 C 2 The photoelectric signal amplification is realized by the action of the composite material, and the detection sensitivity of the 5-formyl cytosine deoxyribonucleotide is improved.
(2) The invention utilizes the aldehyde group specificity covalent reaction of the amino group on the polydopamine and the 5-formacyl cytosine deoxyribonucleotide to improve the specificity of the detection of the 5-formacyl cytosine deoxyribonucleotide.
(3) The detection method is simple, low in cost and capable of realizing miniaturization of instruments, and the efficient and sensitive detection of the 5-formyl cytosine deoxyribonucleotide can be realized only by simply processing the surface of the ITO electrode.
Drawings
FIG. 1: the invention discloses a schematic diagram of photoelectrochemical biosensor construction and 5-formyl cytosine deoxyribonucleotide detection.
FIG. 2: accordion-like Ti prepared in example 1 3 C 2 SEM picture of (g);
FIG. 3: a linear fit of photocurrent intensity to the log of 5-formylcytosine deoxyribonucleotide concentration.
FIG. 4: histogram of changes in photoelectrochemical response under different nucleotide conditions.
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.
Description of the drawings: the "room temperature" in the present invention is in the range of 20 to 30 ℃.
"humid conditions" in the present invention are humidity greater than 90%; the preferred humidity is 95-99%.
The cleaning solution used in the invention comprises the following components: 3-15mM Tris-HCl and 20-60mM KCl, pH 7.4.
The 5-formylcytosine deoxyribonucleotide buffer solution used in the invention comprises the following components: 5-30mM Tris, pH 6.0-8.5.
The DNA dissolving solution used in the present invention comprises the following components: 5-100mM Tris, 1-50mM Ethylene Diamine Tetraacetic Acid (EDTA), pH 5.0-8.5, and the solvent is sterile water.
As described in the background section, although the prior art can detect 5-formylcytosine deoxyribonucleotide, the prior art also has some defects, such as the need of precise and complicated instruments, complicated sample pretreatment, the need of professional operators, low detection sensitivity, poor specificity and the like.
Based on the above, the invention constructs a photoelectrochemical biosensor for detecting 5-formylcytosine deoxyribonucleotide, and the schematic diagram of the photoelectrochemical biosensor for construction and detection is shown in figure 1. The photoelectrochemical biosensor of the invention uses an ITO electrode as a substrate electrode and flower-shaped Bi 2 S 3 And accordion-like Ti 3 C 2 And modifying the surface of the electrode in sequence. And modifying polydopamine to the surface of the electrode by utilizing electrostatic adsorption. The 5-formyl cytosine deoxyribonucleotide is modified on the surface of an electrode by utilizing the reaction of the amino group of polydopamine and the aldehyde group of the 5-formyl cytosine deoxyribonucleotide. And modifying the calcined ZIF8 to the surface of the electrode by utilizing the specific binding between phosphate radical and zinc ions. Wherein, the flower-shaped Bi 2 S 3 Is an excellent photoelectric material, namely accordion-shaped Ti 3 C 2 (see FIG. 2) and Bi 2 S 3 The composite material is formed, the photocurrent is increased, polydopamine is modified on the surface of an electrode by electrostatic adsorption, the surface of the electrode is provided with amino, 5-formylcytosine deoxyribonucleotide is introduced to block the migration of photoproduction electrons, the photocurrent is reduced, C-ZIF-8 is used as an artificial mimic enzyme, and H is subjected to H-mediated isothermal amplification (HAS) to generate a complex with high photoelectric conversion efficiency 2 O 2 Catalyzing the 4-chloro-1-naphthol to be oxidized to generate the benzene-4-chlorohexanedione in the presence of the catalyst. Benzo-4-chlorohexadienone is then deposited on the surface of the electrode as an insulating film, which can quench the response of the photoelectrochemical sensor. Therefore, the detection of 5-formylcytosine deoxyribonucleotide can be realized by utilizing the linear relation between the concentration of 5-formylcytosine deoxyribonucleotide and the intensity of photocurrent.
