CN114910532A - Detection method of nitrite by using methanotrophin in-situ reduction nanogold modified electrode and application of detection method - Google Patents
Detection method of nitrite by using methanotrophin in-situ reduction nanogold modified electrode and application of detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of food safety electrochemical analysis, and particularly relates to preparation of a methane-oxidizing rhzomorph bio-mediated nanogold modified electrode, a nitrite electrochemical detection system constructed by the same and application of the nitrite electrochemical detection system. The modification layer with electric activity and catalysis function is fixed on the surface of the electrode to form the sensing element, nitrite is catalyzed by the modification layer on the surface of the sensing element to generate oxidation reaction to generate nitrate, reaction parameters generated on the surface of the electrode are converted into sensing signals which can be generated by a conduction system, and then the sensing signals are received by a transducer serving as a conversion system, converted into electrochemical signals which can be measured, output after secondary amplification processing by an electronic system, and displayed and recorded by an instrument. The nitrite is quantitatively analyzed and detected according to the linear relation between the electric signal obtained by secondary amplification and the nitrite concentration in a certain range. The method has the advantages of high detection stability, wide detection range and simple sample pretreatment, and can be used for detecting samples in different forms.
Description
Technical Field
The invention relates to the technical field of food safety electrochemical analysis, in particular to a method for detecting nitrite by using a methanotrophin in-situ reduction nanogold modified electrode.
Background
Nitrite is widely used as a preservative and an additive in the food industry, but the nitrite is eaten too much and causes a plurality of adverse effects on human health. The harm to human body caused by excessive nitrite intake has two main aspects: firstly, the nitrite is toxic, can oxidize low-price ferrohemoglobin carrying oxygen in blood into high-iron hemoglobin, and leads the blood to lose the function of carrying oxygen, thereby leading people to have the symptom of oxygen deficiency poisoning, and in severe cases, the life of people can be threatened due to respiratory failure; secondly, it has carcinogenic and teratogenic effects. Therefore, the detection of the content of the nitrite has very important significance on food safety. The commonly used method for detecting nitrite at present comprises the following steps: the spectrophotometry has lower cost, but has poorer sensitivity, is easy to be interfered by ions, needs longer time, and has strict requirements on the color reaction of the solution by the detection result; the fluorescence spectroscopy is to detect the concentration of nitrite by detecting the fluorescence intensity at an excitation wavelength based on a fluorophore generated when a fluorescent probe reacts with the nitrite, but the method is sensitive to environmental factors and has a limited application range; the electrochemical luminescence method has high separation efficiency and low detection limit, but is more complicated, and the reaction process needs higher combustion temperature, so that the risk of the experiment is increased; the colorimetric method is mostly applied to the on-site rapid detection method of nitrite at present, the commonly used method is that a test paper method is used for observing color and comparing the color with a standard colorimetric card so as to carry out qualitative or semi-quantitative analysis on the nitrite, and the method has convenient detection but low sensitivity. The commonly used detection method for nitrite is chromatography in addition to spectroscopy. The high performance liquid chromatography has small sample amount and high accuracy, but the purchase of the instrument is expensive and is easy to be interfered, and the reagent is toxic; the gas chromatography is simple and convenient to operate, has strong anti-interference capability but low recovery rate, and the pretreatment reagent is expensive.
The electrochemical detection method has the advantages of simple and cheap instrument, good reproducibility and repeated utilization, and thus has attracted extensive attention. Various metal oxides can be used as electrodes to detect the content of nitrite, but the exposed electrode surface is easy to react with the environment, so that the detection sensitivity and accuracy are reduced, and therefore the electrodes need to be modified. Ding et al (ACS Applied Electronic Materials,2021,3(2):761-768) prepared a nanocomposite material consisting of copper ion-based metal organogel (MOG-Cu) and multi-walled carbon nanotubes (MWCNTs) for constructing a sensor for electrochemically detecting nitrite. When operating under optimal conditions, the oxidation peak current is directly proportional to the nitrite concentration. Chu et al (Journal of The Electrochemical Society,2021,168(1):017513) prepared a novel antioxidant nitrite Electrochemical sensor based on sodium carboxymethylcellulose functionalized reduced graphene oxide-copper nanoparticles (RGO-CMC @ CuNPs) by utilizing The high hydrophilic property of sodium carboxymethylcellulose. The prepared sensor is used for detecting nitrite by a chronoamperometry method. The sensor was able to retain 91.1% of its initial sensitivity after exposure to air for 25 d.
Chen Guozhen et al (China, 2021, CN 113008958A) adopt a graphene oxide-polyaniline-gold nanoparticle nanocomposite modified electrode to detect nitrite, and the method has good selectivity, high sensitivity and low detection limit, but the synthesis of modified substances is complex.
Penlite et al (China, 2021, CN 113008963A) constructed electrochemical sensors based on Ag-MoS/ionic liquid and hemoglobin layers. The sensor is simple to operate, quick in response and strong in safety protection, but the detection limit is low.
