A method for detecting the extracellular electron transfer current of a single exoelectrogenic bacterium

Abstract

The present invention belongs to the technical field of microbial imaging, and particularly relates to a method for detecting the extracellular electron transfer current of a single electrogenic bacterium. Nano mineral particles are used as probes to allow a single bacterium to bind to the probes. The etching and dissolution process of the probes by the single bacteria is observed through a surface plasmon resonance imaging method. The quantitative relationship between the optical image intensity and the volume of a single particle is used to quantitatively analyze the extracellular electron transfer of the single bacteria. The method has the characteristics of being label-free, interference-free, and highly sensitive, and can remain stable within a continuous observation time of 5 to 6 hours. Compared with the prior art, the method of the present invention can quantitatively obtain the extracellular electron transfer level at the interface of a single bacterium and a natural mineral in situ and without interference, study the native respiratory metabolic behavior of a single bacterium, and provide an effective tool for the performance development of biofuel cells based on electrogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial imaging, and in particular relates to a method for detecting the extracellular electron transfer current of a single electrogenic bacterium. Background Art

[0002] Shewanella is a dissimilatory metal-reducing bacterium that can transfer electrons generated by the respiratory metabolism of organic substrates in cells to the external environment through the extracellular electron transfer (EET) pathway. Electron acceptors are usually mineral species such as Fe(III), Mn(IV), and Pd(II), which accept electrons from bacteria and undergo reduction reactions. Microbial fuel cells use the extracellular electron transfer pathway of Shewanella to convert biochemical energy into electrical energy. Detecting the extracellular electron transfer ability of Shewanella is of guiding significance for the performance optimization of microbial fuel cells.

[0003] Surface plasmon resonance (SPR) technology is an analytical method based on physical optical phenomena. It has the advantages of being label-free, real-time, sensitive, and rapid, and has been widely used in the field of life analysis. The commercial SPR instrument (Biacore) that uses SPR technology to detect interactions between biomolecules is an example. By assembling the SPR detection device with a microscope and recording the image of the reflected light from the sensing interface through the CCD, a microscopic imaging system can be constructed. The scattering of a single nanoparticle can be used to enhance the SPR signal, and the single particle on the microscopic interface can be imaged and analyzed to obtain richer spatial information; and the unique action properties of a single particle can be distinguished from a large amount of averaged information. The surface plasmon resonance imaging method, supplemented by a high numerical aperture objective, has a sensitive response to small changes in the volume of particles inside the evanescent field, and the optical image intensity has a quantitative relationship with the volume of a single particle.

[0004] Most existing studies on bacterial respiratory metabolic dynamics focus on measuring the average level of bacterial electricity production, and are unable to accurately detect the extracellular electron transfer capacity of individual bacteria. At the level of single bacteria, existing ultramicroelectrode methods can measure the electrochemical current intensity of single bacteria at the electrode interface, but cannot quantitatively obtain the level of extracellular electron transfer at the interface between single bacteria and natural minerals in situ and without interference, making it difficult to study the native respiratory metabolic behavior of single bacteria. Summary of the invention

[0005] The present invention solves the above technical problems existing in the prior art and provides a method for quantitatively detecting the extracellular electron transfer current of a single electrogenic bacterium; using nano-mineral particles as probes, a single bacterium is combined with the probe, and the etching and dissolution process of the probe by a single bacterium is observed by surface plasmon resonance imaging. The quantitative relationship between the optical image intensity and the volume of a single particle is used to quantitatively analyze the extracellular electron transfer of a single bacterium.

