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CN109342532B - A core-shell nanorod coated with ferric oxide by a negative nickel carbide layer and its preparation method and application - Google Patents

A core-shell nanorod coated with ferric oxide by a negative nickel carbide layer and its preparation method and application Download PDF

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CN109342532B
CN109342532B CN201811496226.XA CN201811496226A CN109342532B CN 109342532 B CN109342532 B CN 109342532B CN 201811496226 A CN201811496226 A CN 201811496226A CN 109342532 B CN109342532 B CN 109342532B
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曾彩霞
鲁娜
张敏
王娜
张锐
张佳星
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Abstract

本发明属于高分子材料及生物医药领域，具体涉及一种负镍碳化层包覆四氧化三铁的核壳纳米棒及其制备方法和应用。该核壳纳米棒，包括四氧化三铁纳米棒内核与包裹四氧化三铁纳米棒的负镍碳化层外壳。Fe‑NTA络合物与镍盐和多巴胺螯合后煅烧制备该核壳纳米棒。该核壳纳米棒具有与过氧化物酶相似的催化活性，可用于制备类过氧化物酶或过氧化酶模拟酶，还通过生物素‑亲和素系统的桥梁作用，制备将DNA信号检测转换成电学信号检测的电化学传感器，选择性高、特异型好，检测结果更直观、更易于准确统计，且更方便、省时、精度高、准确性好。

Figure 201811496226

The invention belongs to the fields of polymer materials and biomedicine, and in particular relates to a core-shell nanorod coated with a negative nickel carbide layer and ferric tetroxide, and a preparation method and application thereof. The core-shell nanorod includes a ferric tetroxide nanorod inner core and a negative nickel carbide layer shell wrapping the ferric oxide nanorod. The Fe-NTA complex was chelated with nickel salt and dopamine and then calcined to prepare the core-shell nanorods. The core-shell nanorod has a catalytic activity similar to that of peroxidase, and can be used to prepare peroxidase-like or peroxidase-mimicking enzymes, and also through the bridging effect of the biotin-avidin system to prepare and convert DNA signal detection and conversion The electrochemical sensor for electrical signal detection has high selectivity, good specificity, more intuitive detection results, easier and more accurate statistics, and more convenient, time-saving, high precision and good accuracy.

Figure 201811496226

Description

Core-shell nanorod coated with ferroferric oxide by nickel-negative carbonization layer and preparation method and application thereof
Technical Field
The invention belongs to the fields of high polymer materials and biological medicines, and particularly relates to a nickel-negative carbonized layer coated ferroferric oxide core-shell nanorod and a preparation method and application thereof.
Background
Sensitive and efficient detection of trace sequence specific DNA is of great significance in clinical diagnosis, food analysis, bioterrorism and environmental monitoring. Commonly used DNA detection techniques include fluorescence colorimetric detection, molecular imprinting, polymerase chain amplification (PCR), and DNA microarray, however, the applications of these methods are often limited by complicated experimental procedures, expensive instrumentation and limited sensitivity, and some methods are difficult to perform high-sensitivity, low-cost detection of low-abundance nucleic acid samples.
In recent years, many methods have been proposed to replace PCR technology to achieve sensitive detection of DNA, such as fluorescent labels, electrochemical immunosensors, organic electrochemical transistor immunosensors, surface plasmon resonance and electrochemiluminescent aptamer sensors, using enzymes, indicators, nanomaterials in combination with optical and electrochemical technologies as signal amplification labels. Among these methods, the electrochemical biosensor is known as a rapid, inexpensive, and miniaturized detection device. Although these methods have high sensitivity, these methods typically require additional probe modification or conjugation steps. Therefore, designing a DNA detection method which is simpler, more convenient, lower in cost, highly sensitive and highly specific remains a current research hotspot.
DNA nano-self-assembly is a simple and efficient method for signal enhancement by target-probe hybridization. In 2004, Dirks and Pierce first proposed a DNA detection concept with similar PCR sensitivity: hybrid Chain Reaction (HCR). HCR is an enzyme-free method, and hybridization is initiated by an initiator (target DNA) that polymerizes the oligonucleotides into long dsDNA molecules. HCR is an initiator-initiated reaction that greatly reduces interference with background signals. In addition, HCR can be performed under mild conditions, and these advantages make HCR a preferred method for DNA sensing applications.
Fe prepared herein3O4The @ C-Ni is a nano mimic enzyme with horseradish peroxidase-like activity, which is tightly connected with DNA modified with Biotin (Biotin) after being coated with Streptavidin (SA, Streptavidin) to catalyze TMB substrate liquid to generate electrochemical signals.
Thus, the present application combines HCR with electrochemical biological detection techniques, via Fe3O4The @ C-Ni realizes the conversion between HCR and electrochemical signals, and HCR enhances the electrochemical signals and improves the detection sensitivity and specificity.
Disclosure of Invention
The invention provides a nuclear shell nanorod coated with ferroferric oxide by a negative nickel carbonization layer, which has peroxidase-like activity and can catalyze TMB substrate liquid to generate an oxidation-reduction reaction to generate an electrochemical signal; the core-shell nanorod is combined with DNA modified with biotin after being coated with streptavidin, and qualitative and quantitative detection of the DNA is converted into qualitative and quantitative detection of an electrochemical signal based on the peroxidase-like activity of the core-shell nanorod, so that the detection of the DNA is free of additional probe modification or conjugation, and the method is simple, rapid, low in cost, high in sensitivity and good in specificity.
The invention also provides a preparation method of the core-shell nanorod coated with the negative nickel carbonization layer and ferroferric oxide, and the method is simple, convenient and easy to operate, low in cost and suitable for industrial production.
A nuclear shell nanorod coated with a nickel-negative carbonized layer and containing ferroferric oxide comprises a ferroferric oxide nanorod inner core and a nickel-negative carbonized layer shell coating the ferroferric oxide nanorod to form a rod-shaped nuclear shell nano material.
The length of the core-shell nanorod coated with the ferroferric oxide by the nickel-negative carbonization layer is 1-10 mu m, and the width of the core-shell nanorod is 80-100 nm.
A preparation method of a core-shell nanorod coated with ferroferric oxide by a nickel-negative carbonization layer comprises the following steps:
(1) core-shell nanorod (Fe-NTA @ PDA-Ni) with Fe-NTA complex, nickel salt, dopamine and alkaline compound dissolved in water, wherein pH is 8-9 alcohol and water is chelated to generate negative nickel polydopamine layer coated Fe-NTA complex2+)；
(2) Calcining the core-shell nanorod with the Fe-NTA complex coated by the negative nickel polydopamine layer in the inert atmosphere to obtain a core-shell nanorod (Fe) with the ferroferric oxide coated by the negative nickel carbonization layer3O4@C-Ni)。
In the chelating system in the step (1), the concentration of the Fe-NTA complex is 0.8-2 mg/mL, preferably 1 mg/mL; the mass ratio of the Fe-NTA complex to the nickel element is 4.5-6: 1, preferably 5-5.5: 1, more preferably 5.3 to 5.4: 1; the mass ratio of dopamine to nickel elements is 1-2.5: 1, preferably 1.5-2: 1, more preferably 1.6: 1.
and (1) stirring while chelating, and washing the core-shell nanorod coated with the Fe-NTA complex by the nickel-negative polydopamine layer generated by chelating, wherein the specific operation is that absolute ethyl alcohol and distilled water are alternately washed.
