CN113138217B - Riboflavin electrochemical detection method and sensor based on heterozygous biological film - Google Patents
Riboflavin electrochemical detection method and sensor based on heterozygous biological film Download PDFInfo
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Abstract
The invention provides a hybrid biological film-based riboflavin electrochemical detection method and a sensor, which comprise the following steps: (1) Inoculation of Shewanella species intoThe culture medium is added into a container containing reaction buffer solution after being activated; (2) Adding sterile FeCl into the container in the step (1) 3 Solution and sterile Na 2 S 2 O 3 A solution; (3) A reference electrode, a working electrode and a counter electrode are arranged in a container to form a three-electrode system; (4) Under the synthesis condition, shewanella generates FeS nano particles with conductivity and covers the surfaces of cells to form electroactive heterozygous cells, and the electroactive heterozygous cells are adsorbed on the working electrode in the step (3) to form an electroactive heterozygous cell biomembrane which is connected with a signal detection system to form a bioelectrochemical sensor; (5) The invention realizes quantitative and ultrasensitive detection of the riboflavin by loading external voltage on the working electrode, adding an electron acceptor of Shewanella, adding the riboflavin, detecting and recording a current change value.
Description
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to a riboflavin electrochemical detection method and a sensor based on a hybrid biological membrane.
Background
Riboflavin, i.e. vitamin B 2 (VB 2 ) Is a water-soluble B vitamin, and has important significance for human health. The rapid and accurate determination of the content of the riboflavin has important significance in the synthesis of the riboflavin and the production of related food, medicine and health-care product industries. Existing methods for detecting riboflavin include microbiological methods, fluorescence spectrophotometry, high Performance Liquid Chromatography (HPLC), mass spectrometry, and the like. However, these methods have the common disadvantages of long detection period, complicated sample processing, high cost and the like.
The bioelectrochemical sensor uses biological materials or organisms as identification elements to detect signals, and has the advantages of simple instrument requirement, high sensitivity, good specificity and the like. However, in bioelectrochemical systems, the limitation of the contact area between biological cells and non-biological surfaces prevents the surfaces from establishing an effective electron transfer interface. The biological nanometer heterozygous cell is taken as a unique cell combined with nanometer particles, has excellent characteristics of cells and materials, and the combination of electroactive bacteria and conductive nanometer particles generally forms a biological-nanometer electron transfer mechanism, so that the electron transfer rate and coulomb efficiency can be improved.
Under specific potential control conditions, shewanella can reduce sodium fumarate by electrons from extracellular electrodes under the catalysis of its own sodium fumarate reductase and form a current in an external circuit. Riboflavin is used as an electron mediator for the intracellular and extracellular electron transfer processes, and the concentration of Riboflavin is directly related to the electron transfer efficiency of the process, i.e. the magnitude of the external circuit current intensity. Therefore, the riboflavin in the sample can be efficiently and rapidly detected and the concentration thereof can be determined directly by the current change of the reaction system.
The invention builds a novel electrochemical detection method and sensor for riboflavin based on a hybrid biological membrane based on the method, and no report for detecting the riboflavin by using the bioelectrochemical method is found through searching the prior art.
Disclosure of Invention
Aiming at the technical problems, the invention provides a riboflavin electrochemical detection method and a sensor based on a hybrid biological film, which are used for preparing an electroactive hybrid cell biological film for amplifying electrochemical signals for the first time, and the problems of high detection cost, low sensitivity, complex electrode materials and the like in the prior art are solved by utilizing the reducing capability of Shewanella to a sulfur source and an iron source, generating FeS nano particles with conductivity by cells, partially covering the surfaces of the FeS nano particles to form hybrid cells, and adsorbing the hybrid cells on a working electrode to form the electroactive hybrid cell biological film, so that the electron exchange surface area of the electroactive cells is expanded, the signal amplifying performance of a biological sensing system is greatly improved.
