Composition, bio-ink, bio-electrode material and application thereof

Abstract

The invention relates to the field of bioelectrochemistry, in particular to a composition, biological ink, a biological electrode material and application thereof. The invention provides a composition, which comprises a hydrogel and Shewanella oneidensis MR-1, and is characterized in that the hydrogel and the Shewanella oneidensis MR-1 are combined to form the composition, and the hydrogel and the Shewanella oneidensis MR-1 are combined to form the composition. The hydrogel is prepared from polyacrylamide and sodium alginate. A biological electrode material constructed by performing 3D printing on the bio-ink provided by the invention can realize low-detection-limit specific detection on 2, 4-dinitrotoluene, and the purposes of reducing mechanical damage caused by hydraulic shearing through self-repairing and improving system sensitivity and signal-to-noise ratio are achieved through design of ink components.

Description

Technical Field

[0001] The present invention relates to the field of bioelectrochemistry, and in particular to a composition, a bio-ink, a bio-electrode material and applications thereof. Background Art

[0002] Wars and conflicts have brought carcinogenic residues of explosives into the soil, groundwater and air, which can cause serious environmental pollution and health risks. Metal detectors pose a serious threat to the safety of inspectors. The use of whole-cell biosensors can avoid these shortcomings. Their low cost, renewability and portability make them more suitable for on-site monitoring. 2,4-Dinitrotoluene is the most stable and highly volatile compound in explosives and has become an effective alternative strategy for explosives detection. However, China's national standard emission limit for 2,4-Dinitrotoluene is 0.5 mg / L, and current biosensors are difficult to fully meet the detection conditions.

[0003] Bioelectrochemical system is a promising technology for monitoring unknown water samples. The electroactive bacteria Shewanella oneidensis MR-1 is environmentally sensitive. Using it as a base bacteria for engineering has the potential to reduce the detection limit of 2, 4-dinitrotoluene to a certain extent. At the same time, the booming development of 3D bioprinting, an additive manufacturing technology, has brought great convenience to the use of engineered bacteria. Bio-ink can provide bacteria with a suitable microenvironment for growth and prevent bacterial leakage. Regulating its composition can also give it conductivity, self-repair, antifreeze and other properties. Summary of the invention

[0004] In view of this, the present invention provides a composition, a bio-ink, a bio-electrode material and its application. The bio-electrode material constructed by 3D printing of the bio-ink provided by the present invention can achieve low detection limit specific detection of 2, 4-dinitrotoluene, and achieves the purpose of self-repairing to reduce mechanical damage caused by hydraulic shearing and improve system sensitivity and signal-to-noise ratio through the design of ink components.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a composition, comprising: a hydrogel and Shewanella oneidensis MR-1;

[0007] The hydrogel is obtained by cross-linking polyacrylamide and sodium alginate.

[0008] In some embodiments of the present invention, in the above composition, the Shewanella oneidensis MR-1 is obtained by transformation of a recombinant plasmid and / or transfection of a chassis strain;

[0009] The recombinant plasmid comprises: promoter P yqjF , transcriptional activators YhaJ and cymA genes;

[0010] The chassis strain is: the Shewanella oneidensis MR-1 in which the cymA gene is knocked out.

[0011] In some embodiments of the present invention, in the above composition, the Shewanella oneidensis MR-1 comprises an extracellular electron transport pathway that can be controlled by 2, 4-dinitrotoluene.

[0012] In some embodiments of the present invention, in the above composition, the chassis bacteria for introducing the plasmid is Shewanella oneidensis MR-1 in which the key gene CymA for opening the extracellular electron transport pathway is knocked out.

[0013] In some embodiments of the present invention, in the above composition, the upstream genes of the plasmid introduced into the chassis bacteria from the Shewanella oneidensis MR-1 are the promoter yqjF induced by 2, 4-dinitrotoluene and its transcriptional regulatory factor yhaJ.

[0014] In some embodiments of the present invention, in the above composition, the downstream gene of the plasmid introduced into the chassis bacteria from Shewanella oneidensis MR-1 is CymA, a key gene for opening the extracellular electron transfer pathway.

[0015] In some embodiments of the present invention, in the above composition, the mass fraction of the polyacrylamide is 10-50.