In step (1), the basic compound comprises tris or a phosphate, preferably tris.
Adding the alcohol water solution dissolved with nickel salt and dopamine and an alkaline compound or a solution dissolved with the alkaline compound into the alcohol water solution dissolved with the Fe-NTA complex, and simultaneously stirring for treatment. Specifically, an alkaline compound or a solution dissolved with the alkaline compound is added into an alcohol-water solution dissolved with an Fe-NTA complex, and then the alcohol-water solution dissolved with nickel salt and dopamine is added; or adding the alcohol-water solution dissolved with nickel salt and dopamine into the alcohol-water solution dissolved with the Fe-NTA complex, and then adding the alkaline compound or the solution dissolved with the alkaline compound; chelating to generate a core-shell nanorod of the negative nickel polydopamine layer coated Fe-NTA complex. The alcohol water solution dissolved with nickel salt and dopamine and the alkaline compound or the solution dissolved with the alkaline compound are selected to be slowly dropped.
Step (1), the preparation method of the Fe-NTA complex comprises the following steps: and dissolving ammonium ferrous sulfate and nitrilotriacetic acid in water, and carrying out hydrothermal reaction to generate a Fe-NTA complex.
In the hydrothermal reaction system, the concentration of the iron element is 0.15-0.2 mol/L, preferably 0.16-0.17 mol/L; the molar ratio of the iron element to the nitrilotriacetic acid is 1.5-5: 1, preferably 2-2.5: 1, more preferably 2: 1.
carrying out hydrothermal reaction for 5-20 hours at 150-250 ℃. The hydrothermal reaction temperature is preferably 180-200 ℃, and the hydrothermal reaction time is preferably 10-15 hours, and more preferably 12 hours.
And (2) calcining the core-shell nanorod coated with the Fe-NTA complex by the nickel-negative poly dopamine layer at 500-800 ℃ in an inert atmosphere. The inert atmosphere comprises any one or combination of nitrogen, helium, neon or carbon dioxide.
The prepared nuclear shell nanorod coated with ferroferric oxide by the negative nickel carbide layer has catalytic activity similar to that of peroxidase, can be used for preparing peroxidase-like enzyme or peroxidase mimic enzyme, catalyzes oxidation reaction of peroxide (especially hydrogen peroxide) serving as an electron acceptor substrate and a color development indicator (such as 3,3',5,5' -tetramethyl benzidine and TMB) serving as an electron donor, and realizes qualitative and/or quantitative detection of the peroxide by measuring ultraviolet absorption spectrum of a constant proportion oxidation product of the color development indicator. The redox reaction route of the core-shell nanorod catalytic TMB base solution with ferroferric oxide coated by the nickel-negative carbonization layer is as follows:
Figure BDA0001896954260000031
the core-shell nanorod coated with ferroferric oxide by the nickel-negative carbonized layer can also be used for qualitative and/or quantitative detection of bioactive substances for generating hydrogen peroxide based on redox activity similar to peroxidase, wherein the bioactive substances comprise cholesterol, glucose, ascorbic acid, glycine or histidine. The hydrogen peroxide generated by the oxidation of the bioactive substance and a color indicator (3,3',5,5' -tetramethyl benzidine, TMB) are subjected to redox reaction under the catalytic action of the nuclear shell nano rod of ferroferric oxide coated by the negative nickel carbonization layer, and the qualitative and/or quantitative determination of the bioactive substance is realized by qualitatively and/or quantitatively determining the hydrogen peroxide.
When detecting hydrogen peroxide, the method comprises the following steps: uniformly mixing a sample containing hydrogen peroxide, the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer, a color development indicator and a buffer solution, and reacting for 5-30 minutes under the conditions that the pH value is 2-10 and the temperature is 25-65 ℃; separating the ferroferric oxide core-shell nanorod coated with the nickel-negative carbonization layer, detecting the absorption spectrum of the reaction solution, and performing qualitative and/or quantitative determination on the hydrogen peroxide.
Preferably, the pH is 2-5; more preferably, the pH is 3 to 4, and still more preferably 4; the buffer solution is acetic acid-sodium acetate, phosphoric acid-sodium phosphate or phosphoric acid-sodium hydrogen phosphate, and the like, and preferably acetic acid-sodium acetate.
The color indicator is 3,3',5,5' -tetramethyl benzidine (TMB), o-phenylenediamine (OPD), 2' -diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), luminous ammonia or a fluorescent reagent Amplex Red, and 3,3',5,5' -tetramethyl benzidine is preferred. The content of the color indicator in the detection system is 0.05-0.2 mmol/L, preferably 0.1-0.2 mmol/L, and more preferably 0.1 mmol/L.
The content of the core-shell nanorod coated with the ferroferric oxide by the nickel-negative carbonization layer in a detection system is 5-100 mu g/mL, preferably 50-100 mu g/mL, and more preferably 100 mu g/mL.
The reaction temperature is preferably 50 ℃ to 60 ℃, more preferably 60 ℃.
In a preferred embodiment of the invention, 0.2mol/L of acetic acid-sodium acetate buffer solution with pH 4 is used, the color indicator is 3,3',5,5' -tetramethylbenzidine, the detection wavelength of the absorption spectrum is 652nm, the content of 3,3',5,5' -tetramethylbenzidine in the detection system is 0.1mmol/L, and the content of the nickel-negative carbonized layer coated ferroferric oxide core-shell nanorod is 100 μ g/mL.
The electrochemical biosensor mainly comprises an identification system and a signal conversion system closely matched with the identification system, wherein a specific substance to be detected is combined with the identification system to carry out biochemical reaction so as to generate or amplify a biochemical signal; the biochemical signal is then converted by a signal conversion system into an electrical or optical signal that can be qualitative and/or quantitative.
The electrochemical biosensor selectively amplifies target DNA (target DNA) based on a Hybridization Chain Reaction (HCR) with good selectivity and strong specificity to finish signal amplification of the target DNA. Then, peroxidase is labeled on the amplified target DNA based on the bridging action of a biotin-avidin system (BAS). Finally, based on the redox activity of peroxidase, the redox reaction of the peroxidase (especially hydrogen peroxide) as an electron acceptor substrate and a chromogenic indicator (such as 3,3',5,5' -Tetramethylbenzidine (TMB)) as an electron donor is catalyzed, so that a current signal is formed, a biochemical signal is converted into a visual, accurately statistically qualitative and/or quantitative electric signal, and the qualitative and/or quantitative determination of the target DNA is completed.
The nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer is used as peroxidase-like enzyme or peroxidase mimic enzyme based on the redox activity similar to that of peroxidase, can be used for preparing an electrochemical biosensor by the bridge action of a biotin-avidin system, converts biological signals into electrical signals, and generates an oxidation-reduction reaction based on catalysis of the nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer, wherein the oxidation-reduction reaction takes peroxide (especially hydrogen peroxide) as an electron acceptor substrate and a color development indicator (such as 3,3',5,5' -tetramethyl benzidine and TMB) as an electron donor.
An electrochemical biosensor comprises a metal electrode, a DNA probe (capture DNA) attached to the metal electrode, an amplified target DNA formed by amplifying a target DNA (target DNA) captured based on the DNA probe, and a nickel-negative carbonized layer coated ferroferric oxide core-shell nanorod marked on the amplified target DNA based on a biotin-avidin system. The DNA probe is modified with sulfydryl and fixedly attached to the surface of a metal (Au) electrode through a metal (Au) -S bond, Biotin (Biotin) is modified on the amplified target DNA, and Streptavidin (Streptavidin, SA) is modified on the ferroferric oxide core-shell nanorod coated with the nickel-negative carbonized layer.
The metal electrode is a gold electrode.
The DNA sequence of the DNA probe is as follows: 5'-CTT TAG GCC AAG AAT TCT GCT ACC-3', thiol (SH) is modified at the 5' end.
The DNA sequence of the target DNA is: 5'-A TTT GCT CAA CCC ACA TAC CCT GA G GTA GCA GAA TTC TTG GCC TAA AG-3', respectively; is prostate cancer DNA.
The amplification target DNA contains a biotin-modified hairpin structure H1(Biotin-H1) And biotin-modified hairpin Structure H2(Biotin-H2) Biotin-modified Biotin-H1The DNA sequence of (a) is 5 '-Biotin-TTT TTT TCA GGG TAT GTG GGT TGA GCA AAT CAA AGT ATT TGC TCA ACC CAC ATA-3', Biotin-modified Biotin-H2The DNA sequence of (a) was 5 '-A TTT GCT CAA CCC ACA TAC CCT GA TAT GTG GGT TGA GCA AAT ACT TTG TTT TTT-Biotin-3'.
The electrochemical biosensor generates a detectable current signal based on the redox reaction with peroxide (especially hydrogen peroxide) as an electron acceptor substrate, for example, TMB substrate solution containing hydrogen peroxide for electrochemical biological detection converts the detection of target DNA to be detected into the detection of the current signal, and the result is more intuitive, the sensitivity is higher, and the selectivity and the specificity are better.
A method for preparing an electrochemical biosensor, comprising the steps of: (1) placing the metal electrode in a PBS solution dissolved with a sulfhydryl-modified DNA probe and a reducing agent, and fixing the sulfhydryl-modified DNA probe based on a metal-sulfur element bond to prepare the metal electrode for modifying the DNA probe;
(2) sealing the metal electrode of the modified DNA probe by using a sealant, and then placing the metal electrode in a target DNA solution to prepare a metal electrode for capturing target DNA;
(3) placing a metal electrode for capturing target DNA in a hairpin structure H dissolved with biotin modification1And biotin-modified hairpin Structure H2Preparing a metal electrode for amplifying the target DNA in the SPSC buffer solution;
(4) and (3) placing the metal electrode for amplifying the target DNA in the avidin-modified negative nickel carbonization layer-coated ferroferric oxide core-shell nanorod solution to prepare the electrochemical biosensor marked with the negative nickel carbonization layer-coated ferroferric oxide core-shell nanorod.
In the step (1), the metal electrode is a gold electrode, and the metal electrode is polished and cleaned in advance.
In the step (1), the reducing agent comprises a thiol compound, a trialkylphosphine compound or Dithiothreitol (DTT), preferably tris (2-carboxyethyl) phosphine (TCEP) which is a thiol compound.
In the PBS solution in the step (1), the concentration of the PBS solute is 6-10 mM, and 8.8mM is preferred; the concentration of the sulfydryl modified DNA probe is 0.8-10 mu M, preferably 1 mu M; the mol ratio of the sulfhydryl-modified DNA probe to the reducing agent is 1: 300-800, preferably 1: 500.
and (1) placing the metal electrode in a PBS solution in which a sulfhydryl-modified DNA probe and a reducing agent are dissolved, and placing the metal electrode at room temperature overnight to prepare the metal electrode for modifying the DNA probe. The metal electrode of the modified DNA probe was repeatedly rinsed with a PBS solution having a concentration of 10mM and blown dry with nitrogen.
In the step (1), the DNA sequence of the sulfhydryl-modified DNA probe is as follows: 5 '-SH-CTT TAG GCC AAG AAT TCT GCT ACC-3'.
In the step (2), the blocking agent comprises MCH solution, BSA solution, Tween-20 (Tween-20) solution, horse serum solution or fat-free milk (No-fat milk) solution, and preferably MCH solution. The concentration of the MCH solution is 0.8-5 mM, preferably 1 mM. The concentration of BSA solution is 5 wt%, the concentration of Tween-20 solution is 0.2 wt%, the concentration of horse serum solution is 10 wt%, and the concentration of fat-free milk solution is 5 wt%.
In step (2), the concentration of the target DNA solution is 1fM to 1nM, preferably 100fM to 1 nM.
In the step (2), the DNA sequence of the target DNA is: 5'-A TTT GCT CAA CCC ACA TAC CCT GA G GTA GCA GAA TTC TTG GCC TAA AG-3', respectively; is prostate cancer DNA.
In the SPSC buffer solution in the step (3), the concentration of sodium chloride is 0.5-1.5 mM, preferably 0.8-1 mM; the concentration of the disodium hydrogen phosphate is 28-50 mM, and preferably 40 mM; biotin-modified hairpin structure H1(Biotin-H1) And biotin-modified hairpin Structure H2(Biotin-H2) The concentration of (b) is 0.05-1 μ M, and preferably 0.2 μ M.
In step (3), the biotin-modified hairpin structure H1(Biotin-H1) The DNA sequence of (a) is 5 '-Biotin-TTT TTT TCA GGG TAT GTG GGT TGA GCA AAT CAA AGT ATT TGC TCA ACC CAC ATA-3'; biotin-modified hairpin structure H2(Biotin-H2) The DNA sequence of (a) was 5 '-A TTT GCT CAA CCC ACA TAC CCT GA TAT GTG GGT TGA GCA AAT ACT TTG TTT TTT-Biotin-3'.
In the step (4), the concentration of the avidin-modified negative nickel carbonization layer coated ferroferric oxide core-shell nanorod solution is 5-50 mug/mL, and preferably 50 mug/mL.
Step (4), the preparation method of the avidin-modified negative nickel carbonization layer coated ferroferric oxide core-shell nanorod comprises the following steps: and (3) reacting the PBS solution uniformly mixed with the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer and the avidin at the temperature of 30-40 ℃, coating the avidin on the surface of the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer, and preparing the avidin-modified ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer.