Zhang Yu xi, etc. (China, 2020, CN 111796010A) invented a Bi 2 Se 3 The preparation method and application of the @ MWNTs composite material and the detection method of the working electrode and the nitrite have the advantages that the obtained composite material is large in active surface area, high in electron transmission rate and strong in stability, but the detection limit is low.
Wangxue et al (China, 2020, CN 112034026A) use a glassy carbon electrode modified by a titanium dioxide-titanium carbide/hexadecyl trimethyl ammonium bromide/chitosan compound as a working electrode, and have the advantages of high sensitivity, good selectivity, low detection limit and the like when measuring nitrite, but the material synthesis needs to be calcined at high temperature, and the preparation process is complex.
Raypeng et al (china, 2019, CN 110672684 a) produced electrochemical sensors based on a gold/copper @ nickel-metal organic framework composite. The method is used for constructing a sensing system for detecting nitrite in lake water, river water and seawater with high sensitivity, the selectivity of the electrode is obviously improved, but the synthesis of a modifying substance needs to be calcined for 3 hours at 350 ℃, and the process is complicated.
Sungjing et al (China, 2019, CN 112986350A) use Indium Tin Oxide (ITO) conductive glass as a substrate, deposit nano gold-nickel particles by an electrochemical deposition method, and use AuNPs/NiNFs/ITO as a modified electrode to prepare an electrochemical sensor for detecting nitrite.
Disclosure of Invention
The invention aims to provide a method for detecting nitrite by using a methanobactin-situ reduction nanogold modified electrode to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a detection method of a methane-oxidizing rhzomorph in-situ reduction nanogold modified electrode pair nitrite comprises the following specific steps:
s1: mixing 0.011-0.040 mmol/L of 1mL of methane-oxidizing rhzomorph with 3mL of 0.01% chloroauric acid solution, and synthesizing the Mb @ AuNPs solution by adopting an in-situ reduction method;
s2: taking 1mL of Mb @ AuNPs solution, dropwise adding the solution onto a copper net coated with a carbon film by using a rubber head dropper, cooling the solution, naturally drying the solution for 5min, analyzing the dried solution by using a transmission electron microscope, and determining the average particle size of the Mb @ AuNPs;
taking 2mL of Mb @ AuNPs solution into a quartz cuvette, scanning by using an ultraviolet spectrometer within a wavelength range of 200-800 nm by using distilled water as a blank, observing the change condition of a spectral curve, adding 0.40mL and 0.10mL of sodium nitrite solution, 0.10mol/L of sodium citrate buffer solution with pH of 4.5, and observing that the ultraviolet spectral absorption values of the Mb @ AuNPs to nitrites with different concentrations change;
taking 2mL of Mb @ AuNPs solution into a fluorescence cuvette, measuring fluorescence intensity under the excitation wavelength of 275nm, observing the fluorescence spectrum, adding 0.4mL of sodium nitrite solution and 0.1mL of 0.1mol/L sodium citrate buffer solution with the pH of 4.5, and observing that the Mb @ AuNPs shows regular change to the fluorescence intensity of nitrites with different concentrations;
taking a 2mLMb @ AuNPs solution, freeze-drying to prepare Mb @ AuNPs freeze-dried powder, taking a KBr tablet prepared from 1mg of Mb @ AuNPs freeze-dried powder, and placing the KBr tablet in a sample chamber for infrared spectrum analysis;
s3: preparing Mb @ AuNPs with the particle size of 10-22 nm from 3mL of Mb, placing a bare gold electrode in the Mb @ AuNPs solution, performing cyclic voltammetry scanning at 100mV/s within the range of-0.6-2.4V for 40 circles, leaching with double distilled water to obtain Mb @ AuNPs/Au electrodes with different particle sizes, and analyzing the electrochemical performance of the electrodes by a cyclic voltammetry method and an alternating-current impedance method to determine the particle size of the nanogold;
taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing the bare gold electrode in a solution, respectively performing cyclic voltammetry scanning for 40 circles at 50-400 mV/s within the range of-0.6-2.4V, and determining the electrodeposition sweeping speed; taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing a bare gold electrode in the Mb @ AuNPs solution, performing cyclic voltammetry scanning at 100mV/s within the range of-0.6-2.4V, changing the number of cyclic voltammetry scanning cycles to be 10-50, and determining the number of electrodeposition cycles;
s4: taking 2mL of 0.1mol/L acetic acid buffer solution with pH of 4, 0.01mol/L phosphoric acid buffer solution, sodium citrate buffer solution and 2mL of 0.01mol/L sodium nitrite solution, analyzing by using the modified electrode prepared in the step S3, and determining the type of the buffer solution; taking 2mL of 0.01-0.20 mol/L sodium citrate buffer solution with pH of 4 and 2mL of 0.01mol/L sodium nitrite solution, and analyzing by using a prepared modified electrode to determine the concentration of the buffer solution; taking 2mL of 0.05mol/L sodium citrate buffer solution with the pH value of 3-7 and 2mL of 0.01mol/L sodium nitrite solution, and analyzing by using a prepared modified electrode to determine the pH value of the buffer solution; taking 2mL of 0.05mol/L sodium citrate buffer solution with pH of 5 and 0.01mol/L sodium nitrite solution with pH of 2mL, analyzing by using a prepared modified electrode, changing the sweep rate of cyclic voltammetry to be 10-200 mV/s, determining the sweep rate of cyclic voltammetry, and establishing the electrochemical detection method for detecting nitrite.
