Hybrid printed bioelectrochemical sensor and preparation method and application thereof

Abstract

The application discloses a hybrid printing bioelectrochemical sensor and a preparation method and application thereof, and belongs to the technical field of environmental monitoring and electrochemical sensing. The sensor comprises a micro silk screen printing electrode substrate and a three-dimensional biosensing layer deposited on the surface of the substrate; the biosensing layer is formed by in-situ cross-linking and solidification of a composite bio-ink, and the bio-ink comprises chitosan, alpha-cellulose, a conductive polymer, electroactive microorganisms and sodium alginate. The preparation method is as follows: the bio-ink is deposited on the electrode surface layer by layer according to a preset model and is solidified by using a 3D bio-printing technology. The application utilizes the synergistic structure of three-dimensional macroscopic channels and microscopic conductive networks, reduces the interface electron transfer resistance and substrate mass transfer resistance, improves the signal-to-noise ratio of heavy metal ion detection in water, and standardizes the manufacturing process, and is suitable for the construction of an in-situ environmental monitoring network.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and bioelectrochemical sensing technology, specifically relating to a hybrid printed bioelectrochemical sensor, its preparation method, and its application. Background Technology

[0002] With the acceleration of industrialization, heavy metal pollution in water bodies has become increasingly prominent. Heavy metal ions, due to their high toxicity, recalcitrant nature, and tendency to accumulate in organisms, pose a potential threat to ecosystems and public health. Currently, the mainstream detection methods for heavy metals in water bodies mainly rely on physicochemical analysis instruments such as atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry (ICP-MS). While these traditional offline detection technologies offer high analytical accuracy, they are costly, complex to operate and maintain, and require pretreatment of water samples such as acidification and digestion. Furthermore, physicochemical analysis only reflects the absolute physicochemical concentration of heavy metals and cannot directly assess the bioavailability and actual ecotoxicity of heavy metal pollutants in water bodies. Therefore, developing low-cost, portable, and directly biotoxic in-situ rapid sensing technologies is a crucial need in the field of environmental monitoring.

[0003] Bioelectrochemical sensors based on electroactive bacteria (EAB) have attracted much attention in recent years. These sensors utilize electrogenic microorganisms as biorecognition elements, converting the inhibitory effects of target toxic substances (such as heavy metal ions) on microbial respiratory metabolism and transmembrane electron transport pathways into real-time quantitative electrical signals, thus providing a more objective reflection of the ecotoxicological effects of pollutants. To achieve portable monitoring, researchers often use miniature screen-printed electrodes (SPEs) as the hardware substrate for these sensors.

[0004] However, combining two-dimensional planar SPEs with microbial sensing technology for use in complex water bodies faces significant physical and mass transfer limitations. The inherent two-dimensional planar structure of commercial SPEs limits their electrochemically active surface area, restricting the number of electroactive microorganisms that can effectively attach and participate in direct interfacial electron transfer. Under this constraint, the initial baseline current generated by the sensor is typically small, and the system signal-to-noise ratio (SNR) is easily affected by fluctuations in the background electrolyte of the actual water body, making it difficult to meet the requirements for rapid screening of heavy metal pollution in actual wastewater.

[0005] To overcome the limitations of micro two-dimensional interfaces, existing technologies typically employ interface modification and biofilm immobilization techniques. Constructing biofilms on electrode surfaces using traditional natural biofilm attachment or artificial drop-coating methods not only results in long film formation cycles but also often leads to uneven bacterial distribution and poor batch-to-batch reproducibility. Furthermore, exposed shallow bacteria are prone to detachment or inactivation when exposed to the shearing impact of complex water flow, affecting the long-term operational stability of the sensor.

[0006] To increase biomass loading and improve interfacial stability, existing technologies have attempted to use non-conductive polymer gels (such as sodium alginate gel) for thick-layer physical embedding modification, and even introduced conventional 3D bioprinting technology to construct three-dimensional carriers. However, a single insulating thick-layer hydrogel matrix introduces significant substrate mass transfer resistance, hindering the rapid diffusion of target heavy metal ions to the internal bioactive centers. At the same time, the insulating gel network forms an electron transport barrier between the microbial cell membrane and the underlying electrode, resulting in low electron collection efficiency, sensor response lag, and consequently reduced detection sensitivity.

