CN111208180B - Method for improving bioelectrode construction by using reduced graphene oxide nano material - Google Patents
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Abstract
The invention discloses a method for improving the construction of a bioelectrode by using a reduced graphene oxide nano material, which is to modify a reduced graphene oxide suspension on the surface of a prepared nano porous gold/glassy carbon electrode to prepare a reduced graphene oxide/nano porous gold/glassy carbon electrode; and then loading a biological material on the surface of the reduced graphene oxide/nano porous gold/glassy carbon electrode to construct the biological electrode. The detection limit and the detection sensitivity of the bioelectrode prepared by the method are greatly improved in the aspect of sensing performance. The method of the invention can provide more attachment sites by using the reduced graphene oxide modification, is beneficial to the fixation of the biological recognition element on the surface of the electrode, enhances the stability of the electrode to the substance to be detected, effectively overcomes the defects of high detection limit and low sensitivity of the traditional biosensor, and has wide application prospect.
Description
Technical Field
The invention relates to a preparation method of a bioelectrode, in particular to a method for improving the construction of the bioelectrode by using a reduced graphene oxide nano material. Belongs to the technical field of electrochemical analysis and test.
Background
In recent years, biosensors have been developed rapidly, and since a biological recognition element having high selectivity for a substance to be detected in an environment is loaded, the type of sensor has higher sensitivity and accuracy in detecting the substance to be detected in the environment. Therefore, biosensors are a hot point of research in the field of electrochemical analysis and testing. However, due to the weak conductive property of the biological recognition element, the related modified material of the electrode is used as a bridge for connecting the electrode and the biological recognition element, and plays an important role in improving the sensing performance of the biosensor.
In the field of sensory analytical testing, detection limits and sensitivity are important parameters for evaluating a detection system. Although the biological material has high selectivity, the sensor has the problems of high detection limit, low sensitivity and the like due to the weak conductivity of the biological material. With the development of nano materials, the nano materials are usedHas some excellent characteristics such as good electrical conductivity, thermal conductivity and biocompatibility, and is widely used in various fields. Among these nanomaterials, reduced graphene oxide (reduced graphene oxide) is a two-dimensional carbon material with a single atom thickness, and is also a single-atom thin sheet-like carbon nanotube, and in the field of sensors, the reduced graphene oxide (reduced graphene oxide) can be used as an electrode modification material for constructing sensors. Graphene oxide has good conductivity due to reduction (10)6S cm-1) And high charge mobility (200000 cm)2v-1s-1) So that electrons can be efficiently transferred from the biometric element to the electrode surface in a catalytic reaction. Meanwhile, the reduced graphene oxide also has excellent biocompatibility, and more attachment sites are provided for identification of element loads in the construction process of the biosensor. Therefore, the reduced graphene oxide is not only an excellent electron transfer carrier, but also a good carrier for immobilization of biomolecules, and a method for constructing a bioelectrode by utilizing modification of a reduced graphene oxide nano material is expected to overcome the defects. However, the reduced graphene oxide/nanoporous gold/glassy carbon electrode is prepared by retrieving relevant reduced graphene oxide suspension on the surface modification of the prepared nanoporous gold/glassy carbon electrode; no report is found on a method for constructing a bioelectrode by loading a biological material on the surface of a reduced graphene oxide/nanoporous gold/glassy carbon electrode.
Disclosure of Invention
Aiming at the defects that the biosensor in the prior art has low detection limit on a detected object and does not reach the sensitivity standard, the invention aims to provide a method for improving the construction of a bioelectrode by using a reduced graphene oxide nano material.