Further, in the step S1, the volume ratio of the methanotrophin to the chloroauric acid solution is 1: 2.5 to 3.5.
Further, Mb @ AuNPs prepared by the CV and EIS characterization method in the step S3 was analyzed using a modified substance having a particle size of 15.84nm, an electrodeposition sweep rate of 100mV/S, and 40 cycles of electrodeposition.
Further, in the step S4, the nitrite system is optimally detected by CV and Differential Pulse Voltammetry (DPV), and the detection condition is 0.05mol/L sodium citrate buffer solution with pH of 5.
An application of a methanomycin in-situ reduction nanogold modified electrode in a nitrite detection method comprises the following steps:
a. mixing 2mL of 0.05mol/L sodium citrate buffer solution with 2mL of 0-0.010 mol/L sodium nitrite solution, and performing DPV analysis on the mixed solution in 0.05mol/L sodium citrate buffer solution with pH of 5 by using the modified electrode prepared in the step S3;
b. the nitrite solution is in a linear relation with the peak current Ip of the DPV curve within the concentration range of 0-0.010 mol/L, and the standard curve is as follows:
I p ＝0.2116c-0.3074。
the application of the methane-oxidizing rhzomorph in-situ reduction nanogold modified electrode in the detection method of nitrite can also be used for detecting samples with different forms, and comprises the following steps:
(1) pretreatment of the sample: tap water, milk, pickle, potato chips and sauced meat in a laboratory are respectively sampled, and the samples are pretreated, wherein the method comprises the following steps:
a. adding 100mL of 0.1mol/L sodium citrate buffer solution with pH of 4 into 100mL of tap water and 100mL of milk, standing for 30min, and filtering supernatant;
b. cutting 5.0g of pickle and potato chip product sample into small pieces, mixing with 100mL of distilled water, crushing, performing ultrasonic treatment for 30min, and filtering supernatant;
c. finally, 100mL of 0.1mol/L sodium citrate buffer solution with the pH value of 4 is added;
d. adding 10.0g of sauced meat into 150mL of deionized water, placing in a stirrer, stirring for 2min, taking out the suspension, placing in a 50 ℃ water bath kettle, carrying out water bath for 15min, cooling, diluting to 250mL, and filtering;
e. when in use, 3mL of filtrate is taken and centrifuged for 15min at 8000r/min, and the supernatant is diluted to 4 mL.
(2) And (3) actual sample detection:
placing the pretreated sample in a 25X 40mm electrochemical special bottle, respectively adding 1mmol/L, 5mmol/L and 10mmol/L nitrite standard solutions into pickle and pork cooked in soy, respectively adding 0.1mmol/L, 0.5mmol/L and 1mmol/L nitrite standard solutions into milk, tap water and potato chip solutions, detecting the sample solution by using a prepared Mb @ AuNPs/Au electrode through DPV, calculating the measured concentration by using a standard curve, and analyzing the standard recovery rates of different samples; the recovery rate of the Mb @ AuNPs/Au electrode standard addition is between 94.6 and 104.3 percent.
Compared with the prior art, the invention has the beneficial effects that:
a preparation method of a biological mediated gold nanocluster modified electrode, a nitrite electrochemical detection system constructed by the same and application thereof. The modified material methane-oxidizing rhzomorph in-situ reduction nanogold (Mb @ AuNPs) prepared by adopting the biosynthesis method has the characteristics of cleanness, no toxicity, environmental friendliness and the like, methane-oxidizing bacteria (Mb) is easy to obtain, the material synthesis condition is easy to change and optimize, the operation process is simple, the detection time is short, the response is quick, the detection stability is high, the detection range is wide, the sensitivity is high, the sample pretreatment is simple, and the modified material methane-oxidizing rhzomorph in-situ reduction nanogold (Mb @ AuNPs) can be used for detecting samples in different forms.