[0007] In summary, existing microbial sensor fabrication processes struggle to simultaneously achieve high biomass retention, rapid substrate mass transfer, and efficient interfacial electron transport on micro-SPE substrates. In particular, conventional bio-inks face the dual challenges of insufficient structural strength and lack of conductivity in aqueous environments. Therefore, there is an urgent need to develop a novel engineered manufacturing method. By designing multi-component composite bio-inks and combining them with advanced printing processes, this method can overcome the limitations of traditional two-dimensional interfaces and conventional insulating three-dimensional hydrogels. By simultaneously reshaping the microscopic conductive network and designing the macroscopic three-dimensional configuration, a three-dimensional sensing interface with high biocompatibility, high electron transport efficiency, and low mass transfer resistance can be constructed, thereby improving the sensor's detection performance in real aquatic environments. Summary of the Invention

[0008] To address the technical problems of existing micro two-dimensional biosensors, such as limited interfacial mass transfer, high electron transport barriers, and poor device reproducibility due to manual drop-casting, this invention aims to provide a hybrid-printed bioelectrochemical sensor for heavy metal detection and its fabrication method. This sensor utilizes a specific multi-component hydrogel network in synergy with microelectrodes, significantly improving the sensitivity and stability of signal detection.

[0009] The objective of this invention can be achieved through the following technical solutions: A hybrid printed bioelectrochemical sensor includes: a two-dimensional micro-screen-printed electrode substrate, and a three-dimensional biosensing layer tightly attached to the surface of the working area of ​​the substrate; The three-dimensional biosensing layer is a three-dimensional porous gel network formed by layer-by-layer deposition and in-situ cross-linking and curing of composite bio-ink. The raw material components of the composite bio-ink include: chitosan, α-cellulose, conductive polymer, electroactive microorganisms, and sodium alginate; wherein, the sodium alginate is cross-linked to form a hydrogel substrate framework, the chitosan and α-cellulose are interspersed and dispersed in the hydrogel substrate framework to provide structural support, and the conductive polymer constructs a microscopic conductive network inside the framework and contacts the outer membrane of the electroactive microorganisms.

[0010] As a preferred technical solution, the conductive polymer is poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid (PEDOT:PSS); the electroactive microorganism is Shewanella oneidensis MR-1.

[0011] As a preferred technical solution, the concentration ratio of each component in the preparation of the composite bio-ink is as follows: chitosan concentration is 90~110 g / L, α-cellulose concentration is 35~45 g / L, the volume percentage of conductive polymer PEDOT:PSS dispersion is 5~15%, and sodium alginate concentration is 60~80 g / L; the electroactive microbial cell density (OD) in the composite bio-ink before curing is... 600 The value is 3.0~4.0.

[0012] This invention also provides a method for preparing the above-mentioned hybrid printed bioelectrochemical sensor, comprising the following steps: (1) Provide micro screen-printed electrodes and perform surface cleaning pretreatment to serve as a two-dimensional conductive substrate for the sensor; (2) Chitosan, α-cellulose and PEDOT:PSS conductive polymer dispersion were mixed in an aqueous solvent and ultrasonically dispersed evenly. Then, a suspension of Shewanella onychomycosis in the logarithmic growth phase was inoculated and sodium alginate was slowly added under stirring to obtain a uniformly mixed composite bio-ink. (3) Using a 3D bioprinting device, the composite bio-ink obtained in step (2) is deposited layer by layer on the working electrode surface of the microelectrode obtained in step (1) according to a preset three-dimensional model by pneumatic extrusion. (4) Spray an aqueous solution containing a crosslinking agent onto the electrode surface after deposition to perform in-situ ionic crosslinking, so that sodium alginate is cured into a gel. After washing with a buffer solution to remove free components, the sensor is obtained.

[0013] As a preferred technical solution, the process parameters for 3D bioprinting in step (3) are: extrusion pressure of 30~80 PSI, printing speed of 5~15 mm / s, and nozzle inner diameter of 0.2~0.8 mm; more preferably, the extrusion pressure is 55~65 PSI and the printing speed is 8~10 mm / s.

[0014] As a preferred technical solution, the aqueous solution of the crosslinking agent in step (4) is a calcium chloride (CaCl2) aqueous solution with a mass concentration of 30~70 g / L; more preferably, the concentration is 45~55 g / L and the crosslinking curing time is 0.5~5 minutes.

[0015] This invention also provides the application of the aforementioned hybrid printed bioelectrochemical sensor in the rapid screening and quantitative detection of heavy metal pollutants in water.

[0016] As a preferred technical solution, the heavy metal pollutants include Cr 6+ Cu 2+ Ni 2+ or Pb 2+ One or more combinations thereof.