[0006] The technical solution of the present invention is as follows:

[0007] A method for detecting the extracellular electron transfer current of a single exoelectrogenic bacterium comprises the following steps:

[0008] (1) Preparation of an electrogenic bacteria dispersion: dispersing the electrogenic bacteria in a buffer solution; the electrogenic bacteria are dissimilatory metal-reducing bacteria;

[0009] (2) Preparation of nano-mineral probes: dispersing mineral nanoparticles in PDDA solution;

[0010] (3) Assembly of a single electrogenic bacterium and a nano-mineral probe: adding the nano-mineral probe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1), and after culturing, obtaining a complex of a single electrogenic bacterium and the nano-mineral probe;

[0011] (4) Detection of extracellular electron transfer current: Surface plasmon resonance microscopy was used to observe the change in the intensity of the plasmon optical image of the complex of a single electrogenic bacterium and a nano-mineral probe prepared in step (3) ΔI SPR , and thus calculate the total number of electron transfers N during the observation time e :

[0012] N e =-kρΔI SPR N A / M r

[0013] in

[0014] N e represents the total number of electron transfers during the observation time;

[0015] ΔI SPR represents the change in the observed intensity of the plasmon optical image;

[0016] k represents the volume change ΔV of the nano-mineral probe and the intensity change ΔI of the plasmon optical image SPR The proportionality coefficient of the linear relationship between them, ΔV=kΔI SPR ;

[0017] ρ and M r represents the density and relative molecular mass of the nano-mineral probe;

[0018] N A represents Avogadro's constant.

[0019] When a single complex of electrogenic bacteria and nano-mineral probes is deposited on the imaging surface of a gold film substrate, under anaerobic conditions, the electrogenic bacteria transfer the electrons generated by substrate respiratory metabolism to the nano-mineral probes through the cytochrome complex on the outer membrane, generating bioelectric current at the interface between bacteria and minerals. The nano-mineral probes are reduced and dissolved, and their volume decreases, which in turn causes a decrease in the intensity of the plasmon optical image.

[0020] Preferably, the electrogenic bacteria are Shewanella, and the mineral nanoparticles are Fe3O4 nanoparticles or Fe2O3 nanoparticles.

[0021] Preferably, the specific steps of step (1) are:

[0022] Shewanella oneidensis MR-1 was inoculated into LB medium and cultured anaerobically on a shaker; the cultured bacteria were collected by centrifugation, washed three times with buffer and resuspended to remove the residual LB medium.

[0023] More preferably, in step (1), the bacterial culture conditions are 150 rpm, 37° C., 24 h; the centrifugation parameters for centrifugal collection are 5000 rpm, 4 min; and the formula of the buffer solution is 85.5 mM NaCl, 1 mM MgSO4, and 10 mM HEPES.

[0024] Preferably, the specific steps of step (2) are: ultrasonically dispersing the mineral nanoparticles in the PDDA solution, and the amount of the mineral nanoparticles added to the PDDA solution is: adding 5 mg of mineral nanoparticles to every 30 mL of 1 mg / mL PDDA solution.

[0025] Preferably, the particle size of the mineral nanoparticles is 20 nm to 150 nm.

[0026] Preferably, the PDDA solution contains 250 mM NaCl, which is conducive to the spontaneous assembly of polymer chains on the surface of Fe3O4 nanoparticles to form a random conformation.

[0027] Preferably, in step (2), the ultrasonication time is 10 min, the mixture is allowed to stand for 15 min after ultrasonication, and then it is redispersed in water after washing.

[0028] Preferably, the specific steps of step (2) are: dispersing Fe3O4 nanoparticles in a PDDA solution, allowing to stand after ultrasonication, and rinsing three times with secondary water to remove PDDA not bound to the nanoparticles; the obtained PDDA-modified Fe3O4 nanoparticles are redispersed in secondary water and can be stored at room temperature for 2 weeks.

[0029] Preferably, the specific steps of step (3) are: adding the mineral nanoprobe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1), placing it in a shaking table for anaerobic culture, so that the positively charged nanoparticles and the negatively charged bacteria are adsorbed and combined by electrostatic action, and the excess mineral nanoprobe can be removed by magnetic separation to obtain a complex of a single electrogenic bacteria and a nano mineral probe.

[0030] More preferably, in step (3), the volume ratio of the mineral nanoprobe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1) is 0.5 mL:15 mL, and the concentration OD of the electrogenic bacteria dispersion is 600 It is 0.8~1.