Drawings
FIG. 1 is a schematic diagram of a system for preparing a modified electrode and detecting nitrite according to the present invention;
FIG. 2 is a transmission electron microscope scan of Mb @ AuNPs of the present invention;
FIG. 3 is a CV diagram of various electrodes of the present invention in an electrolyte;
FIG. 4 is a diagram of EIS of various electrodes of the invention in electrolyte;
FIG. 5 is a CV diagram of nitrite detection by different Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 6 is an EIS diagram of detection of nitrite by different Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 7 is a CV diagram of nitrite detection using different electrodeposition sweep rates Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 8 is an EIS graph of nitrite detection using different electrodeposition sweep rates Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 9 is a CV diagram of nitrite detection by different electrodeposition turns Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 10 is an EIS diagram of nitrite detection by different electrodeposition turns Mb @ AuNPs/Au electrodes according to the present invention;
FIG. 11 is a CV diagram illustrating the effect of different buffer solutions on the electrochemical behavior of nitrite in accordance with the present invention;
FIG. 12 is a DPV graph of the effect of different buffer solutions of the present invention on the electrochemical behavior of nitrite;
FIG. 13 is a CV diagram of the effect of different buffer solution concentrations on the electrochemical behavior of nitrite in accordance with the present invention;
FIG. 14 is a DPV graph of the effect of different buffer solution concentrations on the electrochemical behavior of nitrite in accordance with the present invention;
FIG. 15 is a CV diagram of the effect of different buffer solutions pH on nitrite electrochemical behavior in accordance with the present invention;
FIG. 16 is a DPV graph of the effect of different buffer solutions pH on nitrite electrochemical behavior in accordance with the present invention;
FIG. 17 is a CV diagram illustrating the effect of cyclic voltammetric sweep rate on nitrite electrochemical behavior in accordance with the present invention;
FIG. 18 is a DPV curve of a nitrite standard curve according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
Referring to fig. 1-18, the present invention provides a technical solution: the invention comprises the following steps:
(1) mixing 1mL of 0.011-0.040 mmol/L methanotrophin with 3mL of 0.01% chloroauric acid solution, and synthesizing an Mb @ AuNPs (methanotrophin @ nanogold) solution by adopting an in-situ reduction method;
(2) taking 1mL of the prepared Mb @ AuNPs solution, dropwise adding the prepared Mb @ AuNPs solution onto a copper net coated with a carbon film by using a rubber head dropper, cooling the solution, naturally drying the solution for 5min, performing transmission electron microscope analysis after drying, and determining the average particle size of the Mb @ AuNPs.
(3) And (2) placing the bare gold electrode in the Mb @ AuNPs solution, performing CV scanning for 900s under the reaction conditions that the electrodeposition sweeping speed is 100mV/s and the electrodeposition turns are 40 within the range of-0.6-2.4V, and leaching with double distilled water to obtain the Mb @ AuNPs/Au electrode.
(4) Preparing Mb @ AuNPs with the particle sizes of 21.57nm, 18.76nm, 15.84nm, 12.92nm and 10.80nm from 3mL of Mb, placing a bare gold electrode in the Mb @ AuNPs solution, performing CV scanning for 40 circles at 100mV/s within the range of-0.6-2.4V, leaching with double distilled water to obtain Mb @ AuNPs/Au electrodes with different particle sizes, and analyzing the electrochemical performance of the electrodes through CV and EIS to determine the particle size of the nanogold gold; taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing a bare gold electrode in the solution, and performing CV scanning for 40 circles at 50mV/s, 100mV/s, 200mV/s, 300mV/s and 400mV/s within the range of-0.6-2.4V respectively to determine the electrodeposition sweeping speed; taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing a bare gold electrode in the Mb @ AuNPs solution, performing CV scanning at 100mV/s within the range of-0.6-2.4V, changing the number of CV scanning circles to be 10, 20, 30, 40 and 50, and determining the number of electrodeposition circles.
(5) Taking 2mL of 0.1mol/L acetic acid buffer solution with pH of 4, phosphoric acid buffer solution, sodium citrate buffer solution and 2mL of 0.01mol/L sodium nitrite solution, analyzing by using the modified electrode prepared in the step (4), and determining the type of the buffer solution; taking 2mL of 0.01mol/L, 0.05mol/L, 0.10mol/L, 0.15mol/L and 0.20mol/L sodium citrate buffer solution with pH of 4 and 2mL of 0.01mol/L sodium nitrite solution, and analyzing by using a prepared modified electrode to determine the concentration of the buffer solution; taking 2mL of 0.05mol/L sodium citrate buffer solution with pH values of 3, 4, 5, 6 and 7 and 2mL of 0.01mol/L sodium nitrite solution, analyzing by using a prepared modified electrode, and determining the pH value of the buffer solution; 2mL of 0.05mol/L sodium citrate buffer solution with pH 5 and 2mL of 0.01mol/L sodium nitrite solution were taken, and analyzed using a prepared modified electrode, and the cyclic voltammetric sweep rates were determined by changing the CV sweep rates to 10mV/s, 30mV/s, 50mV/s, 70mV/s, 100mV/s, 130mV/s, 150mV/s, 170mV/s, and 200 mV/s. Finally, an electrochemical detection method for detecting nitrite is established.