An electrochemical biological detection device comprises a reference electrode, a counter electrode, a working electrode, hydrogen peroxide and TMB substrate solution or TMB substrate solution containing hydrogen peroxide, wherein the electrochemical biosensor prepared by the method is used as the working electrode.
The reference electrode is Ag/AgCl or Hg/HgCl, and the counter electrode is a Pt electrode.
A method of performing an assay using an electrochemical biological assay device, the steps comprising: a reference electrode, a counter electrode and the working electrode of the electrochemical biosensor prepared in the application are placed in a TMB substrate solution containing hydrogen peroxide, and qualitative detection is carried out by using cyclic voltammetry and quantitative detection is carried out by using time-amperometry. The reference electrode is Ag/AgCl or Hg/HgCl, and the counter electrode is a Pt electrode.
The application has the advantages that: the prepared negative nickel carbonization layer coated ferroferric oxide core-shell nanorod has catalytic activity similar to that of peroxidase, can be used for preparing peroxidase-like enzyme or peroxidase mimic enzyme, catalyzes oxidation-reduction reaction of peroxide (especially hydrogen peroxide) serving as an electron acceptor substrate, and carries out qualitative and/or quantitative detection on the peroxide (especially hydrogen peroxide).
Based on the bridge action of a biotin-avidin system, the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer is combined with target NDA, biological signal detection is converted into electrical signal detection based on peroxide (especially hydrogen peroxide) redox reaction, the electrochemical sensor and a detection device based on the electrochemical sensor can be prepared, qualitative and/or quantitative detection of target DNA is carried out, and the biological signal amplification effect of hybridization chain reaction is combined, so that the detection sensitivity is improved. The electrochemical sensor is used for detection, so that the selectivity is high, the special shape is good, the detection result is more visual and easier to accurately count, and the electrochemical sensor is more convenient, time-saving, high in precision and good in accuracy.
Drawings
FIG. 1 shows Fe prepared in example 13O4SEM picture of @ C-Ni.
FIG. 2 is example 2Fe3O4Graph showing the catalytic effect of @ C-Ni as a peroxidase-like enzyme.
FIG. 3 shows Fe at different temperatures in example 23O4Graph of catalytic effect of @ C-Ni.
FIG. 4 shows Fe at different pH values in example 23O4Graph of catalytic effect of @ C-Ni.
FIG. 5 shows different Fe values in example 23O4Graph of catalytic effect of the dosage of @ C-Ni.
FIG. 6 shows example 3 as H2O2Fe with concentration as variable3O4Test chart of kinetics experiment of @ C-Ni.
FIG. 7 shows the concentration of TMB as a variable in example 33O4Test chart of kinetics experiment of @ C-Ni.
FIG. 8 shows naked Au/C-DNA/MCH/T-DNA/H in example 61-H2/Fe3O4Scheme for preparation of @ C-Ni @ SA electrodes.
Fig. 9 is a Cyclic Voltammetry (CV) test result of the self-assembled electrochemical sensor of each of examples 6 to 10.
FIG. 10 shows the different concentrations of Fe in example 113O4Self-assembled Au/C-DNA/MCH/T-DNA/H of @ C-Ni @ SA1-H2/Fe3O4And the electrochemical detection effect graph of the @ C-Ni @ SA electrochemical sensor.
FIG. 11 shows different concentrations of [ H ] in example 121/H2]Self-assembled Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4And the electrochemical detection effect graph of the @ C-Ni @ SA electrochemical sensor.
FIG. 12 shows the capture of target DNA (Au/C-DNA/MCH/T-DNA/H) in example 13, respectively1-H2/Fe3O4@ C-Ni @ SA) and uncaptured target DNA (Au/C-DNA/MCH/H1-H2/Fe3O4@ C-Ni @ SA) in the electrochemical detection of the self-assembled electrochemical sensor.
FIG. 13 shows self-assembled Au/C-DNA/MCH/T-DNA/H of target DNA at different concentrations in example 141-H2/Fe3O4And the electrochemical detection effect graph of the @ C-Ni @ SA electrochemical sensor.
FIG. 14 is Au/C-DNA/MCH/T-DNA/Fe self-assembled at different concentrations of target DNA without HCR amplification in example 153O4And the electrochemical detection effect graph of the @ C-Ni @ SA electrochemical sensor.
FIG. 15 is the Au/C-DNA/MCH/T-DNA/H self-assembly of example 16 different single base mismatched target DNAs1-H2/Fe3O4And the electrochemical detection effect graph of the @ C-Ni @ SA electrochemical sensor.
Detailed Description
The invention is described in detail below with reference to the drawings and specific examples.
Example 1 Fe3O4Synthesis of @ C-Ni
(1) Preparation of Fe-NTA complexes
Uniformly dissolving 2.6g of ammonium ferrous sulfate hexahydrate and 0.6g of nitrilotriacetic acid in 40mL of water, reacting at the constant temperature of 180 ℃ for 720min, cooling to room temperature, filtering to obtain precipitate, and alternately washing with ethanol and distilled water for several times to obtain white precipitate, namely Fe-NTA.
(2)Fe3O4Preparation of @ C-Ni
200mg of tris was uniformly dissolved in 5mL of water to form solution A.
Uniformly dissolving 15mg of Dopamine (DA) and 37.6mg of nickel chloride hexahydrate in a mixed solution consisting of 2mL of ethanol and 1mL of water to form a solution B.
50mg of Fe-NTA was uniformly dissolved in a mixed solution of 30mL of anhydrous ethanol and 20mL of water to form a solution C.
Under the stirring condition, sequentially dripping the solution A and the solution B into the solution C, after the dripping is finished, keeping the pH value of the formed mixed solution at about 8-9, continuing stirring for 20 hours, filtering to obtain a precipitate, and alternately washing the precipitate for a plurality of times by using ethanol and distilled water to obtain a black precipitate, namely Fe-NTA @ PDA-Ni2+。
Mixing Fe-NTA @ PDA-Ni2+Calcining under the protection of nitrogen (500 ℃) to obtain Fe3O4@ C-Ni, SEM picture of which is shown in FIG. 1.
Fe3O4@ C-Ni is rod-shaped, with a length of 1-10 μm and a width of 80-100 nm; the ferroferric oxide nano rod is positioned in Fe3O4In the rod-shaped body of @ C-Ni, Fe is formed3O4The inner core of the @ C-Ni rod-shaped body; the negative nickel carbonization layer wraps the ferroferric oxide nano rod to form Fe3O4The outer shell of the @ C-Ni rod-shaped body.
Example 2Fe3O4Peroxidase-like Activity of @ C-Ni
1. Peroxidase-like activity
(1) 285. mu.L, 290. mu.L and 295. mu.L of 0 were each prepared.2M acetic acid-sodium acetate buffer solution with pH 4.0 is added into each centrifuge tube in turn, 6 uL, 0 uL, 6 uL and 0 uL Fe are added into each centrifuge tube3O4@ C-Ni aqueous solution (5mg/mL), 6. mu.L, 0. mu.L of aqueous hydrogen peroxide solution (0.01M), 3. mu.L of 3,3',5,5' -tetramethylbenzidine anhydrous ethanol solution (TMB,10mM), and the above solutions were mixed uniformly;
(2) reacting the mixed solution prepared in the step (1) at room temperature for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the above mixed solution was measured with an ultraviolet-visible absorption spectrophotometer.