The technical scheme of the invention is as follows: an electrochemical detection method of riboflavin based on a hybrid biological membrane comprises the following steps:
step (1) the Shewanella bacteria are inoculated to a culture medium for activation, and the activated Shewanella bacteria are added into a container containing a reaction buffer solution after being washed and resuspended;
step (2) adding sterile FeCl into the container in the step (1) 3 Solution and sterile Na 2 S 2 O 3 A solution;
step (3) a reference electrode, a working electrode and a counter electrode are arranged in the container to form a three-electrode system;
under the synthesis condition, shewanella generates FeS nano particles with conductivity and covers the surfaces of cells to form electroactive heterozygous cells, the electroactive heterozygous cells are adsorbed on the working electrode of the step (3) to form an electroactive heterozygous cell biomembrane, and the three-electrode system is connected with a signal detection system to form a bioelectrochemical sensor;
and (5) loading external voltage on the working electrode of the bioelectrochemical sensor obtained in the step (4), adding an electron acceptor of Shewanella into the container, then adding a riboflavin sample, detecting and recording a current change value, and calculating the concentration of the riboflavin.
Preferably, the Shewanella (Shewanellaoneidensis MR-1) is purchased from American Type Culture Collection (ATCC); the preserved Shewanella is inoculated to a culture medium for culture, and activated thalli are obtained.
In the above scheme, the reaction buffer comprises the following substances, namely LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride, pH=7; m9 medium: dodecahydrate, 17.8g/L of disodium hydrogen phosphate, 3g/L of monopotassium phosphate, 0.5g/L of sodium chloride and 10.5g/L of ammonium chloride; 18mM sodium lactate, 0.1mM calcium chloride and 1mM magnesium sulfate.
In the above scheme, shewanella in step (1) needs to be activated to OD 600 ＝0.5-4。
In the above scheme, the activated Shewanella in step (1) is resuspended in reaction buffer, its OD 600 The value is controlled between 0.05 and 5.
In the above scheme, the experimental operations in the step (1) to the step (3) should be performed in an anaerobic environment, and the container is kept in an anaerobic state during the synthesis of the hybrid cell biofilm and the detection of the riboflavin.
In the above scheme, the step (2) adds sterile FeCl 3 Solution and sterile Na 2 S 2 O 3 The concentration of the solution is greater than 50 mu m.
In the above scheme, the step (2) is added with sterile FeCl 3 Standing the solution, adding sterile Na 2 S 2 O 3 A solution.
In the above scheme, the working electrode in the step (3) is carbon cloth or carbon felt.
In the above scheme, in the step (3), the working electrode is installed in a container before synthesis of the heterozygous cells, so that a biological film is formed on the surface of the working electrode at the same time of synthesis of the heterozygous cells, and the working electrode is directly used after the biological film is formed or is transferred to a new container in an anaerobic environment for use.
In the scheme, the synthesis condition in the step (4) means that the synthesis time is more than 5 hours and the synthesis temperature is 4-37 DEG C
In the above scheme, the electron acceptor of Shewanella in the step (5) is sodium fumarate solution. The concentration of the sodium fumarate solution is 50 mM-saturated concentration.
In the above scheme, the external voltage applied to the working electrode in the step (5) is negative (less than 0V), sodium fumarate is added as an electron acceptor after balancing, and riboflavin is added for detection after baseline balancing.
The invention also provides a sensor according to the hybrid biological film-based riboflavin electrochemical detection method, wherein the sensor comprises a signal detection system, a three-electrode system and a signal generation system; the signal generating system comprises an electron acceptor of Shewanella, an electroactive hybrid cell biofilm and a reaction buffer solution.
In the above scheme, the electron acceptor of Shewanella is sodium fumarate solution.
Compared with the prior art, the invention has the beneficial effects that: the invention constructs an electroactive heterozygous cell biological film formed by Shewanella and nano ferrous sulfide, and uses the biological film to construct a bioelectrochemical sensor to amplify signals so as to realize quantitative and ultrasensitive detection of riboflavin.