[0016] In some embodiments of the present invention, in the above composition, the molecular weight of the polyacrylamide is 2 million to 14 million.

[0017] In some embodiments of the present invention, in the above composition, the mass fraction of sodium alginate is 3-10%; specifically: 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and most preferably: 7%.

[0018] The present invention also provides biological ink, comprising: the above composition and liquid culture medium.

[0019] In some embodiments of the present invention, in the above-mentioned biological ink, the mass volume ratio of the polyacrylamide to the liquid culture medium is (0.01~0.035) g:1 mL.

[0020] In some embodiments of the present invention, in the above-mentioned biological ink, the mass volume ratio of the polyacrylamide to the liquid culture medium is 0.01 g:1 mL, 0.015 g:1 mL, 0.017 g:1 mL, 0.02 g:1 mL, 0.03 g:1 mL or 0.035 g:1 mL, and the most preferred ratio is: 0.017 g:1 mL.

[0021] In some embodiments of the present invention, in the above-mentioned biological ink, the liquid culture medium is preferably a PBS solution, the PBS solution uses deionized water as a solvent, and its solute components include: potassium dihydrogen phosphate (KH2PO4) 0.27 g / L, disodium hydrogen phosphate (Na2HPO4) 1.42 g / L, sodium chloride (NaCl) 8 g / L, and potassium chloride (KCl) 0.2 g / L.

[0022] The present invention also provides a method for preparing the above-mentioned biological ink, wherein the polyacrylamide, the alginic acid and the liquid culture medium are mixed, degassed, and mixed with the bacterial liquid of Shewanella oneidensis MR-1 to obtain the biological ink.

[0023] In some embodiments of the present invention, in the above preparation method, the bacterial solution OD of Shewanella oneidensis is 600 It is 2~4.

[0024] In some embodiments of the present invention, in the above preparation method, the bacterial solution OD of Shewanella oneidensis is 600 It is 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4, and 2 is most preferred.

[0025] In some embodiments of the present invention, in the above preparation method, the sodium alginate and the polyacrylamide are added to a beaker and mixed evenly, the liquid culture medium is added thereto, and the mixture is gently stirred to avoid the generation of bubbles as much as possible, and then the hydrogel is placed in a vacuum drying oven and degassed at 25°C for 1 day and then placed in a -20 degree refrigerator for freezing, and then freeze-dried for standby use, and when used, the sterilized liquid culture medium is added for rehydration, and then the culture solution of Shewanella oneidensis MR-1 is added, and the mixture is stirred evenly to obtain 3D printing biological ink;

[0026] In some embodiments of the present invention, in the above preparation method, the culture fluid of the engineered Shewanella oneidensis MR-1 is preferably centrifuged to remove the LB medium used for culture before being mixed, and then the obtained bacteria are resuspended in liquid culture medium.

[0027] The present invention also provides a bioelectrode material, comprising: the above-mentioned bio-ink and / or the bio-ink obtained by the above-mentioned preparation method.

[0028] The present invention also provides a method for preparing the above-mentioned bio-electrode material, and the bio-electrode material is obtained by 3D printing and cross-linking the bio-ink.

[0029] In some embodiments of the present invention, in the above-mentioned preparation method, the 3D printed bioelectrode material is preferably a fractal structure in size, the fractal structure is derived from the Hilbert curve, and the mesh diameter is preferably 0.1~1.6 mm, specifically 0.1mm, 0.2mm, 0.4mm, 0.6mm, 0.8mm, 1.0mm, 1.2 mm, 1.4 mm or 1.6 mm, and most preferably 1.0 mm.

[0030] In some embodiments of the present invention, in the above preparation method, the cross-linking uses a 0.5 M calcium chloride solution; the cross-linking time is (5~30) minutes, specifically 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes, and most preferably 10 minutes.

[0031] The present invention also provides the use of the above composition, the above bio-ink, the bio-ink obtained by the above preparation method, the above bio-electrode material and / or the bio-electrode material obtained by the above preparation method in sewage monitoring.

[0032] In some embodiments of the present invention, in the above application, the organic pollutants contained in the sewage preferably include sodium lactate and 2, 4-dinitrotoluene.