FIG. 2 shows a transmission electron microscope scan of Mb @ AuNPs. The prepared Mb @ AuNPs have a spherical structure, and exist stably among particles due to electrostatic repulsion, and the particle size of the Mb @ AuNPs is about 15.84 nm.
As shown in FIG. 3, significant redox peaks were seen on both the bare gold electrode and the Mb @ AuNPs/Au electrode. Compared with a bare gold electrode, the peak current of the Mb @ AuNPs/Au electrode is improved by 0.93 multiplied by 10 -5 A(P<0.05), the peak potential is reduced by 0.015V (P)<0.05). This is due toBecause Mb @ AuNPs have the peroxidase activity of Mb, and the Mb @ AuNPs have the characteristics of large specific surface area, high conductivity and the like, the electron transfer performance of the sensor is improved, and the redox peak current is obviously increased (P)<0.05)。
As shown in fig. 4, the linear part at the low frequency in the fitted impedance spectrum represents the electron rapid diffusion process, and the semicircular part at the high frequency represents the electron transfer limited process. And the value of the electrode electron transfer resistance is equal to the value of the circular arc radius in the impedance map. The electron transfer resistances of the bare gold electrode and the Mb @ AuNPs/Au electrode after fitting are respectively as follows: 6147 Ω, 3363 Ω. This is because the characteristics such as a large specific surface area of Mb @ AuNPs increase the electron transfer path, and the electron transfer efficiency is high, thereby reducing the electron transfer resistance of the electrode.
As shown in CV curves in fig. 5 and 6, as the particle size of Mb @ AuNPs increases, the peak current of the Mb @ AuNPs/Au electrode tends to increase and then decrease, because as the concentration of Mb increases, the particle size of Mb @ AuNPs increases and the electron transfer efficiency increases. Compared with the particle size of 21.57nm, the peak current of the 15.84nm Mb @ AuNPs/Au electrode is increased by 1.097 multiplied by 10 -5 A(P<0.05), the peak potential was lowered by 0.027V (P)<0.05). This is probably due to the agglomeration of the synthesized Mb @ AuNPs caused by too high Mb concentration, which leads to a decrease in the effective surface area of the electrode, a decrease in the peak current, an increase in the reaction time, and an increase in the peak potential. The EIS curve shows that the arc radius of the Mb @ AuNPs/Au electrode with the particle size of 15.84nm is obviously smaller than that of the modified electrode with the rest particle size, which shows that the electron transfer resistance of the modified electrode with the particle size of 15.84nm is smaller, the electron transfer efficiency is higher, and the electron transfer path of the Mb @ AuNPs/Au electrode is increased along with the increase of the particle size of the Mb @ AuNPs; and when the particle size is too large (>15.84nm) to reduce the specific surface area of Mb @ AuNPs due to agglomeration between particles, resulting in a decrease in electron transport paths and an increase in electron transport resistance of the modified electrode. Therefore, an Mb @ AuNPs/Au electrode with a particle size of 15.84nm was selected for subsequent experiments.
In FIGS. 7 and 8, the CV curves show that the peak current tends to increase and decrease as the sweep rate of electrodeposition is increased within the range of 50 to 400 mV/s. Compared with 50mVs, the peak current of the modified electrode prepared with the sweep rate of 100mV/s is increased by 0.33 multiplied by 10 -5 A(P<0.05), no significant change in peak potential; compared with 400mV/s, the peak current of the modified electrode prepared with the sweep rate of 100mV/s is increased by 1.95 multiplied by 10 -5 A(P<0.05), the peak potential was lowered by 0.015V (P)<0.05), because the film forming speed of Mb @ AuNPs on the surface of the electrode is increased along with the increase of the sweep rate, the peak current is increased, and when the sweep rate is too high, the Mb @ AuNPs can not form a compact assembled film on the surface of the electrode, thereby reducing the peak current and reducing the reaction rate. The EIS curve shows that the larger the radius of the arc, the larger the electron transfer resistance. With the increase of the sweep rate, the electron transfer resistance also shows the trend of firstly decreasing and then increasing, and the prepared modified electrode electron transfer resistances are respectively as follows: 3363 Ω, 2637 Ω, 3542 Ω, 4169 Ω, 5326 Ω. As the sweep rate increases, the electron transfer capability tends to increase first and then decrease, and an excessively high sweep rate may result in incomplete formation of the film, thereby decreasing the electron transfer efficiency and decreasing the electron transfer capability. Therefore, an Mb @ AuNPs/Au electrode with an electrodeposition sweep rate of 100mV/s was selected for subsequent experiments.
