CN110161094B

CN110161094B - Method for rapidly detecting free radicals based on electrochemical sensor

Info

Publication number: CN110161094B
Application number: CN201910599998.4A
Authority: CN
Inventors: 孙冲; 王道营; 张牧焓; 徐为民; 邹烨; 李鹏鹏
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-07-04
Filing date: 2019-07-04
Publication date: 2021-09-28
Anticipated expiration: 2039-07-04
Also published as: CN110161094A

Abstract

The invention discloses a method for rapidly detecting free radicals based on an electrochemical sensor, which comprises the following steps: step one, preparation of carboxylated g-C₃N₄Nanoparticles; step two, constructing an electrochemical sensor to obtain ssDNA/carboxylated-g-C₃N₄An NPs/CS electrode; step three, establishing detection^·Standard curve of OH free radical, formulated at different concentrations^·OH radical solution in which ssDNA/carboxylated-g-C is₃N₄The NPs/CS electrode is a working electrode, and methylene blue is used as a signal molecule; step four, extracting meat^·OH free radical, using ssDNA/carboxylated-g-C₃N₄The NPs/CS electrode detects the same. The method is rapid, high in sensitivity and good in selectivity, and can effectively detect the concentration of the hydroxyl radical.

Description

Method for rapidly detecting free radicals based on electrochemical sensor

Technical Field

The invention relates to a meat quality detection method. More particularly, the present invention relates to a method for rapidly detecting radicals based on an electrochemical sensor.

Background

The active oxygen comprises a superoxide anion radical (O)₂ ^·-) Singlet oxygen radicals (¹O₂)1, 1-Diphenyl-2-trinitrophenylhydrazine free radical (DPPH)^·) Hydroxy radical (C)^·OH), etc., which can cause a cell destructive chain reaction due to their high chemical activity. In the presence of active oxygen, the oxygen is,^·OH radicals, which are considered to be the most active and harmful radicals, react with many substances such as sugars, amino acids, phospholipids, nucleic acids, and organic acids. These reactions can cause peroxidation of lipids and proteins, resulting in the loss of some essential amino acids and the production of some toxic substances. In addition, in high concentration^·Oxidative stress caused by OH radicals causes cell mutation, apoptosis, and causes many diseases such as hypertension, arteriosclerosis, diabetes, cataract, arthritis, cancer, etc. Therefore, for in meat^·Quantitative detection of OH radicals is essential. At present, capillary electrophoresis, gas chromatography-mass spectrometry, liquid chromatography, chemiluminescence, fluorescence, colorimetry and the like are used for detection^·OH radicals. However, these methods are time consuming, cumbersome, require specialized instrumentation and personnel, and are not widely used in practical production.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a method for rapidly detecting free radicals based on an electrochemical sensor, which can effectively detect the concentration of hydroxyl free radicals, has a rapid construction method, high sensitivity, good selectivity, and no need of a pretreatment process; the electrochemical sensor has good anti-interference performance to other active oxygen radicals.

To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a method for rapidly detecting radicals based on an electrochemical sensor, comprising the steps of:

step one, preparation of carboxylated g-C₃N₄Grinding nanoparticles, melamine, NaCl and KCl into powder, and calcining for 4h to obtain salt frit(ii) a Grinding, desalting, centrifuging and dialyzing the salt frit, and freeze-drying the prepared solution for later use;

step two, constructing an electrochemical sensor, and carrying out carboxylation on the g-C prepared in the step one₃N₄The nano particles and the chitosan are fixed on a glassy carbon electrode to prepare carboxylated g-C₃N₄NPs/CS electrode, followed by ssDNA modification at carboxylated g-C₃N₄The ssDNA/carboxylated-g-C is prepared on the NPs/CS electrode₃N₄An NPs/CS electrode;

step three, establishing detection^·Standard curve of OH free radical, formulated at different concentrations^·OH radical solution in which ssDNA/carboxylated-g-C is₃N₄The NPs/CS electrode is a working electrode, and methylene blue is used as a signal molecule;

step four, extracting meat^·OH free radical, using ssDNA/carboxylated-g-C₃N₄The NPs/CS electrode detects the same.