The invention discloses a method for improving the construction of a bioelectrode by using a reduced graphene oxide nano material, which comprises the following steps: reducing and oxidizing a graphene suspension on the surface modification of the prepared nano porous gold/glassy carbon electrode to prepare a reduced and oxidized graphene/nano porous gold/glassy carbon electrode; then loading a biological material on the surface of the reduced graphene oxide/nano-porous gold/glassy carbon electrode to construct a biological electrode;
the method is characterized in that:
the preparation method of the nano porous gold/glassy carbon electrode comprises the following steps: polishing a Glassy Carbon Electrode (GCE) by using alumina powder with the diameter of 50nm, and then sequentially placing the GCE in absolute ethyl alcohol and ultrapure water to respectively perform ultrasonic cleaning for 30-90 seconds for later use; placing an Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 10-30 minutes at the temperature of 20-30 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of a polished and cleaned glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare the nano porous gold/glassy carbon electrode;
the method for reducing the graphene oxide suspension on the surface modification of the prepared nano-porous gold/glassy carbon electrode comprises the following steps: fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01-1%, and then carrying out ultrasonic dispersion for 0.5-12 hours at the frequency of 24-80 kilohertz; after ultrasonic treatment, putting the mixture into a refrigerator at 4 ℃ for storage for later use; modifying the prepared reduced graphene oxide suspension to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 0.0796-1.592 microliters per square millimeter in a suspension dropwise manner, and then fixing for 3-24 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode;
the method for loading the biological material on the surface of the reduced graphene oxide/nano-porous gold/glassy carbon electrode comprises the following steps: taking 1-5 ml of the mixture with the density of 2.0 multiplied by 107～5.0×108Centrifuging each/ml constructed overexpression recombinant cell for 5-10 minutes under the condition of 6000 +/-500 revolutions per minute, after the precipitated cell is resuspended by using a phosphate buffer solution, uniformly coating the cell on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode according to the amount of 1.194-2.389 microliter/square millimeter, and then fixing for 3-24 hours at 4 ℃ to obtain the bioelectrode.
In the above method for improving the construction of the bioelectrode by using the reduced graphene oxide nanomaterial, the preferable preparation method of the nanoporous gold/glassy carbon electrode is as follows: placing the Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 30 minutes at the temperature of 30 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of the glassy carbon electrode after polishing and cleaning, and drying for 2 hours in vacuum to prepare the nano porous gold/glassy carbon electrode.
In the above method for improving the construction of the bioelectrode by using the reduced graphene oxide nanomaterial, the preferable method for reducing the graphene oxide suspension on the surface modification of the prepared nanoporous gold/glassy carbon electrode is as follows: fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.5-0.7%, and then carrying out ultrasonic dispersion for 9-10 hours at the frequency of 24-50 kilohertz; after ultrasonic treatment, putting the mixture into a refrigerator at 4 ℃ for storage for later use; modifying the prepared reduced graphene oxide suspension to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 1.0-1.194 microliter per square millimeter by a suspension and dropwise adding mode, and then fixing for 12-15 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode.
In the above method for improving the construction of the bioelectrode by using the reduced graphene oxide nanomaterial, the preferable method for loading the biomaterial on the surface of the reduced graphene oxide/nanoporous gold/glassy carbon electrode is as follows: taking 1-3 ml of the mixture with the density of 3.0 multiplied by 107～3.0×108Centrifuging each/ml constructed overexpression recombinant cell for 10 minutes at 6000 revolutions per minute, after the precipitated cell is resuspended by using phosphate buffer solution, uniformly coating the cell on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode in an amount of 1.317-2.163 microlitres per square millimeter, and then fixing the cell for 12-16 hours at 4 ℃ to construct the bioelectrode; wherein the recombinant cell is a recombinant E.coli overexpressing a sulfide quinone oxidoreductase, or an enzyme or recombinant cell used for set detection purposes.
In the method for improving the bioelectrode construction by using the reduced graphene oxide nano material, before the constructed bioelectrode is subjected to detection operation, 2-4 microliters of Nafion solution with the volume ratio concentration of 0.5% is preferably required to be dripped for pretreatment, and the bioelectrode is stored in a refrigerator at 4 ℃ for later use.
The constructed sulfur-quinone oxidoreductase-overexpressing escherichia coli/reduced graphene oxide/nano-porous gold sulfide detection biosensor is taken as an example to verify that the bioelectrode constructed by the improved method provided by the invention has greatly improved detection limit and detection sensitivity in the aspect of sensing performance. The biological identification device has the outstanding advantages that the proper amount of the fixed reduced graphene oxide nano material can enhance the transmission efficiency of electrons between a biological identification element and an electrode, effectively weaken the obstruction of electron transmission caused by the weak conductivity of the biological identification element, and improve the sensitivity of the biosensor.
The invention has the prominent substantive characteristics and remarkable progress that:
1. the invention provides a method for improving the construction of a bioelectrode by using a reduced graphene oxide nano material, which can effectively solve the problems of high detection limit and low sensitivity of the traditional biosensor based on the characteristics of good conductivity and biocompatibility of the reduced graphene oxide, as shown in figure 1.