Drawings
FIG. 1 is a schematic diagram of the principle of detecting riboflavin by a sensor;
FIG. 2 is a schematic diagram of the structure of the bioelectrochemical sensor of the present invention;
FIG. 3 is a SEM characterization of wild Shewanella bacteria;
FIG. 4 is an SEM characterization of Shewanella-nano ferrous sulfide capsules;
FIG. 5 is a diagram representing the EDS characterization of biologically prepared nano ferrous sulfide;
FIG. 6 is a graph showing the current output signal results after the addition of two standard solutions of riboflavin of different concentrations;
FIG. 7 is a graph showing the current output signal results after successive addition of riboflavin standard solutions of different concentrations;
FIG. 8 is a graph showing the current output signal results after adding the same concentration of riboflavin standard solution to two different reaction systems;
FIG. 9 is a standard graph of the present system for riboflavin detection.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Example 1:
(1) The strain is used: shewanella (Shewanella oneidensis MR-1), available from ATCC American type culture Collection under the accession number ATCC700550.
(2) Shewanella bacteria liquid is obtained: shewanella strain was inoculated into 100mL of LB medium (containing 10g/L of tryptone, 5g/L of yeast extract and 5g/L of sodium chloride, pH=7.0), and cultured at 30℃with shaking at 190rpm for 10 hours to obtain a bacterial liquid. SEM characterization of wild shiva bacteria is shown in figure 3.
(3) Preparing a reaction buffer solution of the bioelectrochemical sensor: 0.735mL of LB liquid medium and 13.965mL of M9 medium were thoroughly mixed, and sodium lactate, calcium chloride, and magnesium sulfate were additionally added to reach final concentrations of 18mM, 0.1mM, and 1mM, respectively.
(4) Obtaining electroactive hybrid cell biofilms: taking 14.7mL of anaerobic reaction buffer solution in a reaction vessel, putting the reaction vessel into a rotor, and activating Shewanella bacteria liquidCentrifuging, adding the centrifuged precipitate into reaction buffer solution (all in N 2 Under protection), shewanella OD 600 =1. At N 2 Under protection, 150uL of filtered sterilized 0.1MFeCL was added 3 The solution was capped with an anaerobic plug, allowed to stand for 1h, and 150uL of sterile 0.1M Na was added 2 S 2 O 3 Solution, mounting electrode. A three electrode system is shown in fig. 1. Magnetically stirring (210 rpm) the cells at a constant temperature of 30 ℃ for 12 hours, and automatically generating FeS nano particles with conductivity and partially covering the surfaces of the cells to form heterozygous cells. SEM characterization of heterozygous cells is shown in figure 4. The heterozygous cells are adsorbed on the working electrode to form an electroactive heterozygous cell biofilm.
(5) Assembly of the bioelectrochemical sensor: the three-electrode working mode is adopted, a saturated calomel electrode is used as a reference electrode, 1X 2cm carbon cloth connected with titanium wires is used as a working electrode, a platinum wire electrode is used as a counter electrode, an electrochemical workstation CHI660E (Shanghai Chen Hua instrument Co., ltd.) is used for control, and an external voltage of-0.6 volt is applied to the working electrode. The biosensor structure is shown in fig. 2.
(6) Addition of sodium fumarate solution: after the output current of the bioelectrochemical sensor is stabilized (about 30 min), adding a sodium fumarate solution with a final concentration of 50mM and a saturated concentration, wherein the addition of the sodium fumarate solution is a premise of detecting riboflavin, sodium fumarate reductase of Shewanella can specifically reduce sodium fumarate in a reaction system under the action of external voltage, and the addition of riboflavin (electronic mediator) accelerates the reduction of sodium fumarate, so that the change of a current value is caused.
(7) Detection of riboflavin-containing samples 2 identical bioelectrochemical sensors were prepared for riboflavin detection according to the steps (2) to (6) above, and a concentration of riboflavin sample was added to each of these 2 sensors, and peak height results of the current response pattern were recorded. The current signal is shown in fig. 6. The riboflavin content was calculated from the standard curve.
