用于灵敏检测痕量镍离子的磁诱导自组装电化学生物传感器及其应用

Magnetic induction self-assembly electrochemical biosensor for sensitively detecting trace nickel ions and application thereof

文档序号：6165 发布日期：2021-09-17 浏览：48次中文

1. A magnetic induction self-assembly electrochemical biosensor for sensitively detecting trace nickel ions is characterized in that the construction process is as follows:

(1) the magnetic glassy carbon electrode adopts Al₂O₃Polishing the powder, and ultrasonically cleaning and drying the powder for later use;

(2) taking 5-10 mu L of alpha-Fe₂O₃/Fe₃O₄Dripping Au nano composite suspension liquid on the surface of the magnetic glassy carbon electrode, and forming a layer of uniform alpha-Fe on the surface of the electrode after the electrode is dried at room temperature₂O₃/Fe₃O₄-an Au film, ready for use;

(3) dripping 5-10 μ L of sulfhydryl modified DNA1 solution containing tris (2-carboxyethyl) phosphine on the electrode surface in step (2), and incubating at 37 deg.C for 1-8h to obtain MGCE/α -Fe₂O₃/Fe₃O₄-Au/DNA1 modified electrode, ready for use;

(4) dropwise adding 5-10 μ L of bovine serum albumin with volume fraction of 0.25% onto the electrode surface obtained in step (3), sealing non-specific sites on the electrode surface, and incubating at 4 deg.C for 30min to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode, ready for use;

(5) dropwise adding 5-10 μ L of methylene blue modified DNA2 solution onto the electrode surface of step (4), wherein the concentration of DNA2 is the same as that of DNA1 in step (3), and incubating at 37 deg.C to obtain MGCE/α -Fe₂O₃/Fe₃O₄And (4) modifying the electrode by Au/DNA1/BSA/DNA2 to obtain the electrochemical biosensor.

2. The magnetically-induced self-assembled electrochemical biosensor for sensitive detection of trace nickel ions according to claim 1, wherein the sequence of DNA1 in step (3) is:

5’-CnAnCGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTAGCAAAAAAAAAAnAnA-SH-3’；

the sequence of the DNA2 in the step (5) is as follows:

5’-MB-AnCnTCACTAT/RA/GGAAGAGATGGACGnTnG-3’。

3. the magnetically induced self-assembled electrochemical biosensor for sensitive detection of trace nickel ions according to claim 2,characterized in that the alpha-Fe in the step (2)₂O₃/Fe₃O₄The concentration of the Au nano-composite suspension is 5-25 mg/mL.

4. The magnetically-induced self-assembled electrochemical biosensor for sensitive detection of trace nickel ions according to any one of claims 1-3, wherein the concentration of the thiol-modified DNA1 solution containing tris (2-carboxyethyl) phosphine in step (3) is 1-20 μ M.

5. The construction method of the magnetically induced self-assembled electrochemical biosensor for sensitive detection of trace nickel ions according to claim 4, wherein the incubation time in step (5) is 30-70 min.

6. The magnetically-induced self-assembled electrochemical biosensor for sensitive detection of nickel ions in trace amounts according to claim 5, wherein the α -Fe in step (2)₂O₃/Fe₃O₄The preparation method of the @ Au nano-composite comprises the following steps:

1) mixing Fe (NO)₃)₃·9H₂O and C₆H₁₀O₈Dissolving the mixture in quantitative double distilled water according to a molar ratio of 1:1.5, and magnetically stirring the mixture for 24 hours at room temperature to obtain a precursor solution which is uniformly dispersed; dehydrating the precursor solution at 60 ℃ by adopting a rotary evaporator to prepare sol; putting the sol into an oven, and drying to obtain fluffy porous dry gel; the dried gel is put into a programmed temperature control furnace and calcined for 2 hours at 450 ℃ to obtain the magnetic alpha-Fe₂O₃/Fe₃O₄A heterogeneous nanoparticle;

2) placing a three-neck flask containing 200mL of trisodium citrate solution with the concentration of 7.5mM on an oil bath, and heating to boil under the reflux condition; dispersing 1mL of alpha-Fe with the concentration of 10mg/mL after ultrasonic treatment₂O₃/Fe₃O₄Adding the heterogeneous nano-particle suspension liquid drop into a three-neck flask, and magnetically stirring for 5 min; 10mL of chloroauric acid solution with the concentration of 10mM is added into a three-necked flask, magnetic stirring is carried out to obtain wine red solution,centrifuging the solution, washing the precipitate, and vacuum drying to obtain alpha-Fe₂O₃/Fe₃O₄-an Au nanocomposite.

7. Use of a magnetically induced self-assembled electrochemical biosensor according to any one of claims 1 to 6 for sensitive detection of trace nickel ions.