The results are shown in FIG. 2, Fe3O4@C-Ni+TMB+H2O2Ultraviolet absorbance of (2) is about TMB + H2O2Twice as high as Fe3O4Four times of @ C-Ni + TMB, the independent TMB can be almost ignored, therefore, the nano material Fe synthesized by the experiment3O4@ C-Ni has peroxidase-like activity.
2. Reaction temperature vs. Fe3O4Effect of the Activity of the @ C-Ni class of enzymes
(1) 290 mu L of 0.2M acetic acid-sodium acetate buffer solution with pH of 4.0 is taken to be arranged in a centrifuge tube, and 6 mu L of Fe is added into the centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), 3. mu.L of aqueous hydrogen peroxide solution (0.1M), and 3. mu.L of 3,3',5,5' -tetramethylbenzidine anhydrous ethanol solution (TMB,20mM) were mixed uniformly and prepared in 9 parts in parallel;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ for 10min respectively;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the above mixed solution was measured with an ultraviolet-visible absorption spectrophotometer.
As shown in FIG. 3, it can be seen that the absorbance at 652nm increases and then decreases with the increase in temperature, and in order to obtain Fe3O4The @ C-Ni was operated under the optimum conditions, and the temperature corresponding to the maximum absorbance was selected to be 60 ℃ as the optimum temperature for the reaction.
3. Reaction pH vs. Fe3O4Effect of the Activity of the @ C-Ni class of enzymes
(1) 290. mu.L of 0.2M acetic acid-sodium acetate buffer solution with pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 were put into each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), 3. mu.L of aqueous hydrogen peroxide solution (0.1M), and 3. mu.L of 3,3',5,5' -tetramethylbenzidine anhydrous ethanol solution (TMB,20mM), and the above solutions were mixed well;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the above mixed solution was measured with an ultraviolet-visible absorption spectrophotometer.
As shown in FIG. 4, it can be seen that the absorbance at 652nm increases and then decreases with the increase in pH, and in order to obtain Fe3O4@ C-Ni was operated under the optimum conditions, and the optimum pH for the reaction was selected to be pH 4.00 corresponding to the maximum absorbance.
4.Fe3O4Effect of @ C-Ni concentration on the Activity of other enzymes
(1) In parallel, 7 portions of 290. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution were placed in each centrifuge tube, and Fe was added to each centrifuge tube in sequence3O4The @ C-Ni aqueous solutions were mixed well to form various final concentrations (0, 5, 10, 15, 20, 50, 100. mu.g/mL), 6. mu.L of aqueous hydrogen peroxide (0.1M), 1.5. mu.L of 3,3',5,5' -tetramethylbenzidine (TMB,10 mM);
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the above mixed solution was measured with an ultraviolet-visible absorption spectrophotometer.
The results are shown in FIG. 5, from which it is clear that Fe is present3O4The concentration of @ C-Ni is linearly related to the absorbance at the wavelength of 652nm, and according to the experience, 20 mu g/mL is selected as Fe3O4The optimum concentration of the @ C-Ni solution.
Example 3 Fe3O4Kinetics experiment of @ C-Ni
1.H2O2Experiment of
[TMB]＝0.20mM
(1) Separately, 290. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution was dispensed in parallel into each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), aqueous hydrogen peroxide solutions of various concentrations (final concentrations of 50, 100, 200, 500, 1000. mu.M, respectively), and 3. mu.L of 3,3',5,5' -tetramethylbenzidine absolute ethanol solution (TMB,20mM) were mixed uniformly, and the concentration of TMB in each mixed solution prepared in parallel was 0.2 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of different hydrogen peroxide concentrations as abscissa and the reciprocal of different reaction rates as ordinate, as shown in fig. 6.
[TMB]＝0.10mM
(1) Separately, 290. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution was dispensed in parallel into each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), aqueous hydrogen peroxide solutions of various concentrations (final concentrations of 50, 100, 200, 500, 1000. mu.M, respectively), and 3. mu.L of 3,3',5,5' -tetramethylbenzidine absolute ethanol solution (TMB,10mM) were mixed uniformly, and the concentration of TMB in each mixed solution prepared in parallel was 0.1 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of different hydrogen peroxide concentrations as abscissa and the reciprocal of different reaction rates as ordinate, as shown in fig. 6.
[TMB]＝0.05mM
(1) Separately, 290. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution was dispensed in parallel into each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), aqueous hydrogen peroxide solutions of various concentrations (final concentrations of 50, 100, 200, 500, 1000. mu.M, respectively), and 1.5. mu.L of 3,3',5,5' -tetramethylbenzidine absolute ethanol solution (TMB,20mM) were mixed uniformly, and the concentration of TMB in each mixed solution prepared in parallel was 0.05 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of different hydrogen peroxide concentrations as abscissa and the reciprocal of different reaction rates as ordinate, as shown in fig. 6.
From the calculation of FIG. 6, the nano mimic enzyme Fe3O4@ C-Ni vs. substrate H2O2The michaelis constant Km is 0.21 and the maximum reaction rate Vm is 2.64.
TMB experiment
[H2O2]＝0.10mM
(1) 5 portions of 285. mu.L of 0.2M acetic acid-sodium acetate buffer solution with pH 4.00 are taken in parallel in a centrifuge tube, and 6. mu.L of Fe is added to the centrifuge tube in turn3O4@ C-Ni aqueous solution (1mg/mL), 3. mu.L of aqueous hydrogen peroxide solution (0.01M), and 3,3',5,5' -tetramethylbenzidine anhydrous ethanol solutions of different concentrations (final concentrations of 200, 300, 400, 600, 800. mu.M, respectively), and the above solutions were mixedH in each mixed solution prepared uniformly and in parallel2O2Is 0.1 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of the concentration of different 3,3',5,5' -tetramethylbenzidine as the abscissa and the reciprocal of the reaction rate as the ordinate, as shown in fig. 7.
[H2O2]＝0.075mM
(1) 5 portions of 285. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution were each placed in parallel in each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), 3. mu.L of aqueous hydrogen peroxide solution (0.01M), and 3,3',5,5' -tetramethylbenzidine anhydrous ethanol solutions of different concentrations (final concentrations of 200, 300, 400, 600, and 800. mu.M, respectively) were mixed uniformly, and H was added to each mixed solution prepared in parallel2O2Is 0.075 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of the concentration of different 3,3',5,5' -tetramethylbenzidine as the abscissa and the reciprocal of the reaction rate as the ordinate, as shown in fig. 7.