[0017] The beneficial effects of this invention are: Breaking mass transfer limitations and enhancing reaction kinetics: This invention utilizes 3D bioprinting technology to construct a macroscopic three-dimensional structure with controllable morphology and self-supporting capabilities. This architecture, through its three-dimensional layered structure and the microporous network within the ink, provides rapid internal diffusion channels for the substrate (target heavy metal ions), completely overcoming the significant mass transfer resistance inherent in traditional two-dimensional bases and dense bulk hydrogel embedding methods, thereby accelerating the sensor's response speed.

[0018] 2. Reshaping the microscopic conductive network and overcoming the interfacial electron transfer barrier: The composite system of this invention deeply interpenetrates the PEDOT:PSS conductive network with a composite macromolecular framework composed of sodium alginate / chitosan / cellulose. PEDOT:PSS forms a continuous electron highway inside the gel, directly bridging the cytochrome of the bacterial outer membrane with the underlying SPE solid electrode, greatly reducing the electron transfer barrier and enabling the efficient collection of originally weak biocurrent signals.

[0019] 3. Balancing high biomass loading and microecological stability: The introduction of chitosan and α-cellulose significantly enhances the mechanical strength and resistance to water flow shearing of the hydrogel; at the same time, this three-dimensional gel network acts as an ideal physical isolation layer (leaking rate <10%), maintaining high hydration capacity while stably anchoring a large number of microorganisms at the electrode interface, greatly extending the service life of the sensor in real complex water bodies.

[0020] 4. Excellent signal-to-noise ratio and extremely low detection limit: Thanks to the dual optimization of mass transfer and electron transport, the baseline current of the sensor in the background solution is significantly improved, and the system signal-to-noise ratio (SNR) is significantly enhanced. Its effect on Cr... 6+ Cu 2+ Ni 2+ and Pb2+ The limits of detection (LODs) were as low as 0.68, 1.35, 13.59, and 13.27 mg / L, respectively, and were effective against common background ions (such as Na+). + , K + Ca 2+ (etc.) It has excellent anti-interference ability and fully meets the needs of early warning of heavy metals in in-situ water environment. Attached Figure Description

[0021] The invention will now be further described with reference to the accompanying drawings.

[0022] Figure 1 This is a schematic diagram of the fabrication process and three-dimensional structure of the hybrid printed bioelectrochemical sensor provided in this embodiment of the invention.

[0023] Figure 2 This is a scanning electron microscope (SEM) image of the internal microstructure of the sensing layer prepared when the volume fraction of the conductive polymer is 10% in Example 3 of the present invention.

[0024] Figure 3 These are electrochemical performance spectra of sensors with different amounts of conductive polymer added, prepared in Example 3 of this invention; wherein, Figure 3 A is the cyclic voltammetry (CV) curve. Figure 3 B is the Nyquist electrochemical impedance spectroscopy (EIS) spectrum.

[0025] Figure 4 The sensor prepared in Example 2 of this invention is used to detect different concentrations of target heavy metal ions (Cr). 6+ Cu 2+ Ni 2+ and Pb 2+ The current drop response and linear fitting relationship are shown in the figure.

[0026] Figure 5 This is a current response curve of the sensor prepared in Example 2 of the present invention under common coexisting ion interference conditions, used to verify the anti-interference performance of the sensor.

[0027] Figure 6 This is a graph showing the physical stability of the composite biohydrogels prepared in Example 3 of this invention with different amounts of conductive polymer added over 7 days; wherein, Figure 6 A shows the results of the bacterial leakage test. Figure 6 B shows the results of the swelling rate test.

[0028] Figure 7 This is a bar chart comparing the signal-to-noise ratio (SNR) of the sensor (experimental group) prepared in Example 2 of this invention, the traditional two-dimensional sensor (control group), and the environmental biosensor in the reference.

[0029] Figure 8 This is a comparison chart of the spiked recovery rate of the sensor prepared in Example 2 of the present invention in deionized water and actual environmental water samples for the target heavy metal.