8. Use of a magnetically induced self-assembled electrochemical biosensor for the sensitive detection of trace nickel ions according to claim 7, characterized in that upon detection: dropwise adding the solution to be detected on the surface of the electrode, and incubating for 30-80min at 37 ℃; the test was carried out using an electrochemical workstation and a three-electrode system, with a magnetic glassy carbon electrode as the working electrode, silver/silver chloride as the reference electrode, a platinum wire electrode as the counter electrode, and 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-The solution is used as electrolyte, an electrochemical biosensor is constructed, and the electrochemical biosensor is tested in a range of-0.1-0.7V by a differential pulse voltammetry method.

9. The use of the magnetically-induced self-assembled electrochemical biosensor for the sensitive detection of trace nickel ions according to claim 8, wherein the solution to be detected is selected from a nickel nitrate solution, a nickel sulfate solution, a nickel bromide solution, or a nickel chloride solution.

10. The use of the magnetically induced self-assembled electrochemical biosensor for the sensitive detection of trace nickel ions according to claim 9, wherein the volume of the solution to be measured is 5-10 μ L.

Background

Nickel (II) (Ni)²⁺) Is an important trace element in biosynthesis, respiration and metabolism, but excessive Ni²⁺Can cause diseases such as cancer, neurasthenia, inflammation, and system disorder. At present Ni²⁺The traditional detection method comprises an adsorption method, a spectrophotometry method, a fluorescence spectrometry method, an inductively coupled plasma mass spectrometry method and the like, but the traditional detection method has the limitations of complex equipment, high operation cost, complex sample preparation, poor specificity and the like. Therefore, it is necessary to search for an effective, simple and highly sensitive Ni²⁺And (3) a detection method. The electrochemical biosensor has the advantages of high detection sensitivity, high specificity, good reproducibility, small required sample amount and short detection time, and becomes a research hotspot for detecting heavy metal ions in recent years.

Electrochemical detection of Ni²⁺The method can be realized by constructing a redox reaction electrochemical sensor and a deoxyribozyme electrochemical sensor, wherein the redox reaction analysis method is more complicated, and the performance of the redox reaction analysis method depends on electrode materials; the deoxyribozyme electrochemical sensor has good selectivity and stability, and is very suitable for detecting heavy metal ions in the environment. With the development of nanotechnology, nanomaterials are widely used to improve the sensitivity of electrochemical sensors. However, there is no strong force between the non-magnetic nano material and the electrode surface, which causes the substrate material to be easily separated from the electrode surface, and the current signal is unstableAnd (4) determining. The magnetic nano material can be stably and rapidly combined on the surface of the magnetic glassy carbon electrode through magnetic force induced self-assembly, and the electrode is simple to regenerate by removing the magnetic core, so that the magnetic glassy carbon electrode has good application potential.

Disclosure of Invention

To realize trace Ni²⁺The invention provides a method for sensitively detecting trace Ni²⁺The magnetically induced self-assembled electrochemical biosensor of (1).

The invention also provides the application of the electrochemical biosensor.

The specific technical scheme of the invention is as follows:

be used for sensitive detection trace Ni²⁺The magnetic induction self-assembly electrochemical biosensor has the following construction process:

(1) the magnetic glassy carbon electrode adopts Al₂O₃Polishing the powder, and ultrasonically cleaning and drying the powder for later use;

(5) dropwise adding 5-10 μ L of methylene blue modified DNA2 solution onto the electrode surface of step (4), wherein the concentration of DNA2 is the same as that of DNA1 in step (3), and incubating at 37 deg.C to obtain MGCE/α -Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode, namely electrochemical cellA biological sensor.

Further, 0.3 μm and 0.05 μm of Al were used for the magnetic glassy carbon electrode in this order₂O₃And (5) polishing the powder.

Further, the sequence of the DNA1 in the step (3) is as follows:

5’-CnAnCGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTAGCAAAAAAAAAAnAnA-SH-3’；

the sequence of the DNA2 in the step (5) is as follows:

5’-MB-AnCnTCACTAT/RA/GGAAGAGATGGACGnTnG-3’。

further, the alpha-Fe in the step (2)₂O₃/Fe₃O₄The concentration of the Au nano-composite suspension is 5-25 mg/mL.

Further, the concentration of the thiol-modified DNA1 solution containing tris (2-carboxyethyl) phosphine in step (3) is 1 to 20. mu.M.

Further, the incubation time of the methylene blue modified DNA2 solution in the step (5) is 30-70 min.

Further, the alpha-Fe in the step (2)₂O₃/Fe₃O₄The preparation method of the @ Au nano-composite comprises the following steps:

2) placing a three-neck flask containing 200mL of trisodium citrate solution with the concentration of 7.5mM on an oil bath, and heating to boil under the reflux condition; dispersing 1mL of alpha-Fe with the concentration of 10mg/mL after ultrasonic treatment₂O₃/Fe₃O₄Adding the heterogeneous nano-particle suspension liquid drop into a three-neck flask, and magnetically stirring for 5 min; 10mL of 10mMAdding chloroauric acid solution into three-neck flask, magnetically stirring to obtain wine red solution, centrifuging the solution, washing precipitate, and vacuum drying to obtain alpha-Fe₂O₃/Fe₃O₄-an Au nanocomposite.