2. Compared with other solvents, the reduced graphene oxide suspension liquid provided by the invention has the advantages that the absolute ethyl alcohol is used as the solvent, the prepared reduced graphene oxide has good dispersibility, and the repeatability of electrode construction is enhanced, as shown in fig. 2. Meanwhile, the method of the invention can provide more attachment sites by using reduced graphene oxide modification, which is helpful for fixing the biological recognition element on the surface of the electrode and enhancing the stability of the electrode to the substance to be detected, as shown in fig. 3.
3. The method optimizes the loading of the reduced graphene oxide suspension, provides a reference of the loading for other different detection systems in practical application as shown in fig. 4, and is beneficial to saving resources and improving the utilization rate of the reduced graphene oxide suspension.
Drawings
FIG. 1 is a current response characterization diagram of a bioelectrode before and after modification of a reduced graphene oxide suspension.
In the figure, a is a bioelectrode which is not loaded with reduced graphene oxide suspension, and b is a bioelectrode which is loaded with quantitative reduced graphene oxide suspension.
Fig. 2 is a characteristic diagram of the dispersibility of a certain amount of reduced graphene oxide suspension prepared from different solvents on the surface of an electrode.
Wherein: the preparation method of the reduced graphene oxide suspension comprises the steps of preparing deionized water (a), a Nafion solution (b) and absolute ethyl alcohol (c) respectively.
FIG. 3 is a sensor constructed by modifying reduced graphene oxide suspensions prepared in the same volume but with different solvents, and detecting 30 μ M sodium sulfide (A) and 50 μ M sodium sulfide (B), respectively.
Fig. 4 shows a sensor constructed by modifying reduced graphene oxide suspensions prepared from absolute ethanol as a solvent in different volumes, and detecting 70 μ M sodium sulfide (a) and 100 μ M sodium sulfide (B), respectively.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents, recombinant E.coli overexpressing sulfide quinone oxidoreductase, enzymes or recombinant cells for setting the detection purpose, and the like used therein are commercially available unless otherwise specified.
Example 1: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01%, and performing ultrasonic dispersion for 0.5 hour at the frequency of 24 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 2: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.5%, and performing ultrasonic dispersion for 6 hours at the frequency of 40 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 3: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass volume ratio concentration of 1%, and performing ultrasonic dispersion for 12 hours at the frequency of 80 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 4: preparation of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with deionized water to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01%, and performing ultrasonic dispersion for 0.5 hour at the frequency of 24 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 5: preparation of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with deionized water to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.5%, and performing ultrasonic dispersion for 6 hours at the frequency of 40 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 6: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with deionized water to prepare a reduced graphene oxide suspension with the mass volume ratio concentration of 1%, and performing ultrasonic dispersion for 12 hours at the frequency of 80 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 7: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with a Nafion solution with the volume ratio of 0.8% to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01%, and then carrying out ultrasonic dispersion for 0.5 hour at the frequency of 24 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 8: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with a Nafion solution with the volume ratio of 0.8% to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.5%, and then carrying out ultrasonic dispersion for 6 hours at the frequency of 40 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 9: preparation and dispersion of reduced graphene oxide suspension
Fully mixing the reduced graphene oxide nano material with a Nafion solution with the volume ratio of 0.8% to prepare a reduced graphene oxide suspension with the mass volume ratio concentration of 1%, and then carrying out ultrasonic dispersion for 12 hours at the frequency of 80 kilohertz; after ultrasonic treatment, the mixture is stored in a refrigerator at 4 ℃ for later use.
Example 10: polishing and cleaning of glassy carbon electrode
The Glassy Carbon Electrode (GCE) was polished with alumina powder having a diameter of 50nm, and then sequentially placed in absolute ethanol and ultrapure water to be respectively ultrasonically cleaned for 30 seconds.
Example 11: polishing and cleaning of glassy carbon electrode
The Glassy Carbon Electrode (GCE) was polished with alumina powder having a diameter of 50nm, and then sequentially placed in absolute ethanol and ultrapure water to be ultrasonically cleaned for 60 seconds, respectively.
Example 12: polishing and cleaning of glassy carbon electrode
The Glassy Carbon Electrode (GCE) was polished with alumina powder having a diameter of 50nm, and then sequentially placed in absolute ethanol and ultrapure water to be ultrasonically cleaned for 90 seconds, respectively.
Example 13: supported catalytic material on glassy carbon electrode
Placing the Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 10 minutes at the temperature of 20 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of the glassy carbon electrode after polishing and cleaning, and drying for 1 hour in vacuum to prepare the nano porous gold/glassy carbon electrode.