[0030] Figure 9 These are laser confocal scanning microscope (CLSM) images of composite biohydrogels with different amounts of conductive polymer added, provided in Example 3 of the present invention, after being stained with propidium iodide (PI); wherein, ae correspond to components with PEDOT:PSS addition amounts of 0, 2%, 5%, 10% and 20% v / v, respectively. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Preparation of multi-component electroactive polysaccharide-based conductive composite bio-ink This embodiment provides a method for preparing composite bio-ink, specifically including the following steps: (1) Preparation of bacterial suspension: Luria-Bertani (LB) liquid medium was prepared, and Shewanella oneidensis MR-1 was inoculated into it and cultured to the logarithmic growth phase under aerobic conditions at 30 °C and 150 rpm. Cells were then collected by centrifugation, washed and resuspended with inorganic salt medium, and the cell density OD of the bacterial suspension was adjusted. 600 The concentration was 20, which was used as a high-concentration bacterial stock solution for later use.

[0033] (2) Preparation of precursor solution: Measure 3.5 mL of inorganic salt culture medium, and add 0.5 g of chitosan (CS) powder, 0.2 g of α-cellulose, and 0.5 mL of PEDOT:PSS conductive polymer dispersion with a solid content of 1.1 wt%. Disperse thoroughly and evenly by mechanical stirring at room temperature to allow the polysaccharide backbone and conductive network to initially interpenetrate, resulting in a high-viscosity conductive composite precursor solution.

[0034] (3) Bacterial suspension remodeling and in situ transient complexation: Take 1.0 mL of the Shewanella oneidensis MR-1 bacterial suspension collected in step (1) and slowly add it dropwise to the precursor solution in step (2), then gently mix. This step utilizes the buffering capacity of the bacterial suspension to adjust the pH and ionic strength of the microenvironment of the system, while simultaneously reducing the final OD of the bacteria in the system. 600 The concentration was controlled at 4.0. Subsequently, under sterile conditions and with vigorous shaking or high-shear stirring, 0.35 g of sodium alginate powder was slowly added to the system. Under the synergistic effect of dynamic shear and steric hindrance of α-cellulose, the positively charged amino groups of chitosan and the negatively charged carboxyl groups of sodium alginate underwent moderate polyelectrolyte complexation. The system was continuously stirred at 25 °C for 5 min, resulting in a sol-gel phase transition, ultimately forming a homogeneous pre-crosslinked hydrogel without macroscopic phase separation or local precipitation, i.e., a composite bio-ink with excellent shear-thinning properties, which was placed in a sterile syringe for later use.

[0035] Example 2: Construction of a hybrid printed bioelectrochemical sensor (3D-Bio-SPE) This embodiment, based on the composite bio-ink prepared in Example 1, combines screen printing and 3D bioprinting technologies to construct an integrated sensing interface: (1) Substrate preparation: Commercially available micro screen-printed electrodes (SPEs) are provided as the bottom conductive substrate of the sensor. Before use, the working area is subjected to routine electrochemical cleaning and drying.

[0036] (2) Three-dimensional deposition: Under aseptic conditions, the bio-ink prepared in Example 1 was pre-loaded into a sterile syringe equipped with a standard 20G bioprinting nozzle. Using an extrusion 3D bioprinting system, at room temperature (20~25 °C), with an extrusion pressure of 60 PSI and a printing speed of 9 mm / s, the bio-ink was precisely deposited layer by layer onto the working electrode surface of the SPE according to the pre-designed three-dimensional model. In a preferred embodiment of the present invention (e.g.) Figure 1 and Figure 2 As shown, the three-dimensional biosensing layer adopts a solid disk-shaped structure, which can maximize the coverage of bio-ink in the working area of ​​the screen-printed electrode, thereby obtaining a higher response current baseline and signal-to-noise ratio; in other optional embodiments, the sensing layer can also be printed as a porous mesh or other geometric contour structure according to actual detection needs.

[0037] (3) In-situ curing: Immediately after printing and deposition, a 50 g / L calcium chloride aqueous solution is atomized and sprayed onto the electrode surface for in-situ ionic crosslinking for 1 min. Sodium alginate molecules in the system rapidly react with Ca... 2+A cross-linked structure is formed and solidified into a three-dimensional hydrogel network. Finally, the functionalized electrode surface is rinsed with sterile buffer to remove residual free cross-linking agent, thus obtaining the 3D-Bio-SPE.