[0033] The present invention also provides a method for monitoring sewage, wherein a sample to be tested is placed in the above-mentioned bioelectrode material and / or the bioelectrode material obtained by the above-mentioned preparation method for sensing detection; the sample to be tested contains organic pollutants; the organic pollutants include: sodium lactate and / or 2, 4-dinitrotoluene.

[0034] In some embodiments of the present invention, in the above method, the bioelectrode material is the main working electrode, the Ag / AgCl electrode is the reference electrode, and the platinum wire electrode is the counter electrode to construct a three-electrode single-chamber battery, and the sewage is monitored in the biobattery.

[0035] In some embodiments of the present invention, in the above method, the concentration of sodium lactate is 50-250 μL / 60 mL, specifically 50 μL / 60 mL, 100 μL / 60 mL, 150 μL / 60 mL, 200 μL / 60 mL or 250 μL / 60 mL.

[0036] In some embodiments of the present invention, in the above method, the concentration of 2, 4-dinitrotoluene is 200-700 μg / L, specifically 200 μg / L, 320 μg / L, 400 μg / L, 600 μg / L or 700 μg / L.

[0037] The present invention provides a composition, comprising: a hydrogel and Shewanella oneidensis MR-1;

[0038] The hydrogel is obtained by cross-linking polyacrylamide and sodium alginate.

[0039] The components of the 3D printing bio-ink provided by the present invention include: sodium alginate, polyacrylamide, engineered Shewanella oneidensis MR-1 and liquid culture medium. The 3D printing bio-electrode material provided by the present invention is made by the 3D printing bio-ink after 3D printing cross-linking treatment. The technical solution provided by the present invention uses sodium alginate-polyacrylamide hydrogel with repairable characteristics as the mechanical continuous phase of the bio-ink, and carries the engineered model electroactive bacteria Shewanella oneidensis as the chassis bacteria to obtain a bio-ink with good printing performance; on this basis, relying on the technical advantages of 3D bio-printing, the production method of the past biofilm forming element is changed, and the bio-ink is printed in the form of a fractal curve to construct a 3D printing bio-electrode material with a high bacterial concentration and excellent mass transfer capacity. The electrode can self-repair after mechanical damage, and can combine 2, 4-dinitrotoluene, and use the substrate sodium lactate to generate current in the presence of 2, 4-dinitrotoluene to achieve specific monitoring of 2, 4-dinitrotoluene in the water environment.

[0040] The experimental results of the embodiments provided by the present invention show that the engineered electroactive microorganisms in the 3D printed bioelectrode material provided by the present invention grow well, the current generated by the electrode material has a good dose-effect relationship with 2, 4-dinitrotoluene in the water environment, the detection limit can be as low as 200 μg / L, and the ink with self-repairing properties can restore its shape and mechanical properties to a certain extent after being subjected to hydraulic shock.

[0041] The experimental results of the embodiments provided by the present invention show that: the final result achieved by the present invention is to obtain a biological ink with self-repairing properties, which can achieve healing after injury within 3 minutes, and printability is an auxiliary property; the result achieved by the prior art is to obtain a printable ink with improved extracellular electronic capacity, and the corresponding characterization properties are that the oxidation / reduction peak current is ranked from large to small as: Control <1% w / v <2% w / v <4% w / v, and the impedance size is ranked from large to small as: Control >1% w / v >2% w / v >4% w / v. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0043] Figure 1 Schematic diagram of the plasmid of the engineered Shewanella oneidensis MR-1 provided in Example 1 of the present invention;

[0044] Figure 2 2 is a dose-effect relationship diagram of free engineered Shewanella oneidensis MR-1 provided in Example 2 of the present invention;

[0045] Figure 3 2 shows a variable temperature nuclear magnetic resonance image provided by Example 3 of the present invention;

[0046] Figure 4 The schematic diagram of self-repairing sodium alginate-polyacrylamide hydrogel repair is shown in Example 4 of the present invention;

[0047] Figure 5 A high- and low-strain alternating repair diagram of the bio-ink provided in Example 5 of the present invention is shown;

[0048] Figure 6 4 shows a viscosity-shear rate scanning diagram of the bio-ink provided in Example 5 of the present invention;