The method for sensitively detecting trace Ni²⁺The use of the magnetically induced self-assembled electrochemical biosensor of (1).

When in detection: dropwise adding the solution to be detected on the surface of the electrode, and incubating for 30-80min at 37 ℃; the test was carried out using an electrochemical workstation and a three-electrode system, with a magnetic glassy carbon electrode as the working electrode, silver/silver chloride as the reference electrode, a platinum wire electrode as the counter electrode, and 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-The solution is used as electrolyte, an electrochemical biosensor is constructed, and the electrochemical biosensor is tested in a range of-0.1-0.7V by a differential pulse voltammetry method.

Further, the solution to be detected is selected from a nickel nitrate solution, a nickel sulfate solution, a nickel bromide solution or a nickel chloride solution.

Further, the volume of the solution to be detected is 5-10 muL.

During detection, an inorganic salt solution to be detected is dripped to the surface of the electrode, incubation is carried out at 37 ℃ for 30-80min, metal ions to be detected activate a catalytic core region of DNA1, DNA2 is broken, and DNA2 fragments with methylene blue leave the surface of the electrode, so that electrochemical signals are changed.

The invention selects nickel ion dependent deoxyribozyme (Ni)²⁺DNAzyme) as biorecognition element in Ni²⁺The catalyst shows good catalytic performance when being present. The dnazymes immobilized on the electrode surface should retain their intrinsic structure and biological activity and be loaded as much as possible to ensure their high sensitivity. Therefore, the substrate material of the electrochemical biosensor should have a large specific surface area, good biocompatibility, and excellent electrical conductivity. The invention prepares alpha-Fe₂O₃/Fe₃O₄-Au nanocomposites as electrode substrate material for signal amplification. alpha-Fe₂O₃/Fe₃O₄The Au nano-composite can stabilize the junction through a magnetic force induced self-assembly technologyCombined on the surface of a Magnetic Glassy Carbon Electrode (MGCE), and the MGCE can be quickly regenerated by removing the magnetic inner core of the electrode. And the gold nanoparticles have good conductivity, so that the electrochemical biosensor has high sensitivity. The electrochemical biosensor is trace Ni²⁺The assays of (a) provide a new sensitive detection method that can be applied to the determination of other metal ions by modifying the biorecognition element.

Compared with the prior art, the invention has the following beneficial effects:

1. the electrochemical biosensor of the invention utilizes nickel ion-dependent deoxyribozyme as a biological recognition element, can specifically recognize nickel ions, and has good selectivity. Based on alpha-Fe₃O₄/Fe₂O₃The Au nano composite is used as an electrode substrate material, has good conductivity and can be stably combined on the surface of an electrode through magnetic force induced self-assembly. The electrochemical biosensor can detect the nickel ion concentration in a linear range of 100pM-10 mu M and has a wider linear range. The detection limit was 55 pM. The electrochemical biosensor constructed by the invention has the advantages of high sensitivity, strong specificity, low detection limit, simple preparation and short detection period.

2. Due to alpha-Fe₂O₃/Fe₃O₄The @ Au nano-composite has magnetism, and can be used for preparing alpha-Fe through a magnetic force induced self-assembly technology₂O₃/Fe₃O₄The @ Au nanocomposite is stably and firmly decorated to the MGCE surface, and alpha-Fe can be rapidly removed by removing the magnetic core of the MGCE₂O₃/Fe₃O₄@ Au, the electrode regeneration is simple and rapid.

3. The invention connects sulfhydryl modified DNA1 to alpha-Fe through Au-S bond by self-assembly technology₂O₃/Fe₃O₄The @ Au nano-composite surface has the advantages of quick and simple operation and no need of using other materials.

4. The method has mild reaction conditions, is easy to control the process, and does not need to train operators; the requirement on required equipment is not high, the operation is simple, and the carrying is convenient; the DNA chain required by each detection is few, and the cost is low; the detection process is rapid and convenient, and the detection result can be obtained in a few minutes.

Drawings

FIG. 1 is a view of α -Fe prepared in preparation example₂O₃/Fe₃O₄A transmission electron micrograph of the @ Au nanocomposite, wherein the scale size in the figure is 50 nm;

FIG. 2 is a graph showing the results of the sensitive detection of Ni traces in example 1²⁺The cyclic voltammetry diagram of the construction process of the magnetically induced self-assembled electrochemical biosensor;

FIG. 3 shows the results of the sensitive detection of Ni traces in example 1²⁺The magnetic induction self-assembly electrochemical biosensor detects Ni²⁺，Ca²⁺，Cu²⁺，Fe³⁺，Pb²⁺，Zn²⁺The peak current of (a);

FIG. 4 shows the results of the sensitive detection of Ni traces in example 1²⁺The magnetic induction self-assembly electrochemical biosensor can detect the concentration and linear range diagram.