[H2O2]＝0.05mM
(1) 5 portions of 285. mu.L of 0.2M pH 4.00 acetic acid-sodium acetate buffer solution were each placed in parallel in each centrifuge tube, and 6. mu.L of Fe was added to each centrifuge tube in sequence3O4@ C-Ni aqueous solution (1mg/mL), 1.5. mu.L of aqueous hydrogen peroxide solution (0.01M), and 3,3',5,5' -tetramethylbenzidine at various concentrations (final concentrations of 200, 300, 400, respectively,600. 800 μ M), the above solutions were mixed well, and H in each mixed solution was prepared in parallel2O2Is 0.05 mM;
(2) reacting the mixed solution prepared in the step (1) in a water bath kettle at the temperature of 60 ℃ for 10 min;
(3) by applying a magnetic field to Fe3O4Separating the @ C-Ni from the reaction solution;
(4) the ultraviolet absorption spectrum of the mixed solution was measured with an ultraviolet-visible absorption spectrophotometer, and a solution curve of the michaelis constant was plotted with the reciprocal of the concentration of different 3,3',5,5' -tetramethylbenzidine as the abscissa and the reciprocal of the reaction rate as the ordinate, as shown in fig. 7.
From the calculation of FIG. 7, the nano mimic enzyme Fe3O4The michaelis constant Km of @ C — Ni for the substrate TMB is 0.35, and the maximum reaction rate Vm is 1.13.
EXAMPLE 4 pretreatment of Au electrodes
(1) Electrode polishing
Pouring a proper amount of 0.05 mu m aluminum oxide powder on the polishing flannelette, uniformly spreading, uniformly drawing 8 characters in the aluminum oxide powder by a vertical Au electrode, and washing the electrode by deionized water (Q water); and (3) placing Au in Q water for 5min by ultrasonic treatment, washing with Q water, performing ultrasonic treatment in an ethanol solution for 5min, washing with Q water, performing ultrasonic treatment in Q water for 5min again, washing with Q water, and drying with nitrogen for later use.
(2) Electrode cleaning
Detection was performed by means of Chenghua electrochemical workstation using a three-electrode system at 0.5M H2SO4And cleaning the electrode.
Slow sweeping (check gold electrode cleanliness)
The potential range is-0.25-1.5V
The scanning rate is 0.1V/S
Scan segment is 2
② quick sweeping (removing impurities on the surface of the electrode, activating the electrode)
The potential range is-0.25-1.5V
The scanning rate is 4V/S
Scan segment is 60
Placing the polished Au electrode in H2SO4The slow sweep was started, and if three consecutive oxidation peaks appeared between 1V and 1.5V, the electrode surface was proved to be clean. If there are no three consecutive oxidation peaks, then a fast sweep is required, with multiple cycles of fast-slow sweep until three consecutive oxidation peaks occur. Wash Au electrode with Q Water, Nitrogen (N)2) And drying the electrode and the electrode cap, covering the electrode cap, and vertically placing the electrode on the foamed plastic for later use.
Example 5 Fe3O4@ C-Ni coating SA (Streptavidin)
1mL of 20. mu.g/mL Fe3O4@ C-Ni aqueous solution and 25. mu.L of 1mg/mL SA-PBS solution were placed in a centrifuge tube, reacted overnight at 37 ℃ in Fe3O4@ C-Ni surface coating SA (Fe)3O4@ C-Ni @ SA) at 10000rpm for 10min, collecting the supernatant, washing with 1 XPBS (0.01M) for several times, dispersing the resultant in 1mL of 1 XPBS, and sonicating uniformly for use.
EXAMPLE 6 preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode
The following operations were carried out according to the preparation route shown in fig. 8:
(1) au electrode modified capture DNA
Adding 10 μ L of 5mM TCEP aqueous solution, 2 μ L of 50 μ M thiol-modified capture DNA aqueous solution (SH-capture DNA), and 88 μ L of 1 XPBS into a centrifuge tube, and reacting at room temperature for 30min to obtain a mixed solution; and dropwise adding 1 mu M of the mixed solution with 6 mu L of the mixed solution on an inverted Au electrode, covering an electrode cap, standing overnight at room temperature, and modifying SH-capture DNA on the Au electrode based on Au-S bonds. The electrodes were repeatedly rinsed with 1 × PBS and blown dry with nitrogen.
(2) Capturing Target DNA
Dripping 10 mu L of 1mM MCH aqueous solution (6-mercaptohexane-1-ol) on an SH-capture DNA modified Au electrode, and reacting for 1h at room temperature; repeatedly washing the electrode with 1 × PBS (0.01M), and drying with nitrogen; then, 6. mu.L of 1nM aqueous target DNA solution was dropped onto each corresponding electrode and reacted at room temperature for 2 hours. The electrodes were repeatedly rinsed with 1 XPBS (0.01M) and blown dry with nitrogen.
(3) Amplification of Target DNA
Centrifugal tubeTo this was added 80. mu.L of 1 XSPSC (1mM NaCl, 50mM Na)2HPO4) Buffer, 10. mu.L of 1. mu.M Biotin-H1Aqueous solution, 10. mu.L of 1. mu.M Biotin-H2Mixing the aqueous solution uniformly; 6 μ L of the above mixture was dropped on the surface of an Au electrode for capturing Target DNA, and reacted at room temperature for 2 hours. The electrodes were repeatedly rinsed with 1 × PBS and blown dry with nitrogen.
(4) Modified Fe3O4@C-Ni@SA
6 mul of 20 mug/mL Fe was added dropwise3O4The @ C-Ni @ SA aqueous solution is put on an Au electrode for amplifying Target DNA, the reaction is carried out for 1H at room temperature, the electrode is washed by 1 XPBS, and Au/C-DNA/MCH/T-DNA/H is prepared1-H2/Fe3O4The results of electrochemical detection using the @ C-Ni @ SA electrode in "electrochemical detection method in example 17" are shown in FIG. 9.
As can be seen from FIG. 9, Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4The redox peak of the @ C-Ni @ SA electrode is significantly higher than that of the other types of electrochemical sensors of examples 7 to 10, which generate significant redox signals.
EXAMPLE 7 preparation of naked Au/C-DNA/MCH/H1-H2/Fe3O4@ C-Ni @ SA electrode
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: after Au electrode modification of capture DNA in step (1), directly amplifying capture DNA without Target DNA capture operation in step (2), and modifying Fe in step (4)3O4@ C-Ni @ SA. The operation of amplifying the capture DNA is as follows: 80 μ L of 1 XSPSC and 10 μ L of 1 μ M Biotin-H were added to the centrifuge tube1、10μL 1μM Biotin-H2Mixing uniformly; 6 mu L of the mixed solution is dripped on the surface of an Au electrode of the modified capture DNA and reacted for 2h at room temperature. The electrode was repeatedly rinsed with 1 XPBS and dried with nitrogen to prepare Au/C-DNA/MCH/H1-H2/Fe3O4The results of electrochemical detection using the @ C-Ni @ SA electrode in "electrochemical detection method in example 17" are shown in FIG. 9.