Example 2:
taking out the Shewanella-nano ferrous sulfide heterozygous system synthesized in the example 1 in an anaerobic workstation, centrifuging at 8000rpm for 5min, discarding the supernatant, and washing 3 times by using anaerobic water in a centrifuging way; centrifuging at 8000rpm for 5min, discarding supernatant, washing with 75% ethanol, 100% ethanol and acetone respectively, and drying the final centrifuged precipitate in an anaerobic workstation, grinding black solid into powder with agate mortar after the precipitation drying is completed, and taking appropriate amount of powder sample for EDS elemental analysis characterization, as shown in figure 5.
Example 3:
the difference from example 1 is that three different concentrations of riboflavin standard solutions were added successively to the same bioelectrochemical sensor. The current signal is shown in fig. 7. The bioelectrochemical sensor can realize continuous monitoring of riboflavin with different concentrations.
Example 4:
the difference from example 1 is that the same concentration of riboflavin standard solution was added to two different reaction systems (one reaction system comprising electron acceptor, electroactive hybrid cell biofilm and reaction buffer solution; the other reaction system comprising electron acceptor, shewanella biofilm and reaction buffer solution; the current signal is shown in FIG. 8).
Example 5:
the difference from example 1 is that a current response is generated by continuously adding riboflavin standard solutions of different concentrations into a bioelectrochemical sensor constructed by electroactive hybrid cell biofilms. The standard curve is plotted on the abscissa of the riboflavin concentration and on the ordinate of the current change generated each time the riboflavin is added, as shown in fig. 9.
FIG. 1 is a schematic diagram of the principle of the sensor for detecting riboflavin. Shewanella self-generates FeS nano particles with conductivity and partially covers the surface of the cells to form heterozygote cells, the heterozygote cells are adsorbed on a working electrode to form an electroactive heterozygote cell biological membrane, and electrons can be transferred into cells from the electrode through nano-sulfide FeS particles, so that the surface area of the electron exchange is increased, and the trans-membrane electron transfer is enhanced. When the working potential is under constant negative pressure, adding sodium fumarate as an electron acceptor, reversely transferring electrons provided by the Shewanella by using electrodes into cells through nano FeS particles on the surfaces of the cells, and reducing the sodium fumarate into sodium succinate by using intracellular fumaric acid reductase; riboflavin is then added, which serves as an electron mediator for Shewanella, indirectly transferring electrons into the cell, providing a reducing power for the reduction of fumaric acid. Therefore, the reaction system consumes more electrons and the reverse current increases.
FIG. 2 is a schematic diagram of a bioelectrochemical sensor structure based on electroactive hybrid cell biofilms. The system comprises a saturated calomel electrode, a carbon cloth working electrode, a platinum wire electrode and an electroactive heterozygous cell.
FIG. 3 is an SEM image of wild Shewanella, from which Shewanella in the shape of ellipses can be seen, and only the biofilm of folds is present on the surface of the bacteria, without the presence of nanoparticulate material.
FIG. 4 is an SEM image of a Shewanella-nano ferrous sulfide hybrid system, from which it can be seen that the surface of the Shewanella is coated with nano ferrous sulfide.
Fig. 5 is an EDS spectrum of biological nano ferrous sulfide showing the co-presence of elemental iron and elemental sulfur, demonstrating that the extracellular nanoparticles are iron sulfides.
FIG. 6 is a graph showing the effect of current response of bioelectrochemical sensors based on electroactive hybrid cell biofilms on the single addition of riboflavin at different concentrations. The bioelectrochemical system constructed by the method can generate obvious current response to the riboflavin, and lays a foundation for detecting the riboflavin. Wherein the addition of 50nM riboflavin produced a current response of 12.87. Mu.A and the addition of 600nM riboflavin produced a current response of 171.59. Mu.A.
FIG. 7 is a graph showing the effect of bioelectrochemical sensors based on electroactive hybrid cell biofilms on the current response of successive addition of riboflavin at different concentrations. This demonstrates that the bioelectrochemical system constructed by the method can perform continuous detection, and that the current responses generated by different concentrations have obvious linear relations. Wherein 50nM riboflavin produced a current response of 12.02 μA, 75nM riboflavin produced a current response of 20.2 μA, and 150nM riboflavin produced a current response of 41.34 μA.