Preferably, the specific steps of constructing the electrochemical sensor in the second step are as follows:

1) pretreating a glassy carbon electrode; polishing the glassy carbon electrode to a mirror surface by using 0.3 mu m and 0.05 mu m of alumina powder in sequence, then performing ultrasonic treatment on the mirror surface for 30s by using absolute ethyl alcohol and ultrapure water in sequence, and drying the mirror surface by using pure nitrogen at room temperature;

2) preparation of carboxylated g-C₃N₄An NPs/CS electrode; taking 3mg/mL carboxylated g-C₃N₄Dripping 5 μ L of nanoparticle solution onto electrode, naturally drying at room temperature, dripping 5 μ L of 1% chitosan solution onto electrode, naturally drying at room temperature to carboxylate g-C₃N₄NPs are fixed on the surface of the electrode;

3) preparation of ssDNA/g-C₃N₄An NPs/CS electrode; Carboxylated-g-C was first treated with EDC/NHS solution₃N₄NPs/CS electrode, activating carboxyl, then, carboxylic-g-C₃N₄NPs/CS electrode is treated with 4 μ M ssDNA solution 5 μ L for 40min, then washed with ultrapure water to remove unbound ssDNA, and naturally dried at room temperature to obtain ssDNA/carboxylated-g-C₃N₄NPs/CS electrode.

Preferably, the standard solution in step three is prepared by the following method: produced by Fenton reaction^·OH free radicals, wherein the molar concentration ratio of ferrous sulfate to hydrogen peroxide is 1:6, and the pH is 3.5; fresh ferrous sulfate and fresh hydrogen peroxide are used to prepare OH free radical solutions with different concentrations.

Preferably, the carboxylation g-C in the first step₃N₄The preparation steps of the nano particles are as follows: weighing melamine, NaCl and KCl according to a proportion, mixing and grinding uniformly, and then placing the powder in a muffle furnace to calcine for 4h at 670 ℃, wherein the heating rate is 10 ℃/min; cooling to room temperature to obtain bright yellow salt frit; grinding the salt clinker into uniform powder, removing salt in the product by using dilute hydrochloric acid, centrifuging, re-dispersing the product into ultrapure water, centrifuging again, and removing large carbon nitride particles; collecting the supernatant, dialyzing with a dialysis bag having a molecular weight of 500 in ultrapure water to remove residual Na⁺，K⁺，Cl^-(ii) a Finally, the dialyzed solution is lyophilized to obtain carboxylated g-C₃N₄Nanoparticles.

Preferably, the ratio of melamine, NaCl and KCl is 4:1: 1.2-1.3.

Preferably, in the third step and the fourth step, the OH radical solutions with different concentrations and the solution to be detected are detected by using square wave voltammetry.

The invention at least comprises the following beneficial effects: (1) carboxylated g-C prepared by the invention₃N₄The nano particles have larger specific surface area, better conductivity and water dispersibility; (2) the aptamer sensor constructed by the invention can detect the hydroxyl radicals, the construction method is quick, the sensitivity is high, the selectivity is good, the pretreatment process is not needed, and the concentration of the hydroxyl radicals can be effectively detected; (3) the invention establishes a linear equation for detecting hydroxyl radicals, and has wide linear range and low detection limit; (4) in the aspect of anti-interference capability, the electrochemical sensor has good anti-interference performance on other active oxygen radicals.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows g-C in the prior art₃N₄Transmission electron micrographs of the material;

FIG. 2 shows the carboxylation g-C according to the invention₃N₄Transmission electron microscopy images of the nanoparticles;

FIG. 3 shows the carboxylation g-C according to the invention₃N₄An infrared spectrum of the nanoparticles;

FIG. 4 shows the carboxylation g-C according to the invention₃N₄Zeta potential maps of the nanoparticles;

FIG. 5 shows (a) chitosan/GCE and (b) bulk-g-C according to one embodiment of the present invention₃N₄/chitosan/GCE,(c)carboxylated-g-C₃N₄/chitosan/GCE and(d)ssDNA/carboxylated-g-C₃N₄Cyclic voltammogram of/chitosan/GCE;

FIG. 6 shows (a) chitosan/GCE and (b) bulk-g-C according to one embodiment of the present invention₃N₄/chitosan/GCE,(c)carboxylated-g-C₃N₄/chitosan/GCE and(d)ssDNA/carboxylated-g-C₃N₄Impedance plot of/chitosan/GCE;

FIG. 7 shows an embodiment of the present invention₂ ^·-，¹O₂，H₂O₂For ssDNA/carboxylated-g-C₃N₄Interference histogram of/chitosan/GCE sensor;

FIG. 8 shows ssDNA/carboxylated-g-C according to one embodiment of the present invention₃N₄of/chitosan/GCE at different concentrations^·OH treatment, combining a square wave voltammogram scanned after methylene blue;