Example 14: supported catalytic material on glassy carbon electrode
Placing the Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 20 minutes at the temperature of 25 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of the glassy carbon electrode after polishing and cleaning, and drying for 1.5 hours in vacuum to prepare the nano porous gold/glassy carbon electrode.
Example 15: supported catalytic material on glassy carbon electrode
Placing the Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 30 minutes at the temperature of 30 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of the glassy carbon electrode after polishing and cleaning, and drying for 2 hours in vacuum to prepare the nano porous gold/glassy carbon electrode.
Example 16: reduced graphene oxide suspension modified nano porous gold/glassy carbon electrode
Modifying the prepared reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01% to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 0.0796 microliters per square millimeter by suspending and dropping, and then fixing for 9 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode.
Example 17: reduced graphene oxide suspension modified nano porous gold/glassy carbon electrode
Modifying the prepared reduced graphene oxide suspension with the mass-volume ratio concentration of 0.5% to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 0.8358 microliters per square millimeter in a suspension and dropwise adding mode, and then fixing for 16 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode.
Example 18: reduced graphene oxide suspension modified nano porous gold/glassy carbon electrode
Modifying the prepared reduced graphene oxide suspension with the mass volume ratio concentration of 1% to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 1.592 microliters per square millimeter by suspending and dropping, and then fixing for 23 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode.
Example 19: biological electrode constructed by loading biological material on electrode surface
Taking 1 ml of bacteria with the concentration of 2.0 multiplied by 108And centrifuging the constructed over-expression sulfide quinone oxidoreductase recombinant escherichia coli for 5 minutes under the condition of 5500 revolutions per minute, after the precipitated cells are resuspended by using a phosphate buffer solution, uniformly coating the cells on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode in an amount of 1.194 microliter per square millimeter, and then fixing the cells for 9 hours at 4 ℃ to construct the biological electrode.
Example 20: biological electrode constructed by loading biological material on electrode surface
Taking 3 ml of bacteria with the concentration of 3.0 multiplied by 108And centrifuging the constructed over-expression sulfide quinone oxidoreductase recombinant escherichia coli for 8 minutes at 6000 rpm, after the precipitated cells are resuspended by using phosphate buffer solution, uniformly coating the cells on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode according to the amount of 1.7915 microliters per square millimeter, and then fixing the cells for 16 hours at 4 ℃ to construct the bioelectrode.
Example 21: biological electrode constructed by loading biological material on electrode surface
Taking 5ml of the strain with the concentration of 5.0 multiplied by 108And centrifuging the constructed over-expression sulfide quinone oxidoreductase recombinant escherichia coli for 10 minutes at 6500 r/min, after the precipitated cells are resuspended by using a phosphate buffer solution, uniformly coating the cells on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode in an amount of 2.389 microliters per square millimeter, and then fixing the cells for 23 hours at 4 ℃ to construct the bioelectrode.
Example 22: construction of a biosensor Using the improved method of the invention
(1) The preparation method of the nano porous gold/glassy carbon electrode comprises the following steps: placing the Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 30 minutes at the temperature of 30 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of the glassy carbon electrode after polishing and cleaning, and drying for 2 hours in vacuum to prepare the nano porous gold/glassy carbon electrode.
(2) The method for reducing the graphene oxide suspension on the surface modification of the nano porous gold/glassy carbon electrode comprises the following steps: fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.6%, and performing ultrasonic dispersion for 10 hours at the frequency of 50 kilohertz; after ultrasonic treatment, putting the mixture into a refrigerator at 4 ℃ for storage for later use; modifying the prepared reduced graphene oxide suspension to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 1.1 microliter per square millimeter by suspending and dropwise adding, and then fixing for 15 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode.
(3) The method for reducing the graphene oxide/nano-porous gold/glassy carbon electrode surface to load the biological material comprises the following steps: taking 2 ml of the extract with the density of 1.0 multiplied by 108And centrifuging each ml of constructed recombinant escherichia coli cells over-expressing the sulfide quinone oxidoreductase at 6000 rpm for 10 minutes, after the precipitated cells are resuspended by using phosphate buffer solution, uniformly coating the cells on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode in an amount of 1.8 microliter per square millimeter, and then fixing the cells for 15 hours at 4 ℃ to construct the biological electrode.
Before the detection operation, the bioelectrode obtained by the construction needs to be pretreated by dripping 4 microliters of Nafion solution with the volume ratio concentration of 0.5 percent and stored in a refrigerator at 4 ℃ for later use.