Detailed Description

In order to make the objects, features and advantages of the present invention more comprehensible, the present invention is further described in conjunction with the following embodiments and accompanying drawings so that those skilled in the art may better understand the technical solutions of the present invention. It is obvious that the described embodiments are only a few embodiments of the invention, and that all other embodiments, which can be derived by a person skilled in the art without inventive step, such as embodiments relating to the basic principles of the invention, are intended to be covered by the scope of protection of the invention, except for the use of the invention without altering the claims.

The starting materials used in the examples below:

tris (2-carboxyethyl) phosphine, abbreviated as TCEP;

bovine serum albumin, abbreviated BSA;

TCEP and BSA were purchased from Shanghai Allantin Biotech Co., Ltd;

DNA1 and DNA2 were purchased from Biotechnology engineering (Shanghai) Inc., and are described in the sequence listing:

the sequence of DNA1 is:

5’-CnAnCGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTAGCAAAAAAAAAAnAnA-SH-3’；

nucleotide sequences in DNA1 are as follows: 1, and the DNA1 is a DNA fragment.

The sequence of DNA2 is:

5’-MB-AnCnTCACTAT/RA/GGAAGAGATGGACGnTnG-3’，

wherein SH represents a mercapto group; MB represents methylene blue; n represents a phosphorothioate linkage modification. A/RA/denotes the insertion of an adenine ribonucleotide between the DNA fragment AnCnTCACTAT and the DNA fragment GGAAGAGATGGACGNTnG.

Preparation example: alpha-Fe₂O₃/Fe₃O₄Preparation of @ Au nanocomposite

1) 13.54g Fe (NO)₃)₃·9H₂O and 10.45g C₆H₁₀O₈Dissolving the precursor solution in 100mL of double distilled water, and magnetically stirring the solution for 24 hours at room temperature to obtain a precursor solution which is uniformly dispersed; dehydrating the precursor solution at 60 ℃ by adopting a rotary evaporator to prepare sol; putting the sol into an oven, and drying to obtain fluffy porous dry gel; the dried gel is put into a programmed temperature control furnace and calcined for 2 hours at 450 ℃ to obtain the magnetic alpha-Fe₂O₃/Fe₃O₄A heterogeneous nanoparticle;

2) placing a three-neck flask containing 200mL of trisodium citrate solution with the concentration of 7.5mM on an oil bath, and heating to boil under the reflux condition; dispersing 1mL of alpha-Fe with the concentration of 10mg/mL after ultrasonic treatment₂O₃/Fe₃O₄Adding the heterogeneous nano-particle suspension liquid drop into a three-neck flask, and magnetically stirring for 5 min; adding 10mL of chloroauric acid solution with the concentration of 10mM into a three-neck flask, magnetically stirring to obtain wine red solution, centrifuging the solution, washing precipitate, and vacuum drying to obtain alpha-Fe₂O₃/Fe₃O₄-an Au nanocomposite.

FIG. 1 shows α -Fe prepared under the conditions described in this example₂O₃/Fe₃O₄-transmission electron microscopy of Au nanocomposite; as can be seen from the electron micrograph, gold nanoparticles having an average grain size of 12nm were tightly bound to α -Fe₂O₃/Fe₃O₄A heterogeneous nanoparticle surface.

Example 1

The method is used for sensitively detecting the trace Ni²⁺The magnetic induction self-assembly electrochemical biosensor has the following construction process:

1) magnetic Glassy Carbon Electrode (MGCE) is prepared by using 0.3 μm and 0.05 μm Al in sequence₂O₃Polishing the powder to a mirror surface, then ultrasonically cleaning the powder in ultrapure water, absolute ethyl alcohol and ultrapure water in sequence, and drying the powder to obtain clean MGCE for later use;

2) will prepare the alpha-Fe prepared in example₂O₃/Fe₃O₄the-Au nano compound is ultrasonically dispersed in ultrapure water to prepare alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄-an Au nanocomposite suspension for use;

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid at room temperature to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

4) Adding equal volume of TCEP with the concentration of 10mM and thiol-modified DNA1 mother solution with the concentration of 100 μ M into Tris-HCl buffer solution (50mM, pH 7.4, 100mM NaCl) and mixing uniformly to prepare thiol-modified DNA1 solution with the concentration of 10 μ M for later use;

5) dripping 8 mu L of sulfhydryl modified DNA1 solution containing TCEP in 4) to MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modifies the electrode surface, incubate for 2h at 37 ℃ to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄Au/DNA1 modified electrode, ready for use.