FIG. 9 shows different concentrations of Fe in one embodiment of the present invention²⁺And the response current.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides a method for rapidly detecting free radicals based on an electrochemical sensor, which comprises the following steps:

1. preparation of carboxylated g-C₃N₄Nanoparticles

8.00g of melamine, 2.50g of NaCl and 3.12g of KCl are mixed in an agate mortar and fully ground to be uniform, and then the powder is placed in a muffle furnace to be calcined for 4 hours at 670 ℃, and the heating rate is 10 ℃/min. After cooling to room temperature, a bright yellow salt frit was obtained. The resulting salt frit is further processed: the salt frit was first ground to a uniform powder, then the product was desalted with dilute hydrochloric acid, centrifuged, and the product redispersed in ultrapure water and centrifuged again to remove large carbon nitride particles. Collecting the supernatant, dialyzing with a dialysis bag having a molecular weight of 500 in ultrapure water to remove residual Na⁺，K⁺，Cl^-. Finally, the dialyzed solution is freeze-dried to obtain the carboxylated carbon nitride nano particles.

2. Construction of electrochemical sensors

2-1. pretreatment of glassy carbon electrode

The Glassy Carbon Electrode (GCE) was polished to a mirror surface with 0.3 and 0.05 μm alumina powder in this order, then sonicated with absolute ethanol and ultrapure water in this order for 30 seconds, and blown dry with pure nitrogen at room temperature.

2-2. carboxylation of g-C₃N₄Preparation of NPs/CS electrode

Dripping 5 mu L of 3mg/mL carbon nitride nanoparticle solution on an electrode, naturally airing at room temperature, dripping 5 mu L of 1% Chitosan (CS) solution on the electrode, naturally airing at room temperature to carboxylate g-C₃N₄NPs are immobilized on the electrode surface.

2-3.ssDNA/g-C₃N₄Preparation of NPs/CS electrode

Carboxylated-g-C was first treated with EDC/NHS (1:1) solution₃N₄NPs/CS electrode, activated carboxyl. Then, 5 μ L,4 μ M ssDNA was bound to the carboxylated carbon nitride nanoparticles via the amino group at its end. After 40min, washing away unbound ssDNA with ultrapure water, and naturally drying the electrode at room temperature to obtain ssDNA/carboxylated-g-C₃N₄NPs/CS electrode.

3. Establishing a detection^·Standard curve for OH radicals

3-1. preparation of Standard solution

The invention utilizes Fenton reaction to produce^·OH free radical: fe²⁺+H₂O₂→Fe³⁺+(OH)^-OH. the molar concentration ratio of ferrous sulfate to hydrogen peroxide is 1:6, pH 3.5. Fresh ferrous sulfate and fresh hydrogen peroxide are used to prepare OH free radical solutions with different concentrations.

3-2, drawing standard curve

The signal molecule detected by the invention is methylene blue, and an electrochemical signal can be obtained by reduction of the methylene blue combined on the ssDNA. Due to the fact that^·OH free radicals damage the ssDNA strand, resulting in the break of ssDNA, and thus, different concentrations of^·OH radicals, which treat ssDNA, cause different degrees of damage to ssDNA, resulting in different amounts of ssDNA binding to methylene blue, and thus different current responses. Recording the peak current value i, and establishing a linear curve according to the relation between the current value and the concentration of OH free radicals.

3-3 extracting meat^·OH free radical, preparing the solution to be detected by the above method, and using the ssDNA/carboxylated-g-C of the present invention₃N₄The NPs/CS electrode detects the same.

4. Before detection, the ssDNA/carboxylated-g-C of the invention₃N₄The NPs/CS electrode also needs to be carried out

4-1, detecting electrochemical performance: first, The synthesized carboxylated carbon nitride nanoparticles were analyzed by transmission electron microscopy (TEM, HITACHI, Japan), Fourier transform infrared spectroscopy (FTIR, VARIAN Cary 5000, USA), ZETA potential (ZP, Nano ZS90, Malvern instruments, UK), X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB250Xi, usa) technique, which demonstrated successful synthesis of the material. The electrochemical properties of the materials were then investigated using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Investigating electrochemical response of sensor to detecting target by Square Wave Voltammetry (SWV) and detecting target with different concentration^·OH free radicals are detected. All electrochemical measurements of the invention were carried out at the electrochemical workstation CHI760E (shanghai, chenhua), three-electrode system: the saturated calomel electrode is used as a reference electrode, the platinum wire electrode is used as a counter electrode, and the glassy carbon electrode is used as a working electrode.