Example 23: detection of sulfide standard substance by biosensor of recombinant microbial cell of nano-porous gold
(1) The biosensor successfully constructed by the method of example 22 was used as a working electrode as a biological detection electrode, a platinum electrode was used as a counter electrode, and a saturated calomel electrode was used as a reference electrode to assemble a biosensor of a three-electrode system; wherein the nanoporous gold/glassy carbon electrode is previously 0.5M H2SO4And (3) scanning for 10 circles by cyclic voltammetry to represent the effective working area of the nano porous gold/glassy carbon electrode by the reduction peak current value.
(2) And (3) sulfide detection: before the biosensor is used for the first time, the biosensor is put into a reaction system of phosphate buffer solution, and a sulfide compound with the final concentration of 0.1-7000 mu M is addedThe standard substance adopts a current-time Curve (Amperometric i-t Curve), selects-0.24V of external voltage, observes the current response of the added sulfide standard substance with known concentration, and makes a concentration-current standard Curve of the current response; observing whether the current value changes along with the concentration of the added sulfide standard substance, so as to judge whether sulfide exists in the solution; the linear detection range of the electrode pair p-phenylenediamine standard is 0.1-7000 mu M, the detection limit is 98.5nM, and the sensitivity is 400.42 mu A cm-2mM-1(ii) a The results of the detection are shown in FIG. 1.
After the electrodes were stored in a 4 ℃ freezer for 4 weeks, the response current remained 88.4%, with lower detection limits and higher sensitivity compared to other sulfide sensor detection systems, as shown in table 1:
TABLE 1
The phosphate buffer solution reaction system in the above steps is: 15mL of phosphate buffer at pH 7.0 with a concentration of 50 mM. The phosphate is preferably sodium phosphate.
Reference to the literature
1.Aziz,M.A.,Almadi,R.,Yamani,Z.H.,Indium tin oxide nanoparticle-modified glassy carbon electrode for electrochemical sulfide detection in alcoholic medium.Anal.Sci.2018,34:599-604.
2.Rossi Salamanca-Neto,C.A.,Scremin,J.,Fatibello-Filho,O.,Clausen,D.N.,Sartori,E.R.,Assessment of the performance of triphenylphosphine for the voltammetric determination of elemental sulphur in cosmetic products.Analyst 2018,143:3600-3606.
3.Ghadiri,M.,Kariminia,H.,Azad,R.R.,Spectrophotometric determination of sulfide based on peroxidase inhibition by detection of purpurogallin formation.Ecotox.Environ.Safe.2013,91:117-121.
4.Vosoughi,A.,Yazdian,F.,Amoabediny,G.,Hakim,M.,Investigating the effect of design parameters on the response time of a highly sensitive microbial hydrogen sulfide biosensor based on oxygen consumption.Biosens.Bioelectron.2015,70:106-114.
5.Savizi,I.S.P.,Kariminia,H.R.,Ghadiri,M.,Roosta Azad,R.,Amperometric sulfide detection using Coprinus cinereus peroxidase immobilized on screen printed electrode in an enzyme inhibition based biosensor.Biosens.Bioelectron.2012,35:297-301.
6.Mirzaei,M.,Amoabediny,G.,Yazdian,F.,Sheikhpour,M.,Ebrahimi,E.,Zadeh,B.E.H.,Animmobilized Thiobacillus thioparus biosensing system for monitoring sulfide hydrogen；optimized parameters in a bioreactor.Process Biochem.2014,49:380-385.
7.Bian,C.C.,Wang,H.M.,Zhang X.L.,Xiao,S.,Liu,Z.,Wang,X.,Sensitive detection of low-concentration sulfide based on the synergistic effect of rGO,np-Au,and recombinant microbial cell.Biosens.Bioelectron.2020,151:111985.