In one embodiment of the present invention, the construction process of the photoelectrochemical biosensor is as follows:
(1) Flower-like Bi 2 S 3 The preparation of (1): mixing 1.0-3.0g Bi (NO) 3 ) 3 ·5H 2 Dissolving O and 0.2-1.0g thiourea in 80mL ethylene glycol, mixing the above solutions under stirring, stirring for 10-60min, transferring the mixture into a reaction kettle, standing at 150-200 deg.C for 20-40h, centrifuging at 6000-12000rpm for 10min, collecting precipitateCentrifugally washing with water and absolute ethyl alcohol for 2-6 times, drying at 40-60 ℃ and collecting solid, wherein the obtained solid is flower-shaped Bi 2 S 3 And (3) nano materials.
(2) Accordion like Ti 3 C 2 The preparation of (1): 40mL 10-40wt% of HF solution and Ti 3 AlC 2 Mixing, stirring for 20-27h, centrifuging at 10000-12000rpm for 10min, collecting precipitate, centrifuging and washing with water and anhydrous ethanol for 2-6 times, drying at 40-60 deg.C, and collecting solid to obtain accordion-shaped Ti 3 C 2 。
(3) Preparation of polydopamine: adding 0.1-1g of dopamine hydrochloride into 90mL of secondary water, mixing the solutions under a stirring state, adding 0.1-1g of NaOH, aging at 55 ℃ for 3-5h, centrifuging at a rotating speed of 6000-12000rpm for 10min, collecting precipitates, centrifuging and washing with water and absolute ethyl alcohol for 2-6 times, drying at 40-60 ℃ and collecting solids, wherein the obtained solids are spherical PDA nano materials.
(4) Preparation of calcined ZIF 8: respectively adding 0.01-0.05g of Zn (NO) 3 ) 2 ·6H 2 Dissolving O and dimethyl imidazole in 120mL of methanol, stirring for 1 hour, collecting precipitate, centrifugally washing with water and ethanol, drying, and collecting solid to obtain the ZIF8 nano material. And calcining the obtained ZIF8 nano material in a tubular furnace for 2 hours to obtain a calcined ZIF8 nano material.
(5)Bi 2 S 3 Preparation of the dispersion: weighing 2-10mg of flower-shaped Bi prepared in the step (1) 2 S 3 Adding the nano material into 2-10mL of deionized water, and performing ultrasonic dispersion for 0.5-3 hours.
(6)Ti 3 C 2 Preparation of the dispersion: weighing 2-10mg of accordion-shaped Ti prepared in the step (2) 3 C 2 Adding the nano material into 2-10mL of deionized water, and performing ultrasonic dispersion for 0.5-3 hours.
(7) Preparation of polydopamine dispersion: and (4) weighing 2-10mg of the polydopamine nano material prepared in the step (3), adding into 2-10mL of deionized water, and performing ultrasonic dispersion for 0.5-3 hours.
(8) Preparation of calcined ZIF8 dispersion: and (5) weighing 2-10mg of the calcined ZIF8 prepared in the step (4), adding into 2-10mL of deionized water, and performing ultrasonic dispersion for 0.5-3 hours.
(9) Preparation of electrode washing buffer: mixing 3-15mM Tris-HCl and 20-60mM KCl with sterilized water as solvent, and adjusting pH to 7.4.
(10) Preparing a nano enzyme reaction solution: to 1mL of NaAc-HAc solution having pH 5.5, 1 to 10. Mu.L of H was added, respectively 2 O 2 And (4) obtaining the nano enzyme reaction solution by using 1-10 mu L of 4-chlorine-1-naphthol solution.
(11) Pretreating an ITO electrode: cutting ITO conductive glass into 5 × 1cm 2 Sequentially using acetone and 1-4M NaOH in alcohol-water solution (V) Anhydrous ethanol :V Secondary water 1-1), ultrasonically treating the ITO electrode with secondary water for 15-60min, then washing with the secondary water, and naturally drying for later use.