As can be seen from FIGS. 1 and 2, the bulk carbon nitride exhibits a two-dimensional planar structure with a particle size on the order of microns, and the carboxylated g-C of the present invention is comparable to the bulk carbon nitride₃N₄The nano particles are uniformly dispersed, the particle size is about 20nm, and an electron microscope picture proves that the carboxylated g-C₃N₄The nanoparticles were successfully synthesized.

As can be seen in FIGS. 3 and 4, 1706cm^-1The peak at (A) is a characteristic absorption peak of C ═ O, 1393cm^-1And 1580cm^-1The peak at (A) is a characteristic absorption peak of-COO, 3000cm^-1To 3500cm^-1The broad peaks at (A) are characteristic absorption peaks for-OH and-NH. g-C₃N₄The Zeta potential value of the material is-0.056 mV, which is close to zero, and the carboxylated g-C₃N₄The Zeta potential of the nano particles is-24 mV, and the nano particles show electronegativity. The above characterization shows that carboxylated g-C₃N₄The nanoparticles were successfully synthesized.

As shown in FIGS. 5 and 6, the cyclic voltammetry test according to the present invention was performed at 1mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-(1:1) at a sweep rate of 100mV/s and a potential of from-0.4V to 0.7V. Electrochemical impedance measurements were performed at 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-(1: 1). As can be seen from the cyclic voltammogram of FIG. 5 and the electrochemical impedance plot of FIG. 6, compared to g-C₃N₄Materials carboxylated g-C prepared by the invention₃N₄The nano particles have better conductive capability and electrochemical response capability, after the ssDNA is modified, the impedance is obviously increased and the current is reduced due to the poor conductive performance of the ssDNA, which shows that sThe sDNA was successfully modified onto the electrode.

4-2. anti-interference detection

The electrochemical sensor prepared by the carboxylated g-C3N4 nano particle is further subjected to anti-interference analysis, except that^·OH free radical, O₂ ^·-、¹O₂，H₂O₂And are all common active oxygen free radicals in meat, therefore, the three free radicals are selected by the invention for interference experimental research. The concentrations of the three radicals are selected to be^·Ten times of the concentration of OH free radicals, square wave voltammetric scanning is carried out, and the current change value is recorded. FIG. 7 is for O₂ ^·-，¹O₂，H₂O₂Interference measurements were performed 3 times per sample. Wherein Δ I ═ I₀–I，I₀Represents ssDNA has not been substituted^·The current value obtained by combining methylene blue during OH treatment, I represents^·Current values obtained with methylene blue after OH treatment. As shown in FIG. 7, the experimental results show that the three radicals have no significant change in current compared to the hydroxyl radical, and the inventors found in their studies that the OH radical has high specificity for the cleavage of ssDNA strands. Therefore, the electrochemical sensor prepared from the carboxylated g-C3N4 nano particles has better anti-interference capability.

Example 1

FIG. 8 shows ssDNA/carboxylated-g-C₃N₄the/chitosan/GCE electrode has 12 concentrations^·OH solution treatment, combined with square wave voltammograms scanned after methylene blue. Wherein the concentration of the OH solution is respectively as follows: (a) untreated, (b)0.002mM, (c)0.01mM, (d)0.05mM, (e)0.1mM, (f)0.3mM, (g)0.5mM, (h)1mM, (i)2mM, (j)4mM, (k)8mM and (l)10 mM. 12 curves from top to bottom in FIG. 8^·The concentration of the OH solution is reduced from the concentration (a) to the concentration (l) in sequence. In example 1 of the present invention, for example, Fe²⁺Representative of concentration^·OH concentration, as can be seen from FIGS. 8 and 9, with Fe²⁺The increase in the concentration of the water increases,^·the more the OH concentration increases, the more the ssDNA is cleaved, the less the amount of bound methylene blue, the lower the current response,Δ I becomes large. Fe can be seen²⁺Has good linear relation with the current change value, and the linear equation is that delta I (mu A) is 0.9512log [ Fe ]²⁺]+2.3941(R²0.9973), linear range of 2 × 10^-6-1.0×10^-2M, detection limit of 8 x 10^-7M (S/N-3). Based on the linear equation, in shrimp meat^·OH radical concentration was measured and the results are shown in Table 1.

Example 2

The procedure of example 1 was followed for the treatment of chicken meat^·OH radical concentration was measured and the results are shown in Table 1.