6) Adding 5 μ L BSA with volume fraction of 0.25% dropwise to MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1 modifying electrode surface, blocking non-specific sites on electrode surface, preventing non-specific adsorption, incubating at 4 deg.C for 30min to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄Au/DNA1/BSA modified electrode, ready for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

8) dripping 8 μ L of methylene blue modified DNA2 solution with the concentration of 10 μ M in 7) to MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode surface, incubating for 60min at 37 ℃ to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode, namely, the modified electrode is used for sensitively detecting trace Ni²⁺The magnetically induced self-assembled electrochemical biosensor of (1).

Test example 1:

the detection process of the modified electrode is as follows:

1) adding 8 μ L of Ni with a concentration of 10 μ M²⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2/Ni²⁺And modifying the electrode.

2) The test was carried out using an electrochemical workstation (purchased from Shanghai Chenghua instruments Co., Ltd., CHI660E) and a three-electrode system, MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2/Ni²⁺The modified electrode was used as a working electrode, silver/silver chloride as a reference electrode, and a platinum wire electrode as a counter electrode, using 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-The solution was used as an electrolyte, and the peak current of DPV was measured to be 3.6. mu.A by Cyclic Voltammetry (CV), alternating impedance spectroscopy (EIS) and Differential Pulse Voltammetry (DPV). Shows that the electrochemical biosensor can be used for trace Ni²⁺Detection of (3).

Test example 2:

the cyclic voltammograms obtained by measuring the characteristics of the various electrodes are shown in FIG. 2. In the figure, cyclic voltammograms of various electrodes are shown in sequence from a to g;

the electrodes corresponding to a-g are as follows: bare MGCE, MGCE/alpha-Fe₂O₃/Fe₃O₄、MGCE/α-Fe₂O₃/Fe₃O₄-Au、MGCE/α-Fe₂O₃/Fe₃O₄-Au/DNA1、MGCE/α-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA、MGCE/α-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2、MGCE/α-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2/Ni²⁺；

Wherein the bare MGCE electrode of a is a clean magnetic glassy carbon electrode;

wherein MGCE/α -Fe of b₂O₃/Fe₃O₄The preparation process of the modified electrode is as follows:

1) will prepare the alpha-Fe prepared in example₂O₃/Fe₃O₄The heteroplasmon nano-particles are dispersed in ultrapure water by ultrasonic wave to prepare alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Heterogeneous nano-particle suspension for later use;

2) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping heterogeneous nano-particle suspension on the surface of a cleaned Magnetic Glassy Carbon Electrode (MGCE), and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄And modifying the electrode.

Wherein c is MGCE/alpha-Fe prepared in step 3) of example 1₂O₃/Fe₃O₄-an Au-modified electrode;

wherein d is MGCE/alpha-Fe prepared in step 5) of example 1₂O₃/Fe₃O₄-Au/DNA1 modified electrodes;

wherein e is MGCE/alpha-Fe prepared in step 6) of example 1₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode;

wherein f is MGCE/alpha-Fe prepared in step 8) of example 1₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode;

wherein g is MGCE/alpha-Fe prepared in step 1) of test example 1₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2/Ni²⁺Modifying the electrode;

as a result of the test, it can be seen from FIG. 2 that the current response value of curve c is higher than that of curve b because the well-conductive gold nanoparticles are successfully modified to alpha-Fe₂O₃/Fe₃O₄Heterogenous nanoparticle surface, evidence of alpha-Fe₂O₃/Fe₃O₄Au is used as an electrode surface substrate material for amplifying current signals, so that the sensitivity of the electrochemical biosensor can be effectively improved;

the peak currents of curve d and curve e decrease in sequence due to poor conductivity and steric hindrance of DNA1 and BSA; from curve f, it is seen that after the DNA2 is modified to the electrode, since methylene blue is close to the surface of the electrode, the electron transfer capability is enhanced, and the current signal is increased;

curve g shows a reduced current response due to Ni²⁺After the dropwise addition onto the electrode, the catalytic core region of the DNA1 is activated, the DNA2 chain is broken, the DNA2 chain fragment with the methylene blue fragment is far away from the surface of the electrode, the electron transfer capacity is reduced, and the current signal is reduced.

Test example 3:

MGCE/alpha-Fe constructed as in example 1₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode as electrochemical biosensor, different kinds of metal ion solution were added dropwise to examine the selectivity of electrochemical sensor, test was performed using electrochemical workstation and three-electrode system, modified magnetic glassy carbon electrode as working electrode, silver/silver chloride as reference electrode, platinum wire electrode as counter electrode, 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-The solution was used as an electrolyte and tested by Cyclic Voltammetry (CV), alternating impedance spectroscopy (EIS) and Differential Pulse Voltammetry (DPV).

Different metal ion modified electrodes are respectively obtained by the following methods:

1. adding 8. mu.L of Ni with the concentration of 1. mu.M²⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain Ni²⁺And modifying the electrode.