EXAMPLE 8 preparation of bare Au/C-DNA/MCH/T-DNA/H1-H2Electrode for electrochemical cell
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: modifying Fe without the step (4)3O4Preparation of Au/C-DNA/MCH/T-DNA/H by following the procedure of @ C-Ni @ SA1-H2The results of electrochemical detection using the electrode according to "electrochemical detection method in example 17" are shown in FIG. 9.
As can be seen from FIG. 9, the signals generated by Au/C-DNA/MCH/T-DNA/H1-H2 are almost negligible.
EXAMPLE 9 preparation of bare Au/C-DNA/MCH/T-DNA electrode
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: without (2) capturing Target DNA, step (3) amplifying Target DNA, and step (4) modifying Fe3O4@ C-Ni @ SA Au/C-DNA/MCH/T-DNA electrodes were prepared and electrochemical detection was carried out by the "electrochemical detection method of example 17", and the results are shown in FIG. 9.
EXAMPLE 10 preparation of bare Au/C-DNA/MCH electrode
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: the step (3) of amplifying Target DNA and the step (4) of modifying Fe are not carried out3O4The operation of @ C-Ni @ SA. Only 10 mu L of 1mM MCH is required to be dripped on the SH-capture DNA modified Au electrode for reaction for 1h at room temperature; the electrode was repeatedly washed with 1 XPBS (0.01M) and dried with nitrogen to prepare an Au/C-DNA/MCH electrode, and electrochemical detection was carried out by the electrochemical detection method of example 17, the results of which are shown in FIG. 9.
As can be seen from fig. 9, the HCR process can enhance the signal and is essential. Fe3O4The @ C-Ni @ SA can catalyze TMB base solution to generate electrochemical signals.
Example 11 self-Assembly of varying concentrations of Fe3O4@ C-Ni @ SA electrochemical sensor
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: step (4) introducing Fe with different concentrations3O4@ C-Ni @ SA, construction of Fe at different concentrations3O4The electrochemical sensor of @ C-Ni @ SA was subjected to electrochemical detection by the "electrochemical detection method of example 17", and the results are shown in FIG. 10.
As can be seen from FIG. 10, with Fe3O4The current signal detected by the self-assembled electrochemical sensor gradually increases when the concentration of @ C-Ni @ SA increases, but when Fe3O4When the concentration of @ C-Ni @ SA reached 50. mu.g/mL, the current signal decreased, so that 20. mu.g/mL was used as Fe3O4Preferred concentrations of @ C-Ni @ SA.
Example 12 self-Assembly of different concentrations [ H ]1/H2]Electrochemical sensor
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: step (3) introduction of [ H ] of different concentrations1/H2](0.05, 0.1, 0.2. mu.M), different concentrations of [ H ] were constructed1/H2]The electrochemical sensor of (1) was subjected to electrochemical detection by the electrochemical detection method of "example 17", and the results are shown in FIG. 11.
As can be seen from FIG. 11, following H1/H2The concentration is increased, the current signal detected by the self-assembled electrochemical sensor is gradually increased, and the concentration is increased at [ H ]1/H2]When the maximum value is reached at 0.2. mu.M, [ H ] is selected1/H2]As a preferred concentration, 0.2 μ M.
Example 13 electrochemical sensor for self-Assembly of + -target DNA
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: step (2) selecting and capturing target DNA and not capturing target DNA, respectively, constructing electrochemical sensors for assembling target DNA and unassembled target DNA, and adopting "example 17 electrochemical detection method"electrochemical detection was carried out, and the results are shown in FIG. 12.
As can be seen from fig. 12, the electrochemical sensor formed by self-assembly with 1nM target DNA can detect a current signal of 2000nA, while the electrochemical sensor formed by self-assembly without target DNA can only detect a current signal of about 90nA, and thus the electrochemical sensor constructed in the present application can realize highly sensitive detection of DNA.
Example 14 electrochemical sensor self-assembling different concentrations of target DNA
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: target DNAs with different concentrations are introduced in the step (2), electrochemical sensors with different concentrations of target DNAs are constructed, electrochemical detection is performed by the electrochemical detection method of example 17, and the result is shown in FIG. 13.
As is clear from FIG. 13, the electrochemical sensor formed by self-assembly with the addition of 1fM target DNA can detect a current signal of about 200nA, which is twice as high as that without the addition of target DNA, and has high detection sensitivity. And the detection sensitivity is greatly improved along with the increase of the target DNA concentration.
Example 15 electrochemical sensor self-assembling different concentrations of target DNA and without HCR amplification
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: step (2) introduces Target DNAs with different concentrations, and does not amplify the Target DNAs in step (3), self-assembles electrochemical sensors with different concentrations of Target DNAs and no HCR amplification, and performs electrochemical detection by the electrochemical detection method of example 17, and the result is shown in FIG. 14.
As can be seen from fig. 14, as the target DNA concentration increases, the current signal detected by the electrochemical sensor gradually increases, and the sensitivity gradually increases. As can be seen from FIG. 13 in example 14, the current signal detected in this example is much lower than that in example 14 in which HCR amplification was performed, due to the absence of HCR amplification, at the same concentration of target DNA.
Example 16 electrochemical sensor for self-assembling Single base mismatched (SNP) target DNA
The preparation method was the same as "example 6" preparation of naked Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA electrode ", except for: step (2) electrochemical detection was carried out using the electrochemical detection method of "example 17" by introducing target DNA with one base mismatch (1MM), target DNA with two base mismatches (2MM), and target DNA with three base mismatches (3MM), respectively, and by using electrochemical sensors that correspond to the electrochemical sensors that form the electrochemical detection methods of one base mismatch, two base mismatch (2MM), and three base mismatch (3MM), respectively, of the self-assembly degree, and the results are shown in FIG. 15.
As can be seen from fig. 15, the electrochemical sensor constructed according to the present invention has high selectivity for target DNA.
Example 17 electrochemical detection method
The different kinds of electrochemical sensors prepared in examples 6 to 16 were detected by the following electrochemical detection method.
Firstly, a three-electrode system is used for detection by means of a Chenghua electrochemical workstation, an Ag/AgCL electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, a self-assembled Au electrode is used as a working electrode, and electrochemical detection is carried out in 1.5mL of TMB base solution containing hydrogen peroxide.
Secondly, performing qualitative detection on the prostatic cancer DNA by using a cyclic voltammetry method, wherein the parameter range is as follows:
the potential range is 0-0.8V
The scanning rate is 0.1V/S
The prostate cancer DNA was quantitatively measured by time-current (I-t) with the following parameters:
the time range is 0-100s
The scanning rate is 0.1V/S
And thirdly, making a current-concentration curve according to the current I in the obtained time-current curve and the concentration of the target DNA.