[0038] Example 3: Comparison of electrochemical properties of the sensing interface with different concentrations of conductive polymers This embodiment included a gradient concentration control experiment: following the methods of Examples 1 and 2, only the volume percentage of PEDOT:PSS in the bio-ink was changed to prepare 3D-Bio-SPEs containing 0%, 2%, 5%, 10%, and 20% v / v PEDOT:PSS, respectively. The electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

[0039] Test results (e.g.) Figure 3 As shown in the figure, the characteristic oxidation peak current at +0.10V significantly increased from 27.0 μA to 166.0 μA as the PEDOT:PSS concentration in the ink increased from 0% to 10%. EIS testing results showed that the 10% v / v component had the lowest charge transfer resistance (Rct), demonstrating that the microscopic conductive network significantly reduced the electron transport barrier. Combined with live / dead bacteria staining (CLSM) results, significant cytotoxicity was observed when the PEDOT:PSS concentration reached 20%. These data indicate that 10% v / v is the optimal ratio that balances electrochemical signal enhancement and biocompatibility.

[0040] Example 4: Quantitative Detection of Heavy Metals Using a Hybrid Printed Bioelectrochemical Sensor This embodiment performs quantitative testing of heavy metal toxicity on the optimally formulated 3D-Bio-SPE prepared in Example 2: (1) A standard three-electrode test system containing an Ag / AgCl reference electrode was constructed. The test substrate contained 15 mM sodium lactate as an electron donor to maintain the basal metabolism of bacteria. A constant polarization potential of 0.2 V (vs. Ag / AgCl) was applied to the 3D-Bio-SPE through an electrochemical workstation, and the dynamic changes of biocurrent were captured in real time by chronoamperometry (it).

[0041] (2) After the system has run for approximately 2000 seconds and the baseline current has reached a steady state, the target heavy metal toxicant (Cr) at different concentration gradients (0~200 mg / L) is quantitatively introduced into the test system. 6+ Cu 2+ Ni 2+ and Pb 2+ ).

[0042] (3) After the target substance is fully exposed for about 4000 s, the steady-state decay of the biocurrent is accurately recorded to quantify its toxic inhibitory effect.

[0043] Test results (e.g.) Figure 4 As shown in the figure, within the steady-state response range of 900–1000 s after exposure, the current decrease of the four heavy metals all exhibited excellent linear correlation with their concentrations (R0). 2 > 0.99). Calculated according to the IUPAC guidelines (3σ / S), this sensor is sensitive to Cr. 6+ Cu 2+ Ni 2+ and Pb 2+ The limits of detection (LOD) were as low as 0.68, 1.35, 13.59, and 13.27 mg / L, respectively. Meanwhile, thanks to the synergy of the three-dimensional mass transfer and conductive network, the signal-to-noise ratio (SNR) of the sensor in this embodiment was significantly improved compared to the two-dimensional control group.

[0044] Example 5: Evaluation of the sensor's anti-interference performance in complex water matrix This embodiment included a coexisting ion interference test: following the test platform and polarization conditions of Example 4, after the baseline current stabilized, common background interfering ions found in water were introduced into the system, including K+ at a concentration of 1.0 g / L. + NO3 - NH4 + Cl - SO4 2- Ions, and Ca at a concentration of 50 mg / L 2+ Mg 2+ ion.

[0045] Test results (e.g.) Figure 5 As shown in the figure, the system baseline current fluctuates very little after the introduction of the aforementioned high concentration of background ions, and no significant false positive or false negative signals are generated. This result confirms that the multi-component composite hydrogel network of the present invention not only has a nanofiltration effect, but also has a strong buffering effect on sudden changes in water salinity, thus endowing the sensor with excellent resistance to electrolyte interference.

[0046] Example 6: Verification of sensor recovery rate in actual environmental water samples This embodiment applies the sensor to the heavy metal spike recovery rate test of actual natural water samples: (1) Water sample collection and pretreatment: Water from a natural lake in Hefei City was collected and filtered using a 0.45 μm water system filter membrane to remove large particulate suspended matter from the water sample, and the treated actual lake water matrix was prepared for use.

[0047] (2) Deionized water control group test: An inorganic salt culture medium was prepared using deionized water, and sodium lactate with a final concentration of 15 mM was added as an electron donor for electrogenic metabolism to construct the control test solution. The sensor prepared in this invention was placed in the deionized water test system. A constant polarization potential of 0.2 V (vs. Ag / AgCl) was applied, and after the system current stabilized to a flat baseline, a standard concentration of the target heavy metal ions (Cr) was quantitatively added. 6+ Cu 2+ Ni 2+ and Pb 2+ (The spiked concentration was set at 200 mg / L). After sufficient exposure, the steady-state decay of the current was recorded.