2. Adding 8 μ L of 100 μ M Ca²⁺The solution is dripped intoExample one modified electrode surface, incubated at 37 deg.C for 80min to obtain Ca²⁺And modifying the electrode.

3. Cu of 8. mu.L at a concentration of 100. mu.M²⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain Cu²⁺And modifying the electrode.

4. Adding 8 μ L of 100 μ M Fe³⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain Fe³⁺And modifying the electrode.

5. Adding 100 μ M Pb in 8 μ L²⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain Pb²⁺And modifying the electrode.

6. Adding 8 mu L of Zn with the concentration of 100 mu M²⁺Dropwise adding the solution to the surface of the modified electrode prepared in the first embodiment, and incubating at 37 ℃ for 80min to obtain Zn²⁺And modifying the electrode.

As seen from FIG. 3, Ca was compared with the control group without any metal ion added²⁺，Cu²⁺，Fe³⁺，Pb²⁺And Zn²⁺When present, the current signal does not change significantly. The reason is that the interfering metal ions cannot be Ni²⁺DNAzymes recognize that the catalytic core region of the deoxyribozyme is not activated. Only Ni²⁺Can be covered with Ni²⁺Specific recognition by DNAzyme, strand DNA2 is cleaved and a fragment of strand DNA2 with methylene blue leaves the electrode surface, resulting in a significant decrease in the current signal. Proving that the electrochemical biosensor is paired with Ni²⁺Has better selectivity.

Test example 4:

MGCE/alpha-Fe constructed as in example 1₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode as electrochemical biosensor, and Ni with different concentrations is dripped²⁺And (4) examining the linear range of the concentration which can be detected by the electrochemical sensor.

Ni²⁺When the concentration is 10 mu M, the DPV peak current is measured to be 3.6 mu A; when the concentration of nickel ions is 1 mu M, the DPV peak current is measured to be 9.1 mu A; the peak DPV current was measured to be 15 at a nickel ion concentration of 100 nM.0 muA; when the concentration of nickel ions is 10nM, the DPV peak current is measured to be 18.2 muA; when the concentration of nickel ions is 1nM, the DPV peak current is measured to be 25.5 muA; the peak current of DPV was found to be 31.3. mu.A at a nickel ion concentration of 100 pM.

As seen in FIG. 4, the current response is dependent on Ni²⁺The concentration is increased and reduced, the concentration range which can be detected by the electrochemical biosensor is 100pM-10 mu M, the detection line is 55pM calculated by linearly fitting data, the linear equation is I (mu A) ═ 5.5272lg C (M) -24.0097, and the correlation coefficient R is²0.996. I represents current and C represents concentration.

Example 2

The method is used for sensitively detecting the trace Ni²⁺The magnetic induction self-assembly electrochemical biosensor has the following construction method:

1) magnetic Glassy Carbon Electrode (MGCE) is prepared by using 0.3 μm and 0.05 μm Al in sequence₂O₃Polishing the powder to a mirror surface, then sequentially carrying out ultrasonic cleaning in ultrapure water, absolute ethyl alcohol and ultrapure water for 1min respectively, and drying to obtain a clean MGCE for later use;

2) will prepare the alpha-Fe prepared in example₂O₃/Fe₃O₄the-Au nano compound is ultrasonically dispersed in ultrapure water to prepare alpha-Fe with the concentration of 5mg/mL₂O₃/Fe₃O₄-an Au nanocomposite suspension for use;

3) adding 8 mu L of alpha-Fe with the concentration of 5mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

5) dripping 8 mu L of sulfhydryl modified DNA1 solution containing TCEP in 4) to MGCE/alpha-Fe₂O₃/Fe₃O₄Au modified electrode surface, incubated at 37 ℃ for 2h to obtain MGCE/α-Fe₂O₃/Fe₃O₄Au/DNA1 modified electrode, ready for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

In the detection, 8 mu L of Ni with the concentration of 10nM²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 2₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min. The DPV peak current is measured to be 14.3 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. alpha-Fe₂O₃/Fe₃O₄Au as electrode substrate material for signal amplification strategies. The detected material has good conductivity, so that the electrochemical biosensor is used for Ni²⁺The detection has higher sensitivity, lower detection limit and wider linear range; and the magnetic material has magnetism, can be quickly and stably combined on the surface of the electrode through magnetic force induced self-assembly, and can realize quick regeneration of the electrode by removing the magnetic core of the electrode.