[0049] Figure 7 FIG. 1 is a schematic diagram of the structure of a 3D bioelectrode material provided in Example 6 of the present invention;

[0050] Figure 8 A schematic diagram showing the biocompatibility of a 3D printed bioelectrode provided in Example 7 of the present invention is shown;

[0051] Fig. 9 1. A graph showing the current-time result of the block-shaped bioelectrode provided in Example 8 of the present invention;

[0052] Fig.10 2 is a graph showing the current-time result of the 3D bioelectrode provided in Example 8 of the present invention;

[0053] Fig.11 FIG. 4 shows the dose-effect curve of the 3D bioelectrode provided in Example 8 of the present invention on 2, 4-dinitrotoluene. DETAILED DESCRIPTION

[0054] The invention discloses a composition, a biological ink, a biological electrode material and applications thereof.

[0055] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0056] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0057] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0058] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0059] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0060] The invention provides a 3D printing biological ink, the components of which include sodium alginate, polyacrylamide and liquid culture medium.

[0061] In the 3D printing bio-ink provided by the present invention, the sodium alginate is preferably provided by Sinopharm Chemical Reagent Co., Ltd. (China), with the national medicine number 30164428, and is in the form of light yellow powder. The mass fraction of the sodium alginate is preferably 3% to 10%, specifically 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and most preferably 7%.

[0062] In the 3D printing bio-ink provided by the present invention, the polyacrylamide is preferably provided by Aladdin® (USA), with product number: P108471, specification is non-ionic, molecular weight is 2 million to 14 million, and the dosage ratio of the polyacrylamide to the liquid culture medium is preferably (0.01 to 0.035) g:1 mL, specifically 0.01 g:1 mL, 0.015 g:1 mL, 0.017 g:1 mL, 0.02 g:1 mL, 0.03 g:1 mL or 0.035 g:1 mL, and most preferably: 0.017 g:1 mL.

[0063] In the 3D printing bio-ink provided by the present invention, the bacterial cell density OD of the engineered Shewanella oneidensis MR-1 in the liquid culture medium is 600 The value is preferably 2 to 4, specifically 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4, and most preferably 2.

[0064] In the 3D bioprinting ink provided by the present invention, the liquid culture medium can maintain the activity of Shewanella oneidensis MR-1, and will not interfere with the bacteria's specific detection of 2, 4-dinitrotoluene; the liquid culture medium is preferably a PBS solution, and the PBS solution uses deionized water as a solvent, and its solute components include: 0.27 g / L of potassium dihydrogen phosphate (KH2PO4), 1.42 g / L of disodium hydrogen phosphate (Na2HPO4), 8 g / L of sodium chloride (NaCl), and 0.2 g / L of potassium chloride (KCl).

[0065] The present invention also provides a method for preparing the 3D printing bio-ink described in the above technical solution, comprising the following steps:

[0066] Sodium alginate and polyacrylamide are added to a beaker and mixed evenly, and liquid culture medium is added thereto, and gently stirred to avoid the generation of bubbles as much as possible. Subsequently, the hydrogel is placed in a vacuum drying oven and degassed at 25°C for 1 day, and then placed in a -20 degree refrigerator for freezing. After the degassing, it is freeze-dried for use. When used, sterilized liquid culture medium is added for rehydration, and then engineered Shewanella oneidensis MR-1 culture fluid is added, and stirred evenly to obtain 3D printing bio-ink;

[0067] The components of the culture solution include engineered Shewanella oneidensis MR-1 and liquid culture medium.

[0068] In the bio-ink preparation method provided by the present invention, the culture solution of the engineered Shewanella oneidensis MR-1 is preferably centrifuged to remove the LB culture medium used for culture before being mixed, and then the obtained bacteria are resuspended in liquid culture medium.

[0069] In the method for preparing the bio-ink provided by the present invention, the specific mixing process is preferably to first rehydrate the sodium alginate-polyacrylamide hydrogel freeze-dried product with a sterile liquid culture medium and then mix it evenly with the culture medium of the engineered Shewanella oneidensis MR-1 to ensure that the OD 600 The value is 2.0.

[0070] The present invention also provides a 3D printed bio-electrode material, which is made by subjecting the 3D printed bio-ink described in the above technical solution to a 3D printing cross-linking treatment.