Example 3

The procedure of example 1 was used to prepare sausages^·OH radical concentration was measured and the results are shown in Table 1.

TABLE 1 electrochemical sensor test values (μmol/g)

The values obtained were all determined in parallel five times for each sample and averaged.

Comparative example

Using analytical instruments to treat shrimp meat, chicken meat and sausage respectively^·OH radical concentration was measured and the results are shown in Table 2.

TABLE 2 analytical instrumentation reference values (μmol/g)

As can be seen from tables 1 and 2: the data measured by the electrochemical sensor prepared by using the carboxylated g-C3N4 nano particles are identical with the data measured in an analytical instrument. Therefore, the electrochemical aptamer sensor has potential practical application value.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The method for rapidly detecting the free radicals based on the electrochemical sensor is characterized by comprising the following steps:

step one, preparation of carboxylated g-C₃N₄Nano particles, namely grinding melamine, NaCl and KCl into powder and then calcining for 4 hours to prepare salt fusion cakes; grinding, desalting, centrifuging and dialyzing the salt frit, and freeze-drying the prepared solution for later use;

step three, establishing detection^•Standard curve of OH free radical, formulated at different concentrations^•OH radical solution in which ssDNA/carboxylated-g-C is₃N₄The NPs/CS electrode is a working electrode, and methylene blue is used as a signal molecule;

step four, extracting meat^•OH free radical, using ssDNA/carboxylated-g-C₃N₄The NPs/CS electrode detects the same;

the specific steps for constructing the electrochemical sensor in the second step are as follows:

1) pretreating a glassy carbon electrode; polishing a glassy carbon electrode to a mirror surface by using 0.3 mu m and 0.05 mu m of alumina powder in sequence, then performing ultrasonic treatment on the mirror surface by using absolute ethyl alcohol and ultrapure water in sequence for 30s, and drying the mirror surface by using pure nitrogen at room temperature;

2) preparation ofCarboxylated g-C₃N₄An NPs/CS electrode; taking 3mg/mL carboxylated g-C₃N₄Dripping 5 muL of nano particle solution onto an electrode, naturally airing at room temperature, dripping 5 muL of 1% chitosan solution onto the electrode, and naturally airing at room temperature to enable the carboxylated g-C₃N₄NPs are fixed on the surface of the electrode;

3) preparation of ssDNA/g-C₃N₄An NPs/CS electrode; Carboxylated-g-C was first treated with EDC/NHS solution₃N₄NPs/CS electrode, activating carboxyl, then, carboxylic-g-C₃N₄After the NPs/CS electrode is treated by 4 mu M ssDNA solution 5 mu L for 40min, the unbound ssDNA is washed away by ultrapure water, and the ssDNA/carboxylated-g-C can be obtained after the electrode is naturally dried at room temperature₃N₄An NPs/CS electrode;

the carboxylation g-C in the step one₃N₄The preparation steps of the nano particles are as follows: weighing melamine, NaCl and KCl according to a proportion, mixing and grinding uniformly, and then placing the powder in a muffle furnace to calcine for 4 hours at 670 ℃, wherein the heating rate is 10 ℃/min; cooling to room temperature to obtain bright yellow salt frit; grinding the salt clinker into uniform powder, removing salt in the product by using dilute hydrochloric acid, centrifuging, re-dispersing the product into ultrapure water, centrifuging again, and removing large carbon nitride particles; collecting the supernatant, dialyzing with a dialysis bag having a molecular weight of 500 in ultrapure water to remove residual Na⁺， K⁺，Cl^-(ii) a Finally, the dialyzed solution is lyophilized to obtain carboxylated g-C₃N₄Nanoparticles.

2. The method for rapidly detecting free radicals based on the electrochemical sensor as claimed in claim 1, wherein the free radical solution in the third step is prepared by the following method: produced by Fenton reaction^•OH free radicals, wherein the molar concentration ratio of ferrous sulfate to hydrogen peroxide is 1:6, and the pH is 3.5; using freshly prepared ferrous sulfate and freshly prepared hydrogen peroxide to prepare different concentrations^•OH radical solution.

3. The method for rapidly detecting radicals based on an electrochemical sensor as claimed in claim 1, wherein the ratio of melamine, NaCl and KCl is 4:1: 1.2-1.3.

4. The method for rapidly detecting radicals based on the electrochemical sensor as claimed in claim 1, wherein in the third step and the fourth step, the square wave voltammetry is used for measuring the concentrations of the radicals^•And detecting the OH free radical solution and the solution to be detected.