In fig. 9 and 10, the peak current of the CV curve tends to increase and then decrease as the number of electrodeposition turns increases, and the increase in the number of electrodeposition turns increases the deposition amount of the modifying substance on the electrode, so that the film formed on the electrode by Mb @ AuNPs tends to be complete as the number of electrodeposition scan turns increases, thereby increasing the electron transfer efficiency, promoting the electron transfer, and increasing the response current; however, as the number of scanning cycles of electrodeposition continues to increase, the film thickness increases, and the resistance to electron transfer in the film becomes large, resulting in a decrease in response current. When the number of electrodeposition turns for preparing the Mb @ AuNPs/Au electrode is 40 turns, higher response current is obtained, and the corresponding peak current is 9.94 multiplied by 10 -5 A is improved by 2.01 multiplied by 10 compared with the deposition turns of 10 -5 A(P<0.05), 1.04X 10 times higher than that of the deposition of 50 turns -5 A(P<0.05); the peak potential did not change significantly with the number of deposition cycles. In the EIS curve, the electron transfer resistance tends to decrease and then increase with the number of deposition turns, because the Mb/AuNPs film formed on the electrode tends to be intact and thus increases with the number of electrodeposition scan turnsThe electron transfer path accelerates the electron transfer rate and thus the electron transfer resistance is reduced, but as the number of scanning cycles of electrodeposition continues to increase, the film thickness increases, impeding the transfer of electrons and thus increasing the electron transfer resistance. Therefore, an Mb @ AuNPs/Au electrode with 40 cycles of electrodeposition was selected for subsequent experiments.
In FIGS. 11 and 12, the oxidation peak current was observed in the nitrite system containing 0.1mol/L of an acetic acid buffer solution, a phosphoric acid buffer solution and a sodium citrate buffer solution, and the highest current response was obtained in the case where the system was a sodium citrate buffer solution (2.33X 10) -4 A. The DPV curve had the same tendency as the CV curve, and the difference in peak potential was reduced by 0.044V and 0.009V, respectively, as compared with the acetic acid buffer solution and the phosphoric acid buffer solution, indicating that the reaction progress was accelerated by using the citric acid buffer solution. The shape of the peak in the nitrite system containing sodium citrate buffer solution was ideal and a sharp peak was observed. Therefore, sodium citrate buffer solution was selected for further electrochemical studies.
In FIGS. 13 and 14, the peak currents of the CV and DPV curves both increased and decreased with the increase of the concentration of the buffer solution, and the peak current obtained from the DPV curve was 1.55X 10 when the buffer solution was 0.01 to 0.05mol/L -5 A is remarkably increased to 1.638 multiplied by 10 -5 A(P<0.05)，△I p Is 0.088X 10 -5 A, the peak potential change of the cyclic voltammetry curve is obvious and is reduced from 0.855V to 0.836V (P)<0.05)，△E p Is 0.019V. Under acidic conditions, protons participate in the electrocatalytic reaction of nitrite. Therefore, when the concentration of the buffer solution is too low, the quantity of protons in the solution is small, so that the electrooxidation of nitrite is hindered, the peak current is low, and the peak potential is high. As the concentration of the buffer solution increases, the number of protons in the solution increases, the peak current increases, and the peak potential decreases. When the buffer solution is 0.05-0.2 mol/L, the peak current in the DPV curve is 1.638 multiplied by 10 -5 A is reduced to 0.9707X 10 -5 A(P<0.05)，△I p Is 0.6673X 10 -5 A, the peak potential of the CV curve is increased from 0.836V to 0.85V (P)<0.05)，△E p 0.014V, when the concentration of the buffer solution is too high, the electron transfer between the solution and the electrode surface is inhibited and conduction is promotedResulting in a decrease in peak current and an increase in peak potential. Therefore, 0.05mol/L sodium citrate buffer solution was selected for further electrochemical analysis of nitrite.
In FIGS. 15 and 16, the pH of the reaction medium is an important factor affecting the electrocatalytic oxidation of nitrite by the modified electrode. In buffer solutions of different pH values, the electrochemical behavior of nitrite on Mb @ AuNPs/Au electrodes changes. CV curves show the oxidation peak potential E as the pH increases over the range of pH 3, 4, 5, 6, 7 pa Moving to a negative potential, the protons thus participate in the electrocatalytic reaction of nitrite and are irreversibly reduced. The DPV curve shows that the peak current reaches a maximum at pH 5, P due to nitrite ka The value is 3.3, so most of the nitrite ions are protonated in the acidic solution. Whereas the peak current decreases with increasing solution pH, since electrocatalytic oxidation of nitrite becomes more difficult due to lack of protons. At pH<5, the decrease in current may be due to NO at very low pH 2- Conversion to NO - . The optimum peak current was obtained at pH 5, therefore, 0.05mol/L buffer solution at pH 5 was selected for the subsequent experiments.
As can be seen from fig. 17, the oxidation peak current increases with an increase in the CV sweep rate. Oxidation peak current I p And sweeping velocity v 1/2 The linear relation is good, and the linear equation is as follows:
I p ＝0.2038v 1/2 -0.1716 (1)
from the figure, a distinct redox peak can be observed, and equation (1) (R) 2 0.9972) indicates that the Mb @ AuNPs/Au electrode is a reversible reaction in the electrolyte that is diffusion controlled.