[0071] In the bioelectrode material provided by the present invention, the 3D printed bioelectrode material is preferably a fractal structure in size, the fractal structure is derived from the Hilbert curve, and the mesh diameter is preferably 0.1~1.6 mm, specifically 0.1mm, 0.2mm, 0.4mm, 0.6mm, 0.8mm, 1.0mm, 1.2 mm, 1.4 mm or 1.6 mm, and most preferably 1.0 mm.

[0072] The present invention also provides a method for preparing a 3D printed bioelectrode material, comprising the following steps:

[0073] The 3D printing bio-ink described in the above technical solution is loaded into the nozzle of the 3D bio-printer, 3D printing is performed according to a preset spatial structure, and then the block material obtained by 3D printing is placed in a calcium chloride solution with a concentration of 0.5 M for cross-linking.

[0074] In the method for preparing the bioelectrode material provided by the present invention, the 3D bioprinter is preferably provided by ALLEVI.

[0075] In the method for preparing the bioelectrode material provided by the present invention, the crosslinking agent used in the crosslinking treatment is preferably a 0.5 M calcium chloride solution, and the crosslinking time is preferably (5-30) minutes, specifically 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes, and most preferably 10 minutes.

[0076] The present invention also provides a sewage sensing method, comprising the following process:

[0077] The sewage is sensed and monitored in the electrode chamber of the bioelectrochemical reactor; the electrode material of the bioelectrochemical reactor is the 3D printed bioelectrode material described in the above technical solution or the 3D printed bioelectrode material prepared by the preparation method described in the above technical solution.

[0078] In the sewage sensing method provided by the present invention, the 3D printed bio-electrode described in the above scheme is first used as the main working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode to construct a three-electrode single-chamber battery to monitor sewage in the bio-battery.

[0079] In the sewage sensing method provided by the present invention, the organic pollutants contained in the sewage preferably include sodium lactate and 2, 4-dinitrotoluene (DNT); the sodium lactate is provided by Sangon (China) with a concentration of 60% and a serial number of A604046, and its concentration in sewage is preferably 50-250 μL / 60 mL, specifically 50 μL / 60 mL, 100 μL / 60 mL, 150 μL / 60 mL, 200 μL / 60 mL or 250 μL / 60 mL; the 2, 4-dinitrotoluene is provided by Sinopharm Chemical Reagent Co., Ltd. (China) with a national medicine number of CSDS 121-14-2, and is a light yellow to yellow needle-shaped crystalline solid, and its concentration in sewage is preferably 200-700 μg / L, specifically 200 μg / L, 320 μg / L, 400 μg / L, 600 μg / L or 700 μg / L.

[0080] The components of the bio-ink provided by the present invention include: sodium alginate, polyacrylamide, engineered Oneida Shewanella and liquid culture medium. The bio-electrode provided by the present invention is made of the 3D printed bio-ink through extrusion 3D printing. The technical solution provided by the present invention uses sodium alginate-polyacrylamide hydrogel with repairable properties as the mechanical continuous phase of the bio-ink, and carries the engineered model electroactive bacteria Oneida Shewanella as the chassis bacteria to obtain a bio-ink with good printing performance; on this basis, relying on the technical advantages of 3D bio-printing, the production method of the previous film-forming element is changed, and the bio-ink is printed in the form of a fractal curve to construct a 3D printed bio-electrode material with a high bacterial concentration and excellent mass transfer capacity. The electrode can combine with 2, 4-dinitrotoluene, and use the substrate sodium lactate to generate current in the presence of 2, 4-dinitrotoluene to achieve specific monitoring of 2, 4-dinitrotoluene in the water environment.

[0081] In Examples 1 to 8 of the present invention and Verification Example 1, all the raw materials and reagents used can be purchased from the market.