Claims (1)
1. A method for detecting sulfide by using a reduced graphene oxide nano material improved bioelectrode comprises the following steps:
(1) reducing and oxidizing a graphene suspension on the surface modification of the prepared nano porous gold/glassy carbon electrode to prepare a reduced and oxidized graphene/nano porous gold/glassy carbon electrode;
wherein: the preparation method of the nano porous gold/glassy carbon electrode comprises the following steps: polishing a Glassy Carbon Electrode (GCE) by using alumina powder with the diameter of 50nm, and then sequentially placing the GCE in absolute ethyl alcohol and ultrapure water to respectively perform ultrasonic cleaning for 30-90 seconds for later use; placing an Au/Ag alloy sheet with the thickness of 100 +/-10 nm in pure nitric acid, corroding for 10-30 minutes at the temperature of 20-30 ℃ to prepare a nano porous gold film, fixing the nano porous gold film on the surface of a polished and cleaned glassy carbon electrode, and drying for 1-2 hours in vacuum to prepare the nano porous gold/glassy carbon electrode; the method for reducing the graphene oxide suspension on the surface modification of the prepared nano-porous gold/glassy carbon electrode comprises the following steps: fully mixing the reduced graphene oxide nano material with analytically pure absolute ethyl alcohol to prepare a reduced graphene oxide suspension with the mass-volume ratio concentration of 0.01-1%, and then carrying out ultrasonic dispersion for 0.5-12 hours at the frequency of 24-80 kilohertz; after ultrasonic treatment, putting the mixture into a refrigerator at 4 ℃ for storage for later use; modifying the prepared reduced graphene oxide suspension to the surface of the prepared nano porous gold/glassy carbon electrode according to the amount of 0.0796-1.592 microliters per square millimeter in a suspension dropwise manner, and then fixing for 3-24 hours under the conditions of no wind, no dust and room temperature to prepare the reduced graphene oxide/nano porous gold/glassy carbon electrode;
(2) loading a biological material on the surface of the reduced graphene oxide/nano-porous gold/glassy carbon electrode to construct an improved biological electrode, namely a biosensor of the recombinant microbial cells of the nano-porous gold;
(3) detecting sulfide by using a biosensor of a recombinant microbial cell of nano-porous gold:
the method is characterized in that:
the method for loading the biological material on the surface of the reduced graphene oxide/nano-porous gold/glassy carbon electrode comprises the following steps: taking 1-3 ml of the mixture with the density of 3.0 multiplied by 107～3.0×108Centrifuging each/ml constructed overexpression recombinant cell for 10 minutes at 6000 revolutions per minute, after the precipitated cell is resuspended by using phosphate buffer solution, uniformly coating the cell on the surface of the prepared reduced graphene oxide/nano porous gold/glassy carbon electrode in an amount of 1.317-2.163 microliter per square millimeter, and then fixing the cell for 12-16 hours at 4 ℃ to construct the improved biological electrode; wherein the recombinant cell is a recombinant E.coli overexpressing a sulfide quinone oxidoreductase;
the method for detecting the sulfide by using the biosensor of the recombinant microorganism cell of the nano-porous gold comprises the following steps: the constructed improved biological electrode is used as a biological detection electrode as a working electrode, a platinum electrode is used as a counter electrode, a saturated calomel electrode is used as a reference electrode, and a biosensor of a three-electrode system is assembled; wherein the nanoporous gold/glassy carbon electrode is previously 0.5M H2SO4Scanning for 10 circles by cyclic voltammetry, and representing the effective working area of the nano porous gold/glassy carbon electrode by the reduction peak current value; when the biosensor is used for detection, the electrode is put into a reaction system of phosphate buffer solution, and 0.1-7000 mu M of phosphate buffer solution is addedSulfide, adopting a current-time Curve (Amperometric i-t Curve), selecting an external voltage of-0.24V, and making a concentration-current standard Curve of current response; observing whether the current value changes along with the concentration of the added sulfide, so as to judge whether the sulfide exists in the solution; the linear detection range of the electrode to the sulfide is 0.1-7000 mu M, the detection limit is 98.5nM, and the sensitivity is 400.42 mu A cm-2mM-1。
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| CN106770567A (en) * | 2016-11-25 | 2017-05-31 | 江苏丰格测控技术有限公司 | A kind of Platinum Nanoparticles cobalt/porous gold/graphene composite material and preparation method thereof |
| CN109030595A (en) * | 2017-06-09 | 2018-12-18 | 清华大学 | Biological sensor electrode and biosensor |
| CN110568037A (en) * | 2019-09-02 | 2019-12-13 | 江南大学 | construction method and application of electrochemical cell sensor for acetamiprid and imidacloprid combined toxicity evaluation |
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| CN106018851A (en) * | 2016-05-23 | 2016-10-12 | 济南大学 | Method for preparing steroid immunosensor based on sheet black phosphorus and application |
| CN106770567A (en) * | 2016-11-25 | 2017-05-31 | 江苏丰格测控技术有限公司 | A kind of Platinum Nanoparticles cobalt/porous gold/graphene composite material and preparation method thereof |
| CN109030595A (en) * | 2017-06-09 | 2018-12-18 | 清华大学 | Biological sensor electrode and biosensor |
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