Example 3

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

1) magnetic Glassy Carbon Electrode (MGCE) was sequentially applied with 0.3 μm and 0.05 μm Al₂O₃Polishing the powder to a mirror surface, then ultrasonically cleaning the powder in ultrapure water, absolute ethyl alcohol and ultrapure water in sequence, and drying the powder to obtain clean MGCE for later use;

2) will prepare the alpha-Fe prepared in example₂O₃/Fe₃O₄the-Au nano compound is ultrasonically dispersed in ultrapure water to prepare alpha-Fe with the concentration of 25mg/mL₂O₃/Fe₃O₄-an Au nanocomposite suspension for use;

3) adding 8 mu L of alpha-Fe with the concentration of 25mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

6) Dropwise adding 5 μ L of BSA with volume fraction of 0.25% onto the electrode surface, blocking non-specific sites on the electrode surface to prevent non-specific adsorption, and incubating at 4 deg.C for 30min to obtain MGCE/α -Fe₂O₃/Fe₃O₄Au/DNA1/BSA modified electrode, ready for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

In the detection, 8 mu L of Ni with the concentration of 10nM²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 3₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min.

The DPV peak current is measured to be 9.2 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. alpha-Fe₂O₃/Fe₃O₄Au as electrode substrate material for signal amplification strategies. The detected material has good conductivity, so that the electrochemical biosensor is used for Ni²⁺The detection has higher sensitivity, lower detection limit and wider linear range; and the magnetic material has magnetism, can be quickly and stably combined on the surface of the electrode through magnetic force induced self-assembly, and can realize quick regeneration of the electrode by removing the magnetic core of the electrode.

Example 4

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

4) Adding equal volume of TCEP with the concentration of 10mM and thiol-modified DNA1 mother solution with the concentration of 100 μ M into Tris-HCl buffer solution (50mM, pH 7.4, 100mM NaCl), and mixing to obtain thiol-modified DNA1 solution with the concentration of 1 μ M for later use;

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 1. mu.M of methylene blue modified DNA2 solution for use;

8) dripping 8 muL of methylene blue modified DNA2 solution with the concentration of 1 muM in 7) into MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode surface, incubating for 60min at 37 ℃ to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode for use.

In the detection, 8. mu.L of Ni with the concentration of 10. mu.M is added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 4₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min.

The DPV peak current is measured to be 27.2 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. DNA1 is sterically hindered, poorly conductive, and negatively charged, blocking [ Fe (CN)₆]^3-/4-Reaching the electrode surface resulting in a reduced current signal.

Example 5

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-an Au-modified electrode.

4) Adding equal volume of TCEP with the concentration of 10mM and thiol-modified DNA1 mother solution with the concentration of 100 μ M into Tris-HCl buffer solution (50mM, pH 7.4, 100mM NaCl) and mixing to prepare thiol-modified DNA1 solution with the concentration of 20 μ M for later use;

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 20. mu.M of methylene blue modified DNA2 solution for use;

8) dripping 8 μ L of 20 μ M methylene blue modified DNA2 solution in 7) to MGCE/α -Fe₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode surface, incubating for 60min at 37 ℃ to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode for use.

In the detection, 8. mu.L of Ni with the concentration of 10. mu.M is added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 5₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min.

The DPV peak current is measured to be 3.5 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. DNA1 is sterically hindered, poorly conductive, and negatively charged, blocking [ Fe (CN)₆]^3-/4-Reaching the electrode surface resulting in a reduced current signal.

Example 6

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-an Au-modified electrode.

5) dripping 8 mu L of sulfhydryl modified DNA1 solution containing TCEP in 4) to MGCE/alpha-Fe₂O₃/Fe₃O₄Au modified electrode surface, incubation at 37 ℃2h to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄Au/DNA1 modified electrode, ready for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

In the detection, 8. mu.L of Ni with a concentration of 100pM was added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 6₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min.

The DPV peak current is measured to be 12.7 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. DNA2 was dripped onto the electrode surface and base-complementary paired with DNA1, due to the proximity of methylene blue to the electrode surface, electron transfer capacity was enhanced and current signal was increased.

Example 7

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

2) will prepare the alpha-Fe prepared in example₂O₃/Fe₃O₄-Au nanocomposite ultrasoundDispersing in ultrapure water to prepare alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄-an Au nanocomposite suspension for use;

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-an Au-modified electrode.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

8) dripping 8 μ L of methylene blue modified DNA2 solution with the concentration of 10 μ M in 7) to MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA modified electrode surface, incubating at 37 ℃ for 70min to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au/DNA1/BSA/DNA2 modified electrode for use.

In the detection, 8. mu.L of Ni with a concentration of 100pM was added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 7₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 80 min.

The DPV peak current is measured to be 30.5 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. DNA2 was dripped onto the electrode surface and base-complementary paired with DNA1, due to the proximity of methylene blue to the electrode surface, electron transfer capacity was enhanced and current signal was increased.

Example 8

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

6) Adding dropwise 5 μ L of BSA with volume fraction of 0.25% to the electrode surface, blocking non-specific sites on the electrode surface to prevent non-specific adsorption, incubating at 4 deg.C for 30 ℃min to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄Au/DNA1/BSA modified electrode, ready for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

In the detection, 8. mu.L of Ni with the concentration of 1. mu.M is added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 8₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 30 min.