(12) Flower-like Bi 2 S 3 Fixing: 20-80 mu L of Bi 2 S 3 And dropwise adding the dispersion liquid to the surface of the pretreated ITO electrode, and irradiating and drying by using an infrared lamp. Then, the electrode is washed 1 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as Bi 2 S 3 /ITO。
(13) Accordion Ti 3 C 2 Fixing: mixing 20-80 μ L of Ti 3 C 2 Dropping the dispersion to Bi 2 S 3 Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode is washed 1 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as Ti 3 C 2 /Bi 2 S 3 /ITO。
(14) Fixing polydopamine: dropping 20-80. Mu.L of PDA dispersion to Bi 2 S 3 Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode is washed 1 to 5 times with a washing liquid. And (5) drying by nitrogen. The prepared electrode is marked as PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
(15) Immobilization of 5-formylcytosine deoxyribonucleotide: dropping 10-50 μ L of 5-formylcytosine deoxyribonucleotide to PDA/Ti 3 C 2 /Bi 2 S 3 The ITO electrode surface reacts for 0.5 to 3 hours under the humid condition of 37 ℃, and then is cleaned for 1 to 5 times. The prepared electrode was labeled 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
(16) C-ZIF8 immobilization: adding 10-60. Mu.L of C-ZIF8 dispersion to 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The ITO electrode surface reacts for 0.5-5h under the humid condition of 37 ℃, and then is cleaned for 1-5 times. The mixture is put into 13mL of nano enzyme reaction solution to be stirred for reaction, and the prepared electrode mark is C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
In the construction process of the photoelectrochemical sensor, the steps supplement each other, the sequence is strictly limited, each step serves for the next fixed modification, and the lack of the previous step can cause the failure of the subsequent modification. The material fixedly modified on the surface of the ITO electrode can be a commercially available product or can be prepared by itself, and the invention is not particularly limited as long as the performance meets the use requirement.
In another embodiment of the present invention, there is provided a process for detecting 5-formylcytosine deoxyribonucleotides by using the above-mentioned photoelectrochemical biosensor, comprising:
(1) Preparing an electrode detection solution: preparing Tris buffer solution with pH of 5.5-8.5 and concentration of 5-500mM with sterilized water, adding Ascorbic Acid (AA) of 5-500mM into the solution, and making the obtained solution as electrode detection solution.
(2) C-ZIF8/5fdCTP/PDA/Ti prepared with different concentrations of 5-formylcytosine deoxyribonucleotide 3 C 2 /Bi 2 S 3 The ITO electrode is used as a working electrode, the saturated calomel electrode is used as a reference electrode, the Pt wire is used as an auxiliary electrode, a three-electrode system is formed to detect photoelectrochemical signals, a light source is visible light, an application potential is-0.5-0.5V, and photocurrent is recorded in electrode detection liquid.
(3) Establishing photocurrent intensity and C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The relation between ITO concentration and the C-ZIF8/5fdCTP/PDA/Ti in the sample to be detected 3 C 2 /Bi 2 S 3 The content of ITO was measured.
With C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 Increase of ITO, electrode MeterThe quantity of the face C-ZIF8 is increased, so that the amount of benzene-4-chlorohexanedione generated by catalyzing the oxidation of 4-chloro-1-naphthol is increased, the photocurrent is reduced, and the detection of the 5-formylcytosine deoxyribonucleotide can be realized according to the linear relation between the concentration of the 5-formylcytosine deoxyribonucleotide and the photocurrent intensity.
The detection range of the photoelectric chemical biosensor for 5-formyl cytosine deoxyribonucleotide is 0.001-200nM, and the detection limit is 0.33pM.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1
(1) Flower-like Bi 2 S 3 Preparation of
0.78g of Bi (NO) is added 3 ) 3 ·5H 2 Dissolving O and 0.49g of thiourea in 80mL of deionized water in sequence, mixing the solutions under stirring, stirring for 30min, transferring the mixture into a reaction kettle, standing at 160 ℃ for 25h, centrifuging at a rotating speed of 9000rpm for 10min, collecting precipitate, alternately centrifuging and washing with water and absolute ethyl alcohol for 4 times, drying at 50 ℃ and collecting solid, wherein the obtained solid is flower-shaped Bi 2 S 3 And (3) nano materials.
(2) Accordion like Ti 3 C 2 Preparation of (2)
40mL 40wt% of HF solution and 1g Ti 3 AlC 2 Mixing, stirring for 25h, centrifuging at 11000rpm for 10min, collecting precipitate, sequentially centrifuging and washing with water and anhydrous ethanol for 4 times, drying at 50 deg.C, and collecting solid to obtain accordion-shaped Ti 3 C 2 。
(3) Preparation of polydopamine: adding 0.5g of dopamine hydrochloride into 90mL of secondary water, mixing the solutions under a stirring state, adding 0.5g of NaOH, aging at 55 ℃ for 4 hours, centrifuging at 9000rpm for 10min, collecting precipitates, sequentially and alternately centrifuging and washing with water and absolute ethyl alcohol for 4 times, drying at 50 ℃ and collecting solids, wherein the obtained solids are spherical PDA nano materials.