[0031] More preferably, in step (3), the anaerobic culture conditions are 150 rpm, 37° C., 30 min.

[0032] Preferably, the specific steps of step (4) are: dispersing the complex of a single electrogenic bacteria and a nano-mineral probe in a deoxygenated working solution, diluting it, and depositing it on the imaging surface of a gold film substrate, and using a surface plasmon resonance microscope to observe changes in the plasmon optical image intensity of the complex of a single electrogenic bacteria and a nano-mineral probe.

[0033] More preferably, in step (4), the formula of the working solution is 85.5 mM NaCl, 1 mM MgSO4, 10 mM HEPES, and 20 mM sodium lactate. Sodium lactate is an electron donor necessary for bacterial respiration.

[0034] More preferably, in step (4), the working solution is deoxygenated by passing nitrogen gas into the solution for 30 minutes.

[0035] More preferably, in step (4), the dilution factor is 500 to 1000 times.

[0036] More preferably, in the step (4), the preparation method of the imaging surface of the gold film substrate is: on the surface of a BK7 glass sheet with a thickness of 0.17 mm, 2 nm Cr is first evaporated as an adhesive substrate, and then 47 nm Au is evaporated as a substrate for exciting surface plasmon resonance by an electron beam evaporation method; before use, the gold film is pre-burned with a hydrogen flame to remove organic pollutants that may exist on the surface.

[0037] Preferably, the sample cell of the surface plasmon resonance microscope is a sealed sample cell, which is adhered to a gold film substrate, and an oxygen-free environment is maintained in the sample cell.

[0038] Compared with the prior art, the advantages of the present invention are as follows:

[0039] The present invention quantitatively detects the extracellular electron transfer current of a single Shewanella, and can simultaneously detect the extracellular electron transfer currents of multiple single bacteria in a visual field, thereby obtaining the kinetic information of electron transfer.

[0040] The present invention is based on surface plasmon resonance imaging, which is a label-free method. Compared with the current ultramicroelectrode technology for detecting the electrochemical current intensity of single bacteria on the electrode interface, it can quantitatively obtain the level of extracellular electron transfer at the interface between single bacteria and natural minerals in situ and without interference, and study the native respiratory metabolic behavior of single bacteria, providing an effective tool for the performance development of biofuel cells based on electricity-producing bacteria.

[0041] The method of the present invention is label-free, interference-free and highly sensitive, and can remain stable during a continuous observation time of 5 to 6 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is an example of a scanning electron microscope image of a single Shewanella and nano-mineral probe complex in Example 1.

[0043] Figure 2 Schematic diagram of the detection method of the present invention.

[0044] Figure 3 The plasmon optical image of a single Shewanella and nano-mineral probe complex in Example 1 and the corresponding scanning electron microscope characterization image.

[0045] Figure 4 The single Shewanella of Example 1 was etched into a nano-mineral probe within 10 min, causing a change in plasmon intensity.

[0046] Figure 5 A is the three Shewanella in Example 2 etching the nano-mineral probe within 300 min, causing the plasmon intensity to decrease; Figure 5 B is the number of electron transfers of the three Shewanella bacteria on the nano-mineral probe interface in Example 2 when the three Shewanella bacteria etched the nano-mineral probe within 300 min; Figure 5 CE is the extracellular electron transfer current of the three Shewanella in Example 2 etching the nano-mineral probe within 300 minutes, the three single bacteria at the nano-mineral probe interface, and the corresponding electron transfer kinetic information.

[0047] Figure 6 A is a representative scanning electron microscope characterization image of a single Shewanella and Fe2O3 nano-mineral probe complex in Example 3; Figure 6 B is the three Shewanella in Example 3 that etched the Fe2O3 nanomineral probe within 300 minutes, the extracellular electron transfer current of the three single bacteria on the nanomineral probe interface, and the corresponding electron transfer kinetic information. DETAILED DESCRIPTION