The DPV peak current is measured to be 25.1 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. Dropwise adding Ni²⁺After the solution, the catalytic core region of DNA1 strand is activated, resulting in the cleavage of DNA2 strand and the fragment of DNA2 strand with methylene blue leaves the electrode surface, resulting in a decrease in current signal.

Example 9

The method is used for sensitively detecting the trace Ni²⁺The construction method of the magnetic induction self-assembly electrochemical biosensor comprises the following steps:

3) adding 8 mu L of alpha-Fe with the concentration of 20mg/mL₂O₃/Fe₃O₄Dripping the-Au nano-composite suspension liquid on the surface of the cleaned MGCE, and drying the liquid to obtain MGCE/alpha-Fe₂O₃/Fe₃O₄-Au modified electrode for use.

7) Diluting 100. mu.M of methylene blue modified DNA2 stock solution with Tris-HCl buffer (50mM, pH 7.4, 100mM NaCl) to obtain 10. mu.M of methylene blue modified DNA2 solution for use;

In the detection, 8. mu.L of Ni with the concentration of 1. mu.M is added²⁺The solution was added dropwise to MGCE/α -Fe prepared in example 9₂O₃/Fe₃O₄Au/DNA1/BSA/DNA2 modified electrode surface and incubated at 37 ℃ for 70 min.

The DPV peak current is measured to be 9.9 muA by adopting a Differential Pulse Voltammetry (DPV) within the range of-0.1-0.7V. Dropwise adding Ni²⁺After solution, the catalytic core region of DNA1 strand is activated, resulting in cleavage of DNA2 strand with a subunitDNA2 strand fragments of methyl blue left the electrode surface, resulting in a decrease in the current signal.

Example 1, example 2, and example 3 were compared, and Ni was detected at a concentration of 10nM²⁺In example 1, alpha-Fe₂O₃/Fe₃O₄The maximum current value was 18.2. mu.A at a concentration of 20mg/mL of Au nanocomposite;

in example 2, α -Fe₂O₃/Fe₃O₄alpha-Fe at 5mg/mL of Au nanocomposite₂O₃/Fe₃O₄The Au nano-composite is too small in quantity and poor in conductivity, so that the current signal is reduced and is 14.3 muA;

in example 3, α -Fe₂O₃/Fe₃O₄alpha-Fe at a concentration of 25mg/mL of Au nanocomposite₂O₃/Fe₃O₄The amount of Au nano-composite is too large, the steric hindrance is large, and the current signal is reduced to 9.2 muA. Thus, alpha-Fe₂O₃/Fe₃O₄The Au nanocomplex concentration was 20mg/mL as the optimum concentration.

In examples 1, 4 and 5, Ni was detected at a concentration of 10. mu.M²⁺When the concentration of thiol-modified DNA1 was increased, the DPV current response was decreased due to the increase in the amount of DNA1 and the increase in steric hindrance, and due to the presence of a negatively charged DNA1 phosphate backbone and [ Fe (CN) ]₆]^3-/4-The electrostatic repulsion between the probes increases. When the concentration of the DNA1 solution reached 10. mu.M, the current value tended to level off, the concentration of the DNA1 solution continued to increase, and the current value was substantially constant, at which time the amount of DNA1 on the electrode surface reached a saturated state. Therefore, 10. mu.M was used as the optimum concentration.

In examples 1, 6 and 7, Ni was detected at a concentration of 100pM²⁺When the incubation time of the DNA2 is prolonged, the DPV current signal is increased and then tends to be stable, the DNA2 is dripped on the surface of the electrode and is subjected to base complementary pairing with the DNA1, and the methylene blue is close to the surface of the electrode, so that the electron transfer capacity is enhanced, and the current signal is increased; saturation was reached when the incubation of DNA2 reached 60 min. Thus, it is possible to provideThe DNA2 incubation time was 60min as the optimum time.

In examples 1, 8 and 9, Ni was detected at a concentration of 1. mu.M²⁺In time, the current signal gradually decreases with the increase of the shearing time, and becomes stable at 80 min. Dropwise adding Ni²⁺After the solution, the catalytic core region of DNA1 strand is activated, resulting in the cleavage of DNA2 strand and the fragment of DNA2 strand with methylene blue leaves the electrode surface, resulting in a decrease in current signal. When the shearing time is 30min, the shearing is not complete, and the current signal is large. Therefore, a shear time of 80min was selected as the optimum time.

SEQUENCE LISTING

<110> national energy group scientific and technological research institute Co., Ltd

Jiangsu university

<120> magnetic induction self-assembly electrochemical biosensor for sensitively detecting trace nickel ions and application thereof

<130> 20210611

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 71

<212> DNA

<213> artificial

<220>

<223> DNA1

<220>

<221> misc_feature

<222> (1)..(71)

<223> n = phosphorothioate bond

<400> 1

cnancgtcca tctctgcagt cgggtagtta aaccgacctt cagacatagt gagtagcaaa 60

aaaaaaanan a 71

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