FIG. 8 is a graph showing the effect of bioelectrochemical sensors constructed with wild Shewanella and electroactive hybrid cell biofilms, respectively, on current response to the same concentrations. It can be seen that the current response generated by the latter is about 2 times that of the former after adding 2 mu m riboflavin, which is enough to show that the electroactive hybrid cell biofilm module plays a significant role in signal amplification.
FIG. 9 is a standard graph generated from the current response of an electroactive hybrid cell biofilm-based bioelectrochemical sensor to different concentrations of riboflavin production. We examined the current responses of 1nM to 2000nM riboflavin, respectively, and it can be seen that there is a good linear relationship between the current responses and their corresponding riboflavin concentrations, with a minimum concentration of 1nM, sufficient to demonstrate excellent sensitivity.
It should be understood that although the present disclosure has been described in terms of various embodiments, not every embodiment is provided with a separate technical solution, and this description is for clarity only, and those skilled in the art should consider the disclosure as a whole, and the technical solutions in the various embodiments may be combined appropriately to form other embodiments that will be understood by those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (7)
1. The electrochemical detection method of the riboflavin based on the heterozygous biological membrane is characterized by comprising the following steps of:
step (1), the Shewanella bacteria are inoculated to a culture medium for activation and then added into a container containing a reaction buffer solution;
step (2) adding sterile FeCl into the container in the step (1) 3 Standing the solution, adding sterile Na 2 S 2 O 3 A solution;
step (3) a reference electrode, a working electrode and a counter electrode are arranged in the container to form a three-electrode system, and the working electrode is arranged in the container before synthesis of heterozygous cells, so that a biological film is formed on the surface of the working electrode while the heterozygous cells are synthesized, and the working electrode is directly used after the biological film is formed or is transferred to a new container for use in an anaerobic environment;
step (4) under the synthesis conditions that the synthesis time is more than 5h and the synthesis temperature is 4-37 ℃, shewanella generates FeS nano particles with conductivity and covers the surfaces of cells to form electroactive heterozygous cells, the electroactive heterozygous cells are adsorbed on the working electrode of the step (3) to form an electroactive heterozygous cell biological film, and the three-electrode system is connected with a signal detection system to form a bioelectrochemical sensor;
and (5) loading external voltage on the working electrode of the bioelectrochemical sensor obtained in the step (4), adding an electron acceptor of Shewanella into the container, then adding a riboflavin sample, and detecting and recording a current change value.
2. The electrochemical detection method of riboflavin based on a hybrid biofilm according to claim 1, wherein said activated Shewanella in step (1) is added to the OD of the reaction buffer solution 600 The value is controlled between 0.05 and 5.
3. The electrochemical detection method of riboflavin based on a hybrid biofilm according to claim 1, wherein the experimental operations in step (1) to step (3) should be performed in an anaerobic environment, and the container is kept in an anaerobic state during both the synthesis of the hybrid cell biofilm and the detection of riboflavin.
4. The electrochemical detection method of riboflavin based on a hybrid biofilm according to claim 1, wherein said electron acceptor of shiwanella in step (5) is sodium fumarate solution.
5. The electrochemical detection method of riboflavin based on a hybrid biofilm according to claim 1, wherein in said step (5), a negative voltage is applied to the working electrode, sodium fumarate is added as an electron acceptor after equilibration, and riboflavin is added for detection after baseline equilibration.
6. A sensor for a hybrid biofilm based riboflavin electrochemical detection method according to any of claims 1-5, wherein said sensor comprises a signal detection system, a three electrode system and a signal generation system; the signal generating system comprises an electron acceptor of Shewanella, an electroactive hybrid cell biofilm and a reaction buffer solution.
7. The sensor of the hybrid biofilm-based electrochemical detection method of riboflavin of claim 6, wherein said electron acceptor of shiwanella is sodium fumarate solution.
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