Example 1:
and (3) mixing 2mL of 0.05mol/L sodium citrate buffer solution with 2mL of 0-0.010 mol/L sodium nitrite solution under the optimal condition, and performing DPV analysis on the mixed solution by using a modified electrode. FIG. 18 shows that the linear relationship between the concentration of nitrite solution in the range of 0-0.010 mol/L and the peak current Ip of DPV curve is as follows:
I p ＝0.2116c-0.3074 (2)
shows that the nitrite concentration in the range shows a good linear relationship with the peak current (R) 2 0.9848) that satisfy the analytical requirements. The detection limit was calculated to be 4.43nmol/L, i.e., 3.257ng/kg, and the quantitation limit was calculated to be 13.42nmol/L, with the electrode having a lower detection limit and quantitation limit. The nitrite is detected 10 times by adopting the Mb @ AuNPs/Au electrode prepared under the same condition, the standard deviation (RSD) of the peak current is 2.75 percent, the nitrite is detected after the Mb @ AuNPs/Au electrode is placed for 7 days, and the peak current value changes by delta I p The standard deviation (RSD) of the modified electrode is 2.66%, and the modified electrode has good precision and stability for detecting nitrite.
Example 2:
putting a sample to be detected into a 25 x 40mm electrochemical special bottle, respectively adding 1mmol/L, 5mmol/L and 10mmol/L nitrite standard solutions into pickle and pork sauce sample solutions, respectively adding 0.1mmol/L, 0.5mmol/L and 1mmol/L nitrite standard solutions into milk, tap water and potato chip solutions, detecting the sample solution by using a prepared Mb @ AuNPs/Au electrode through DPV, calculating the concentration to be detected by using a standard curve, and analyzing the standard adding recovery rates of different samples. The test results are shown in table 1, and the experimental results show that the addition standard recovery rate of the Mb @ AuNPs/Au electrode is 94.6-104.3%, the relative standard deviation in the group is below 5%, the calculated detection limit is 3.257ng/kg and is lower than the national standard of 0.4mg/kg and 20mg/kg, and the Mb @ AuNPs/Au electrode has high accuracy and high reliability.
TABLE 1 nitrite content spiking recovery experiment
While there have been shown and described the fundamental principles and essential features of the invention and advantages thereof, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof; the present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for detecting nitrite by using a methanomectin in-situ reduction nanogold modified electrode pair is characterized by comprising the following specific steps:
s1: mixing 0.011-0.040 mmol/L of 1mL of methane-oxidizing rhzomorph with 3mL of 0.01% chloroauric acid solution, and synthesizing the Mb @ AuNPs solution by adopting an in-situ reduction method;
s2: taking 1mL of Mb @ AuNPs solution, dropwise adding the solution onto a copper net coated with a carbon film by using a rubber head dropper, cooling the solution, naturally drying the solution for 5min, analyzing the dried solution by using a transmission electron microscope, and determining the average particle size of the Mb @ AuNPs;
taking 2mL of Mb @ AuNPs solution into a quartz cuvette, scanning by using an ultraviolet spectrometer within a wavelength range of 200-800 nm by using distilled water as a blank, observing the change condition of a spectral curve, adding 0.40mL and 0.10mL of sodium nitrite solution, 0.10mol/L of sodium citrate buffer solution with pH of 4.5, and observing that the ultraviolet spectral absorption values of the Mb @ AuNPs to nitrites with different concentrations change;
taking 2mL of Mb @ AuNPs solution into a fluorescence cuvette, measuring fluorescence intensity under the excitation wavelength of 275nm, observing the fluorescence spectrum, adding 0.4mL of sodium nitrite solution and 0.1mL of 0.1mol/L sodium citrate buffer solution with the pH of 4.5, and observing that the Mb @ AuNPs shows regular change to the fluorescence intensity of nitrites with different concentrations;
taking a 2mLMb @ AuNPs solution, freeze-drying to prepare Mb @ AuNPs freeze-dried powder, taking a KBr tablet prepared from 1mg of Mb @ AuNPs freeze-dried powder, and placing the KBr tablet in a sample chamber for infrared spectrum analysis;
s3: preparing Mb @ AuNPs with the particle size of 10-22 nm from 3mL of Mb, placing a bare gold electrode in the Mb @ AuNPs solution, performing cyclic voltammetry scanning at 100mV/s within the range of-0.6-2.4V for 40 circles, leaching with double distilled water to obtain Mb @ AuNPs/Au electrodes with different particle sizes, and analyzing the electrochemical performance of the electrodes by a cyclic voltammetry method and an alternating-current impedance method to determine the particle size of the nanogold;
taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing the bare gold electrode in the solution, respectively performing cyclic voltammetry scanning for 40 circles at the voltage of-0.6-2.4V and at the voltage of 50-400 mV/s, and determining the electrodeposition sweeping speed; taking 3mL of prepared Mb @ AuNPs with the particle size of 15.84nm, placing a bare gold electrode in the Mb @ AuNPs solution, performing cyclic voltammetry scanning at 100mV/s within the range of-0.6-2.4V, changing the number of cyclic voltammetry scanning cycles to be 10-50, and determining the number of electrodeposition cycles;
s4: taking 2mL of 0.1mol/L acetic acid buffer solution with pH of 4, 0.01mol/L phosphoric acid buffer solution, sodium citrate buffer solution and 2mL of 0.01mol/L sodium nitrite solution, analyzing by using the modified electrode prepared in the step S3, and determining the type of the buffer solution; taking 2mL of 0.01-0.20 mol/L sodium citrate buffer solution with pH of 4 and 2mL of 0.01mol/L sodium nitrite solution, and analyzing by using a prepared modified electrode to determine the concentration of the buffer solution; taking 2mL of 0.05mol/L sodium citrate buffer solution with the pH value of 3-7 and 2mL of 0.01mol/L sodium nitrite solution, and analyzing by using a prepared modified electrode to determine the pH value of the buffer solution; taking 2mL of 0.05mol/L sodium citrate buffer solution with pH of 5 and 0.01mol/L sodium nitrite solution with pH of 2mL, analyzing by using a prepared modified electrode, changing the sweep rate of cyclic voltammetry to be 10-200 mV/s, determining the sweep rate of cyclic voltammetry, and establishing the electrochemical detection method for detecting nitrite.