(4) Preparation of calcined ZIF8
3.36g of Zn (NO) 3 ) 2 ·6H 2 Dissolving O in 40mL of methanol, slowly adding 120mL of methanol aqueous solution containing 8g of 2-methylimidazole under the stirring state, continuously stirring for 24h, centrifuging at the rotating speed of 9000rpm for 10min, collecting precipitate, centrifuging and washing for 4 times by using water and absolute ethyl alcohol, drying at 50 ℃, and collecting solid to obtain the ZIF8 nano material. And (3) calcining the collected ZIF8 nano material in a tubular furnace at 600 ℃ for 2 hours at the heating rate of 5 ℃/min to obtain a calcined ZIF8 material (C-ZIF 8).
(5)Bi 2 S 3 Preparation of the Dispersion
Weighing 6mg of flower-shaped Bi prepared in the step (1) 2 S 3 And adding the mixture into 5mL of deionized water, and ultrasonically dispersing for 1 hour.
(6)Ti 3 C 2 Preparation of the Dispersion
Weighing 6mg of accordion-shaped Ti prepared in the step (2) 3 C 2 Added to 6mL of deionized water, and ultrasonically dispersed for 1 hour.
(7) Preparation of polydopamine dispersion:
and (4) weighing 6mg of the polydopamine nano material prepared in the step (3), adding the polydopamine nano material into 6mL of deionized water, and performing ultrasonic dispersion for 2 hours.
(8) Preparation of C-ZIF8 dispersions
And (3) weighing 6mg of the C-ZIF8 prepared in the step (4), adding the weighed C-ZIF8 into 5mL of deionized water, and performing ultrasonic dispersion for 1 hour.
(9) Preparation of nano enzyme reaction solution
To 1mL of NaAc-HAc solution at pH 5.5, 10. Mu.L of 15mM H was added 2 O 2 10 mu L of 10mM 4-chloro-1-naphthol solution to obtain the nano enzyme reaction solution.
(10) Preparation of electrode washing buffer
10mM Tris-HCl and 50mM KCl were mixed with sterile water as a solvent, and the pH was adjusted to 7.4.
(11) ITO electrode pretreatment
Cutting ITO conductive glassAt a height of 5X 1cm 2 Sequentially using acetone, 1M NaOH in alcohol-water solution (V) Anhydrous ethanol :V Secondary water = 1), ultrasonically treating the ITO electrode with secondary water for 20min, then washing with the secondary water, and naturally airing for later use.
(12) Flower-like Bi 2 S 3 Is fixed to
40 mu L of Bi 2 S 3 And (4) dropwise adding the dispersion liquid to the surface of the pretreated ITO electrode prepared in the step (11), and irradiating and drying by using an infrared lamp. Then, the electrode was washed 3 times with the electrode washing buffer prepared in step (10). And (5) drying by nitrogen. The prepared electrode is marked as Bi 2 S 3 /ITO。
(13)Ti 3 C 2 Is fixed
Mixing 40. Mu.L of Ti 3 C 2 Dropwise adding the dispersion to Bi 2 S 3 Drying the surface of the ITO electrode by irradiation of an infrared lamp. Then, the electrode was washed 3 times with the electrode washing buffer prepared in step (10). And (5) drying by nitrogen. The prepared electrode is marked as Ti 3 C 2 /Bi 2 S 3 /ITO。
(14) Immobilization of polydopamine
40 μ L of PDA dispersion was added dropwise to Ti 3 C 2 /Bi 2 S 3 Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode was washed 3 times with the electrode washing buffer prepared in step (10). And (5) drying by nitrogen. The prepared electrode is marked as PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
(15) Immobilization of 5-formylcytosine deoxyribonucleotides
Add 20. Mu.L of 5-formylcytosine deoxyribonucleotide dropwise to PDA/Ti 3 C 2 /Bi 2 S 3 The ITO electrode surface is reacted for 1h under the humid condition of 37 ℃, and then washed for 3 times by the electrode washing buffer solution prepared in the step (10). The prepared electrode was labeled 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
(16) Immobilization of C-ZIF8
40 μ L of C-ZIF8 dispersion was added dropwise to 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 Reacting the surface of the ITO electrode with 37 ℃ in a humid condition for 2h, then washing the surface of the ITO electrode with the electrode washing buffer solution prepared in the step (10) for 3 times, and placing the prepared electrode in the nano enzyme reaction solution prepared in the step (9) to stir for 15min. The prepared electrode is marked as C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 /ITO。
Example 2: photoelectrochemical detection