The current-voltage curves of the electrochemical sensors for detecting self-assembly of different types by using the electrochemical workstation are shown in FIG. 9, and only Au/C-DNA/MCH/T-DNA/H1-H2/Fe3O4@ C-Ni @ SA produced significant redox signals. Au/C-DNA/MCH/T-DNA/Fe3O4The smaller signal produced by @ C-Ni @ SA demonstrates that the HCR process can enhance the signal, and is essential. The signals generated by Au/C-DNA/MCH/T-DNA/H1-H2 are almost negligible, which indicates that Fe3O4The @ C-Ni @ SA can catalyze TMB base solution containing hydrogen peroxide to generate an electrochemical signal.
The foregoing description of the embodiments is provided to enable any person skilled in the art to make or use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of inventive faculty. Therefore, the present invention is not limited to the above-mentioned embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (9)

1. The application of the nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer is specifically used for preparing peroxidase or peroxidase mimics, and is characterized in that the nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer comprises a ferroferric oxide nanorod inner core and a negative nickel carbide layer shell coating the ferroferric oxide nanorod, and the preparation method comprises the following steps:
(1) chelating alcohol water solution dissolved with Fe-NTA complex, nickel salt, dopamine and alkaline compound and having pH = 8-9 to generate a core-shell nanorod coated with Fe-NTA complex by a nickel-negative polydopamine layer;
(2) and calcining the core-shell nanorod coated with the Fe-NTA complex by the negative nickel polydopamine layer in an inert atmosphere to obtain the core-shell nanorod coated with ferroferric oxide by the negative nickel carbonization layer.
2. The application of the core-shell nanorod coated with ferroferric oxide by a negative nickel carbide layer according to claim 1, wherein the core-shell nanorod coated with ferroferric oxide by a negative nickel carbide layer is used for qualitatively and/or quantitatively detecting hydrogen peroxide or a bioactive substance capable of generating hydrogen peroxide.
3. A method for detecting hydrogen peroxide is characterized in that a sample containing hydrogen peroxide, a nuclear shell nanorod coated with ferroferric oxide by a nickel-negative carbonized layer, a color indicator and a buffer solution are uniformly mixed and react for 5-30 minutes under the conditions that the pH is = 2-10 and the temperature is 25-60 ℃; separating the core-shell nanorod coated with the ferroferric oxide by the nickel-negative carbonization layer, detecting the absorption spectrum of the reaction liquid, and performing qualitative and/or quantitative determination on the hydrogen peroxide;
the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer comprises a ferroferric oxide nanorod inner core and a negative nickel carbonization layer shell coating the ferroferric oxide nanorod, and the preparation method comprises the following steps:
(1) chelating alcohol water solution dissolved with Fe-NTA complex, nickel salt, dopamine and alkaline compound and having pH = 8-9 to generate a core-shell nanorod coated with Fe-NTA complex by a nickel-negative polydopamine layer;
(2) and calcining the core-shell nanorod coated with the Fe-NTA complex by the negative nickel polydopamine layer in an inert atmosphere to obtain the core-shell nanorod coated with ferroferric oxide by the negative nickel carbonization layer.
4. The nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer is used for preparing an electrochemical biosensor and is characterized in that the nuclear shell nanorod coated with the ferroferric oxide by the negative nickel carbide layer comprises a ferroferric oxide nanorod inner core and a negative nickel carbide layer shell coating the ferroferric oxide nanorod, and the preparation method comprises the following steps:
(1) chelating alcohol water solution dissolved with Fe-NTA complex, nickel salt, dopamine and alkaline compound and having pH = 8-9 to generate a core-shell nanorod coated with Fe-NTA complex by a nickel-negative polydopamine layer;
(2) and calcining the core-shell nanorod coated with the Fe-NTA complex by the negative nickel polydopamine layer in an inert atmosphere to obtain the core-shell nanorod coated with ferroferric oxide by the negative nickel carbonization layer.
5. An electrochemical biosensor is characterized by comprising a metal electrode, a DNA probe attached to the metal electrode, amplified target DNA formed by amplifying target DNA captured based on the DNA probe, and a nuclear shell nanorod of ferroferric oxide coated with a nickel-negative carbonized layer marked on the amplified target DNA based on a biotin-avidin system;
the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer comprises a ferroferric oxide nanorod inner core and a negative nickel carbonization layer shell coating the ferroferric oxide nanorod, and the preparation method comprises the following steps:
(1) chelating alcohol water solution dissolved with Fe-NTA complex, nickel salt, dopamine and alkaline compound and having pH = 8-9 to generate a core-shell nanorod coated with Fe-NTA complex by a nickel-negative polydopamine layer;
(2) and calcining the core-shell nanorod coated with the Fe-NTA complex by the negative nickel polydopamine layer in an inert atmosphere to obtain the core-shell nanorod coated with ferroferric oxide by the negative nickel carbonization layer.
6. A method for preparing an electrochemical biosensor, comprising the steps of: (1) placing the metal electrode in a PBS solution dissolved with a sulfhydryl-modified DNA probe and a reducing agent, and fixing the sulfhydryl-modified DNA probe based on a metal-sulfur element bond to prepare the metal electrode for modifying the DNA probe;
(2) the metal electrode for modifying the DNA probe is sealed by a sealant and then placed in a DNA solution to prepare a metal electrode for capturing target DNA;
(3) placing a metal electrode for capturing target DNA in a hairpin structure H dissolved with biotin modification1And biotin-modified hairpin Structure H2Preparing a metal electrode for amplifying the target DNA in the SPSC buffer solution;
(4) placing a metal electrode for amplifying a target DNA in an avidin-modified negative nickel carbonization layer-coated ferroferric oxide core-shell nanorod solution to prepare an electrochemical biosensor marked with a negative nickel carbonization layer-coated ferroferric oxide core-shell nanorod;
the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer comprises a ferroferric oxide nanorod inner core and a negative nickel carbonization layer shell coating the ferroferric oxide nanorod, and the preparation method of the ferroferric oxide core-shell nanorod coated with the negative nickel carbonization layer comprises the following steps:
i) chelating alcohol water solution dissolved with Fe-NTA complex, nickel salt, dopamine and alkaline compound and having pH = 8-9 to generate a core-shell nanorod coated with Fe-NTA complex by a nickel-negative polydopamine layer;
ii) calcining the core-shell nanorod coated with the Fe-NTA complex by the negative nickel polydopamine layer in an inert atmosphere to obtain the core-shell nanorod coated with ferroferric oxide by the negative nickel carbonization layer.
7. The nickel-negative carbonization layer coats the ferroferric oxide core-shell nanorod.
8. An electrochemical biological detection device, comprising a reference electrode, a counter electrode and a working electrode, wherein the electrochemical biosensor of claim 5 is used as the working electrode, and further comprises hydrogen peroxide and TMB substrate solution or TMB substrate solution containing hydrogen peroxide.
9. An electrochemical biological detection method for detecting target DNA, which is characterized by comprising the following steps: a reference electrode, a counter electrode and the working electrode of the electrochemical biosensor according to claim 5 are placed in a TMB substrate solution containing hydrogen peroxide, and qualitative detection is performed using cyclic voltammetry and quantitative detection is performed using time-amperometry.
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