2. The method for detecting nitrite by using the methane-oxidizing rhzomorph in-situ reduction nanogold-modified electrode according to claim 1, which is characterized in that: in the step S1, the volume ratio of the methanotrophin to the chloroauric acid solution is 1: 2.5 to 3.5.
3. The method for detecting nitrite by using methanomectin in-situ reduction nanogold modified electrode according to claim 1, which is characterized by comprising the following steps of: and the Mb @ AuNPs prepared by analyzing in the step S3 through CV and EIS characterization methods adopts a modified substance with the particle size of 15.84nm, the electrodeposition sweep rate of 100mV/S and the number of electrodeposition turns of 40 turns.
4. The method for detecting nitrite by using the methane-oxidizing rhzomorph in-situ reduction nanogold-modified electrode according to claim 1, which is characterized in that: in the step S4, a nitrite system is optimally detected by CV and DPV methods, and the detection condition is 0.05mol/L of sodium citrate buffer solution with pH of 5.
5. The application of the methane-oxidizing rhzomorph in-situ reduction nanogold modified electrode in the detection method of nitrite is characterized in that: the application comprises the following steps:
a. mixing 2mL of 0.05mol/L sodium citrate buffer solution with 2mL of 0-0.010 mol/L sodium nitrite solution, and performing DPV analysis on the mixed solution in 0.05mol/L sodium citrate buffer solution with pH of 5 by using the modified electrode prepared in the step S3;
b. the nitrite solution is in a linear relation with the peak current Ip of the DPV curve within the concentration range of 0-0.010 mol/L, and the standard curve is as follows:
I p ＝0.2116c-0.3074。
6. the application of the methane-oxidizing rhzomorph in-situ reduction nanogold modified electrode in the detection method of nitrite is characterized in that: the application can also be used for detecting samples with different forms, and the steps are as follows:
(1) pretreatment of the sample: tap water, milk, pickle, potato chips and sauced meat in a laboratory are respectively sampled, and the samples are pretreated, wherein the method comprises the following steps:
a. adding 100mL of 0.1mol/L sodium citrate buffer solution with pH of 4 into 100mL of tap water and 100mL of milk, standing for 30min, and filtering supernatant;
b. cutting 5.0g of pickle and potato chip product sample into small pieces, mixing with 100mL of distilled water, crushing, carrying out ultrasonic treatment for 30min, taking supernatant and filtering;
c. finally, 100mL of 0.1mol/L sodium citrate buffer solution with the pH value of 4 is added;
d. adding 10.0g of sauced meat into 150mL of deionized water, placing in a stirrer, stirring for 2min, taking out the suspension, placing in a 50 ℃ water bath kettle, carrying out water bath for 15min, cooling, diluting to 250mL, and filtering;
e. when in use, 3mL of filtrate is taken and centrifuged for 15min at 8000r/min, and the supernatant is diluted to 4 mL.
(2) And (3) detecting an actual sample:
placing the pretreated sample in a 25 x 40mm special electrochemical bottle, respectively adding 1mmol/L, 5mmol/L and 10mmol/L nitrite standard solutions into pickle and meat sample solutions, respectively adding 0.1mmol/L, 0.5mmol/L and 1mmol/L nitrite standard solutions into milk, tap water and potato chip solutions, detecting the sample solution by using a prepared Mb @ AuNPs/Au electrode through DPV, calculating the measured concentration by using a standard curve, and analyzing the standard recovery rates of different samples; the recovery rate of the Mb @ AuNPs/Au electrode standard addition is between 94.6 and 104.3 percent.
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