[0048] (3) Add sodium lactate quantitatively to the actual lake water matrix treated in step (1) to achieve a final concentration of 15 mM, so as to unify the substrate concentration and replenish the carbon source lacking in the environmental water sample. Place the sensor in the actual lake water system treated in step (1). Apply the same constant polarization potential, and after the sensor reaches a flat baseline in the complex matrix, add the same amount and concentration of target heavy metal ions as in step (2). After sufficient exposure, record the steady-state decay of its current.

[0049] (4) Data Analysis and Calculation: Based on the standard curve fitted in Example 4, the actual detected concentrations of the deionized water group and the actual lake water group were calculated by reverse calculation. The formula for calculating the spiked recovery rate is: (actual detected concentration / theoretical spiked concentration) × 100%.

[0050] Test results (e.g.) Figures 7-9 As shown in the figure, the spiked recoveries of each target heavy metal in the actual lake water matrix are consistently between 90% and 120%. This result strongly demonstrates that the sensor of this invention can overcome the complex interference of real natural water matrix and meet the practical application requirements of in-situ online environmental monitoring.

[0051] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A hybrid printed bioelectrochemical sensor, characterized in that, include: Two-dimensional micro-screen printed electrode substrate; And, a three-dimensional biosensing layer attached to the surface of the working area of ​​the substrate; The three-dimensional biosensing layer is a three-dimensional porous gel network formed by layer-by-layer deposition and in-situ cross-linking and curing of composite bio-ink. The composite bio-ink comprises the following raw material components: chitosan, α-cellulose, conductive polymer, electroactive microorganisms, and sodium alginate; In this structure, sodium alginate is cross-linked to form a hydrogel substrate framework, chitosan and α-cellulose are interspersed and dispersed in the hydrogel substrate framework to provide structural support, and the conductive polymer constructs a microscopic conductive network inside the framework and contacts the outer membrane of the electroactive microorganism.

2. The hybrid printed bioelectrochemical sensor according to claim 1, characterized in that, The conductive polymer is poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid.

3. The hybrid printed bioelectrochemical sensor according to claim 1, characterized in that, The electroactive microorganism is Oneida Shiwab.

4. The hybrid printed bioelectrochemical sensor according to claim 1, characterized in that, The concentrations or contents of each component in the composite bio-ink are as follows: The concentration of chitosan is 90~110 g / L; The concentration of α-cellulose is 35~45 g / L; The volume percentage of the conductive polymer dispersion is 5-15%; The concentration of sodium alginate is 60~80 g / L; Before curing, the electroactive microbial cell density OD 600 The value is 3.0~4.

0.

5. A method for preparing a hybrid printed bioelectrochemical sensor according to any one of claims 1-4, characterized in that, Includes the following steps: The micro screen-printed electrodes are pretreated by surface cleaning to serve as a two-dimensional conductive substrate; Chitosan, α-cellulose and conductive polymer dispersions were mixed in an aqueous solvent, ultrasonically dispersed, and then inoculated with an electroactive microbial suspension in the logarithmic growth phase. Sodium alginate was then added under stirring conditions to obtain a composite bio-ink. Using a 3D bioprinting device, the composite bio-ink is deposited layer by layer on the surface of the working electrode by pneumatic extrusion. After deposition, an aqueous solution containing a crosslinking agent is sprayed onto the electrode surface to perform in-situ ionic crosslinking, thereby solidifying sodium alginate. After washing, the sensor is obtained.

6. The method for fabricating a hybrid printed bioelectrochemical sensor according to claim 5, characterized in that, The process parameters for 3D bioprinting in step (3) are: extrusion pressure of 30~80 PSI, printing speed of 5~15 mm / s, and nozzle inner diameter of 0.2~0.8 mm.

7. The method for fabricating a hybrid printed bioelectrochemical sensor according to claim 5, characterized in that, The aqueous solution of the crosslinking agent mentioned in step (4) is a calcium chloride aqueous solution with a mass concentration of 30~70 g / L.

8. The method for fabricating a hybrid printed bioelectrochemical sensor according to claim 5, characterized in that, In step (4), the cross-linking curing time is 0.5 to 5 minutes.

9. The application of the hybrid printed bioelectrochemical sensor according to any one of claims 1 to 4 or the hybrid printed bioelectrochemical sensor prepared by the preparation method according to any one of claims 5 to 8 in the rapid screening and quantitative detection of heavy metal pollutants in water.

10. The application according to claim 9, characterized in that, The heavy metal pollutants include Cr 6+ Cu 2+ Ni 2+ or Pb 2+ One or more combinations thereof.

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