The C-ZIF8/5fdCTP/PDA/Ti prepared in example 1 was used with an electrochemical workstation as the signal acquisition instrument and a 500W xenon lamp as the visible light source (with an additional UV-filtering lens) 3 C 2 /Bi 2 S 3 The method comprises the following steps of taking an ITO electrode as a working electrode, taking a saturated calomel electrode as a reference electrode, taking a Pt electrode as a counter electrode, taking Tris buffer solution containing Ascorbic Acid (AA) as an electrode detection solution, taking 0V voltage as a working voltage, carrying out detection research on an object to be detected by adopting an i-t technology, and establishing a relation between the photocurrent intensity and the concentration of 5-formylcytosine deoxyribonucleotide (FIG. 3). The concentration range of the 5-formyl cytosine deoxyribonucleotide is 0.001-200nM, the detection limit is 0.33pM, and the detection sensitivity is greatly improved.
Example 3: test for Selectivity
Selectivity is an important index of photoelectrochemical sensor performance, and in order to study the specificity of the constructed sensor, the selectivity of the sensor was studied by selecting 5-hydroxycytosine ribonucleotides (5 hmC), 5-methylcytosine ribonucleotides (5 mC), and four different bases as interferents. And the change value of the photocurrent of the sensor (delta I = I) constructed by the participation of different interference reagents 2 -I 1 ，I 1 Is C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 Current value of/ITO, I 2 Is C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The photocurrent values of the electrodes after ITO was treated with different interferents) were compared. The results show that the current value change of the sensor constructed by the interferent is obviously lower than that of 5-formylcytosine deoxyribonucleotide, and the constructed sensor has good specificity (figure 4).
Example 4: stability test
Preparation of 7-branched C-ZIF8/5fdCTP/PDA/Ti by the method of example 1 3 C 2 /Bi 2 S 3 The photocurrent was measured in the detection solution using an ITO electrode (5-formylcytosine deoxyribonucleotide concentration: 5 to 50 nM). The relative standard deviation of the photocurrent obtained was 3.67%, indicating good reproducibility of the method. Mixing C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The ITO sensor continuously measures for 7 periods, and the photoelectrochemical signal is detected in the detection liquid, so that the standard deviation of the obtained photocurrent is 1.42 percent, and the method has good stability.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. The photoelectrochemical biosensor for detecting 5-formylcytosine deoxyribonucleotide is characterized by comprising an electrode and flower-shaped Bi modified on the surface of the electrode in sequence 2 S 3 Accordion-like Ti 3 C 2 Polydopamine, 5-formylcytosine deoxyribonucleotide and calcined ZIF8.
2. The method for preparing the photoelectrochemical biosensor of claim 1, comprising the steps of:
(1) Will contain flower-shaped Bi 2 S 3 The dispersion liquid is dripped on the electrode after pretreatment, and Bi is obtained after drying 2 S 3 An electrode;
(2) Will contain accordion-shaped Ti 3 C 2 Dropwise adding the dispersion of (2) to Bi obtained in step (1) 2 S 3 Drying on the electrode to obtain Ti 3 C 2 /Bi 2 S 3 An electrode;
(3) Dispersing the mixture containing polydopamineDropwise adding the solution to the Ti obtained in the step (2) 3 C 2 /Bi 2 S 3 Drying on the electrode to obtain PDA/Ti 3 C 2 /Bi 2 S 3 An electrode;
(4) Dropwise adding 5-formyl cytosine deoxyribonucleotide to PDA/Ti obtained in step (3) 3 C 2 /Bi 2 S 3 The reaction was carried out on an electrode to give 5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 An electrode;
(5) Dropwise adding the calcined ZIF 8-containing dispersion to the 5fdCTP/PDA/Ti obtained in step (4) by utilizing specific binding between phosphate and zinc 3 C 2 /Bi 2 S 3 On the electrode, after the reaction is finished, the mixture is placed in a nano enzyme reaction solution to be stirred to obtain C-ZIF8/5fdCTP/PDA/Ti 3 C 2 /Bi 2 S 3 The electrode is a photoelectrochemical biosensor for detecting 5-formyl cytosine deoxyribonucleotide.