[0048] The surface plasmon resonance microscope structure used in the present invention is disclosed in Shaopeng Wang, Xinping Huang, Xiaonan Shan, Kyle J. Foley, and Nongjian Tao, Anal. Chem. 2010, 82 (3), 935-941; Yimin Fang, Wei Wang, Xiang Wo, Yashuang Luo, Shaowei Yin, Yixian Wang, Xiaonan Shan and Nongjian Tao, J. Am. Chem. Soc. 2014, 136 (36), 12584-12587, and includes a light source emission module, a surface plasmon resonance excitation module, a focus control module, and an imaging collection module. The light source emission module mainly includes a monochromatic light source and a spatial position adjustment component for adjusting the direction of the light beam. The surface plasmon resonance excitation module is based on an inverted microscopic imaging mode and mainly includes a p-polarizer, a microscope objective lens, an imaging surface of a gold film substrate, and a sealed sample pool. The focus control module includes an electric microscope focus controller. The imaging collection module mainly includes an image sensor for collecting imaging signals.

[0049] Embodiment 1:

[0050] Using 150nm Fe3O4 nanoparticles as nanomineral probes, the intensity of the plasmon optical image of a single Shewanella bacterium was detected within 10 minutes, and the extracellular electron transfer caused the decrease.

[0051] Shewanella was inoculated into LB medium and placed in a shaker (150 rpm, 37°C) for 24 h of anaerobically culture. The cultured bacteria were collected by centrifugation at 5000 rpm for 4 min, washed three times with buffer (85.5 mM NaCl, 1 mM MgSO4, 10 mM HEPES) and resuspended. The OD 6000.8~1. Take 5mg of Fe3O4 nanoparticles with a particle size of 150nm, disperse them in 30mL of PDDA solution with a concentration of 1mg / mL (containing 250mM NaCl), ultrasonicate for 10min, let it stand for 15min, rinse it three times with secondary water, and redisperse it in 1mL of secondary water. Add 0.5mL of PDDA-modified nanoparticle dispersion to 15mL of washed resuspended bacterial solution and place it in a shaker (150rpm, 37℃) for anaerobic culture for 30min. Excess nanoparticles are removed by magnetic separation. The complex of a single Shewanella and nanomineral probe is dispersed in a deoxygenated working solution (85.5mM NaCl, 1mM MgSO4, 10mM HEPES, 20mM sodium lactate), diluted 1000 times, and deposited on the imaging surface of the gold film substrate. The preparation method of the imaging surface of the gold film substrate is as follows: on the surface of a 0.17 mm thick BK7 glass sheet, 2 nm Cr is first evaporated as an adhesive substrate, and then 47 nm Au is evaporated as a substrate for exciting surface plasmon resonance by electron beam evaporation. The gold film is pre-burned with a hydrogen flame before use.

[0052] Figure 1 This is a scanning electron microscope image of a single Shewanella and nano-mineral probe complex. Here are images of 6 single bacteria. A single Fe3O4 nanoparticle with a particle size of 150nm is adsorbed on the surface of a single Shewanella, confirming the successful construction of the complex.

[0053] like Figure 2 , this embodiment uses a surface plasmon resonance imaging system. The light source is a superluminescent diode with a central wavelength of 670nm. The inverted microscope objective is a high numerical aperture objective (60×, NA=1.49), which is used with lenticular oil that matches the refractive index of the glass sheet. The p-polarized incident light beam enters the substrate at a specific angle to excite surface plasmon resonance. The sealed sample pool is firmly adhered to the imaging surface of the gold film substrate with good airtightness. The sample pool is filled with nitrogen to maintain an oxygen-free environment. The sample temperature is maintained at 25°C. The imaging collection module uses a fast CCD camera to record the intensity of the plasmon optical image. When a complex of a single Shewanella and a nanomineral probe is deposited on the imaging surface of the gold film substrate, the plasmon optical image corresponding to the same bacterium is consistent with the scanning electron microscope characterization image as shown in the figure. Figure 3 shown.

[0054] Under anaerobic conditions, Shewanella transfers electrons generated by the respiratory metabolism of the sodium lactate substrate to the nano-mineral probe through the cytochrome complex on the outer membrane, generating bioelectric current at the interface between the bacteria and the mineral. The nano-mineral probe is reduced and dissolved, and its volume decreases, which in turn causes a decrease in the intensity of the plasmon optical image. Within 10 minutes, a single Shewanella etched the nano-mineral probe and obtained a sequence of plasmon optical images. The change in the plasmon intensity of a single bacterium is shown in Figure 2. Figure 4 As shown; the inset shows the image at the corresponding time point minus the first image, and the local dark pattern indicates a decrease in plasmon intensity.

[0055] Embodiment 2:

[0056] Same as Example 1, except that a focus control module including an electric microscope focus controller is used to avoid focus drift and maintain the long-term stability of the imaging system. Within 300 minutes, three Shewanella etched nanomineral probes were simultaneously detected in the same field of view to obtain a plasmon optical image sequence. The last image minus the first image is as follows Figure 5 As shown in A, local dark patterns of varying degrees indicate varying degrees of decrease in plasmon intensity.

[0057] The decrease in the intensity of the plasmon optical image is calculated by the following relationship to generate the electron transfer number:

[0058] N e =-kρΔI SPR N A / M r

[0059] in

[0060] N e represents the total number of electron transfers during the observation time;

[0061] ΔI SPR represents the change in the observed intensity of the plasmon optical image;

[0062] k represents the volume change ΔV of the nano-mineral probe and the intensity change ΔI of the plasmon optical image SPR The proportionality coefficient of the linear relationship between them, ΔV=KΔI SPR ;

[0063] ρ and M r represents the density and relative molecular mass of the nano-mineral probe;

[0064] N A represents Avogadro's constant.

[0065] Within 300 min, the number of electron transfers of the three bacteria on the nano-mineral probe interface was as follows: Figure 5 B. By multiplying the number of electron transfers by the charge carried by a single electron (elementary charge), the extracellular electron transfer currents of the three single bacteria on the nano-mineral probe interface were quantitatively calculated, and the electron transfer kinetic information of the three single bacteria was obtained at the same time, as shown in Figure 5 As shown in CE.

[0066] Embodiment 3:

[0067] Same as Example 2, except that the nano-mineral probe is a Fe2O3 nanoparticle with a particle size of 20-30 nm. A complex of a single Shewanella bacterium and the Fe2O3 nano-mineral probe is deposited on the imaging surface of the gold film substrate. A representative scanning electron microscope characterization image is shown in FIG. Figure 6 As shown in A, multiple Fe2O3 nanoparticles with a particle size of about 30nm are adsorbed on the surface of a single Shewanella. Within 300min, three Shewanella-etched Fe2O3 nanomineral probes were detected simultaneously in the same field of view. The decrease in the intensity of the plasmon optical image was calculated and the electron transfer number was generated by the following relationship:

[0068] N e =-kρΔI SPR N A / M r

[0069] in

[0070] N e represents the total number of electron transfers during the observation time;

[0071] ΔI SPR represents the change in the observed intensity of the plasmon optical image;

[0072] k represents the volume change ΔV of the nano-mineral probe and the intensity change ΔI of the plasmon optical image SPR The proportionality coefficient of the linear relationship between them, ΔV=KΔI SPR ;

[0073] ρ and M r represents the density and relative molecular mass of the nano-mineral probe;

[0074] N A represents Avogadro's constant.

[0075] Within 300 min, the electron transfer numbers of the three single bacteria on the Fe2O3 nano-mineral probe interface were obtained. The electron transfer numbers were multiplied by the charge carried by a single electron (elementary charge), and the extracellular electron transfer currents of the three single bacteria on the Fe2O3 nano-mineral probe interface were quantitatively calculated. At the same time, the electron transfer kinetic information of the three single bacteria was obtained, such as Figure 6 As shown in B.

[0076] Based on the above experiments, the surface plasmon resonance imaging method of the present invention for quantitatively detecting the extracellular electron transfer current of a single Shewanella can directly obtain the extracellular electron transfer level at the interface between a single bacterium and a natural mineral, and further obtain the kinetic information of electron transfer.