3. The method according to claim 2, wherein in the step (1), the flower-like Bi-containing material is 2 S 3 The dispersion of (A) is prepared by mixing flower-like Bi 2 S 3 Dispersing the nano material in deionized water to obtain the nano material; the drying is irradiation of an infrared lamp;
flower-like Bi 2 S 3 The nano material is prepared by the following method:
adding Bi (NO) 3 ) 3 ·5H 2 Dissolving O and thiourea in ethylene glycol, carrying out hydrothermal reaction, collecting precipitate, centrifugally washing with water and ethanol, drying, and collecting solid to obtain flower-shaped Bi 2 S 3 A nano-material.
4. The production method according to claim 2, wherein in the step (2), the Ti contains accordion-like Ti 3 C 2 The dispersion of (A) is prepared by mixing accordion-shaped Ti 3 C 2 Dispersing in deionized water to obtain the product; the drying is irradiation of an infrared lamp;
the accordion-shaped Ti 3 C 2 The preparation method comprises the following steps:
mixing Ti 3 AlC 2 Mixing the aqueous solution and HF solution, stirring at 60 deg.C for 24 hr to obtain a mixture containing accordion-shaped Ti 3 C 2 Centrifuging to collect precipitate, centrifuging with water and ethanol, drying, and collecting solid to obtain accordion-shaped Ti 3 C 2 。
5. The preparation method according to claim 2, wherein in the step (3), the dispersion liquid containing the polydopamine is obtained by dispersing polydopamine nano-material in deionized water; the drying is irradiation of an infrared lamp;
the polydopamine nano-material is prepared by the following method:
mixing dopamine and secondary water, adding a sodium hydroxide solution, stirring at 50 ℃ for 5 hours to form a polydopamine dispersion, centrifuging, collecting precipitates, washing with water by centrifugation, drying, and collecting solids to obtain the polydopamine nano-material.
6. The method according to claim 2, wherein in the step (4), the reaction is carried out for 0.5 to 3 hours under a humid condition at 37 ℃ and then washed 1 to 5 times.
7. The method according to claim 2, wherein in the step (5), the calcined ZIF 8-containing dispersion is obtained by dispersing the calcined ZIF8 nanomaterial in deionized water; the reaction is carried out for 0.5 to 5 hours under the humid condition of 37 ℃, and then the washing is carried out for 1 to 5 times;
the calcined ZIF8 nano material is prepared by the following method:
1) Zn (NO) 3 ) 2 ·6H 2 Dissolving O and dimethyl imidazole in deionized water, stirring for 1 hour, collecting precipitate, centrifugally washing with water and ethanol, drying, and collecting solid to obtain a ZIF8 nano material;
2) Calcining the ZIF8 nano material obtained in the step 1) in a tubular furnace for 2 hours to obtain a calcined ZIF8 nano material, namely C-ZIF8;
the nano enzyme reaction solution is prepared by adding H into NaAc-Hac buffer solution 2 O 2 Mixing the solution and 4-chloro-1-naphthol solution.
8. Use of the photoelectrochemical biosensor of claim 1 to detect 5-formylcytosine deoxyribonucleotides.
9. The method for detecting 5-formylcytosine deoxyribonucleotide using the photoelectrochemical biosensor according to claim 1, comprising the steps of:
the photoelectrochemical biosensor of claim 1 is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a Pt wire is used as an auxiliary electrode, a three-electrode system is formed for photoelectrochemical signal detection, a detection solution is a Tris buffer solution containing 5-500mM ascorbic acid, the pH of the Tris buffer solution is 5.5-8.5, and the relationship between the current and the concentration of 5-hydroxymethylcytosine nucleotide is established to detect the content of 5-hydroxymethylcytosine nucleotide.
10. The method according to claim 9, wherein the detection method used is a current-time method, and the potential applied is-0.5-0.5V.
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