[0077] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the basis of the above embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting the extracellular electron transfer current of a single exoelectrogenic bacterium, characterized in that: The following steps are involved: (1) Preparation of electrogenic bacteria dispersion: dispersing the electrogenic bacteria in a buffer solution; The electrogenic bacteria are dissimilatory metal-reducing bacteria; (2) Preparation of nano-mineral probes: dispersing mineral nanoparticles in PDDA solution; (3) Assembly of a single electrogenic bacterium and a nano-mineral probe: adding the nano-mineral probe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1), and after culturing, obtaining a complex of a single electrogenic bacterium and the nano-mineral probe; (4) Detection of extracellular electron transfer current: Surface plasmon resonance microscopy was used to observe the change in the intensity of the plasmon optical image of the complex of a single electrogenic bacterium and a nano-mineral probe prepared in step (3) ΔI SPR , and thus calculate the total number of electron transfers N during the observation time e : N e =-kρΔI SPR N A / M r in N e represents the total number of electron transfers during the observation time; ΔI SPR represents the change in the observed intensity of the plasmon optical image; k represents the volume change ΔV of the nano-mineral probe and the intensity change ΔI of the plasmon optical image SPR The proportionality coefficient of the linear relationship between them, ΔV=kΔI SPR ; ρ and M r represents the density and relative molecular mass of the nano-mineral probe; N A represents Avogadro's constant; The electrogenic bacteria are Shewanella, and the mineral nanoparticles are Fe3O4 nanoparticles or Fe2O3 nanoparticles.

2. The method according to claim 1, characterized in that The formula of the buffer solution is 85.5 mM NaCl, 1 mM MgSO4, and 10 mM HEPES.

3. The method according to claim 1, characterized in that The specific steps of step (2) are: ultrasonically dispersing the mineral nanoparticles in the PDDA solution, and the amount of the mineral nanoparticles added to the PDDA solution is: 5 mg of the mineral nanoparticles are added to every 30 mL of PDDA solution with a concentration of 1 mg / mL.

4. The method according to claim 1, characterized in that The particle size of the mineral nanoparticles is 20nm-150nm; the PDDA solution contains 250mM NaCl.

5. The method according to claim 1, characterized in that The specific steps of step (3) are as follows: adding the mineral nanoprobe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1), placing the mixture in a shaking table for anaerobic culture, removing the excess mineral nanoprobe by magnetic separation, and obtaining a complex of a single electrogenic bacteria and a nano-mineral probe; the volume ratio of the mineral nanoprobe obtained in step (2) to the electrogenic bacteria dispersion obtained in step (1) is 0.5 mL:15 mL, and the concentration OD of the electrogenic bacteria dispersion is 0.5 mL:15 mL. 600 It is 0.8~1.

6. The method according to claim 1, characterized in that The specific steps of step (4) are: dispersing the complex of a single electrogenic bacteria and a nano-mineral probe in a deoxygenated working solution, diluting it, and depositing it on the imaging surface of a gold film substrate, and using a surface plasmon resonance microscope to observe changes in the intensity of the plasmon optical image of the complex of a single electrogenic bacteria and a nano-mineral probe.

7. The method according to claim 6, characterized in that In step (4), the formula of the working solution is 85.5 mM NaCl, 1 mM MgSO4, 10 mM HEPES, and 20 mM sodium lactate.

8. The method according to claim 6, characterized in that In the step (4), the preparation method of the imaging surface of the gold film substrate is: on the surface of a BK7 glass sheet with a thickness of 0.17 mm, 2 nm Cr is first evaporated as an adhesive substrate, and then 47 nm Au is evaporated as a substrate for exciting surface plasmon resonance by an electron beam evaporation method; before use, the gold film is pre-burned with a hydrogen flame to remove organic pollutants that may exist on the surface.

9. The method according to claim 1, characterized in that The sample cell of the surface plasmon resonance microscope is a sealed sample cell, which is adhered to a gold film substrate, and an oxygen-free environment is maintained in the sample cell.

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