[0082] The present invention will be further described below in conjunction with embodiments:

[0083] Example 1

[0084] Construction of engineered Shewanella oneidensis MR-1:

[0085] The cymA gene-knockout Shewanella oneidensis MR-1 was used as the host strain. The Shewanella strain was cultured in LB medium at 30°C and 150 rpm. All plasmid constructions were carried out in Escherichia coli. The Escherichia coli strain was cultured in LB medium at 37°C and 150 rpm. When necessary, 50 μg / mL kanamycin was added. The promoter P was amplified from Escherichia coli mg1655. yqjF The coding sequence of the transcription activator YhaJ was amplified from Shewanella MR-1, and the gene sequence encoding the tetraheme cytochrome cymA was cloned into the pYYDT expression vector through two rounds of construction after gene purification to construct the expression vector pYYDT-P yqjF -YhaJ-cymA. The plasmid spectrum of the resulting gene pathway is as follows Figure 1 The constructed recombinant plasmid was transformed into competent cells turbo (derived from Escherichia coli K-12 strain), and then transformed into S. onedensis MR-1 ΔcymA by electroporation at 2.47 kV.

[0086] Example 2

[0087] Previous studies have shown that after knocking out CymA, the degradation ability of Shewanella oneidensis MR-1 to high concentrations of methyl orange was significantly reduced. Preliminary experiments confirmed that 240 mg / L of methyl orange was used to quantitatively characterize the CymA pathway activation ability of the engineered Shewanella oneidensis MR-1 constructed in Example 1. 10-1600 μg / L DNT was selected for the toxicant detection experiment, and the results were as follows: Figure 2 The results showed that the removal rate of 10-160 μg / L DNT was improved compared with the control group, but the linear relationship was poor. The removal rate of 800-1600 μg / L was not much different from that of the control group. The test results appeared in 15 minutes, and the linear relationship was excellent. 2 =0.9771. Compared with other literatures that use Escherichia coli as the base bacteria for 2, 4-dinitrotoluene biosensors, the detection limit of the strain constructed in this study is much lower, as low as 200μg / L. The concentration of DNT in wastewater is about 0.04~48.6 mg / L, and the Chinese national standard emission limit of DNT is 0.5 mg / L. The strain constructed in this study can fully meet the detection needs.

[0088] Example 3

[0089] Sodium alginate and polyacrylamide were added to a beaker and mixed evenly. Then, liquid medium was added thereto, and gently stirred evenly to avoid the generation of bubbles as much as possible. Subsequently, the hydrogel was placed in a vacuum drying oven and degassed at 25 °C for 1 day for standby. In order to explore the original driving force of polyacrylamide and sodium alginate in the formation of hydrogels, variable temperature 1H nuclear magnetic resonance characterization was carried out using a Bruker 400 MHz liquid superconducting nuclear magnetic resonance spectrometer. The solvent was D2O, and the temperature varied in increments of 10 °C between 25 °C and 45 °C. The results are as Figure 3 shown that as the temperature increased, the hydrogen bonds between PAM and SA were broken and the signals gradually weakened, indicating that hydrogen bond interaction played a certain role in the gel formation process, and the existence of dynamic hydrogen bonds was also the reason for the self-healing property of the hydrogel.

[0090] Example 4

[0091] Before testing, the hydrogel obtained in Example 3 was prepared into a cylinder with a mold and cut into two parts. One part maintained its original properties, and the other part was stained with rhodamine B. Since the fresh fracture surface had a certain viscosity when cutting the hydrogel, no additional external force was required to connect the two fractured parts of the hydrogel. The two parts were gently placed against each other so that the fracture surfaces contacted each other. The results are as Figure 4 shown that it only took 3 minutes for the two cut sections to be connected to complete self-healing.

[0092] Example 5

[0093] Rheological characterization was carried out on the polyacrylamide-sodium alginate hydrogel prepared in Example 3 using a rheometer (TA Instruments, USA). The hydrogel prepared in Example 3 was denoted as PAMSA-25, where 25 means the mass fraction of polyacrylamide. Hydrogels SA, PAMSA-10, and PAMSA-50 prepared by adding polyacrylamide with different mass fractions to sodium alginate were used as control groups. The results are as Figure 5 、 6 shown. Figure 5 shows the self-healing performance of the polyacrylamide-sodium alginate hydrogel under alternating cycles of different strains (low, 1% and high, 500%) at 25 °C. Figure 6 is the viscosity-shear rate scan chart of different hydrogels. Figure 5 It shows that under the alternating action of high and low strains, the hydrogel exhibits excellent self-recovery performance: the gel-like behavior (G’> G’’) at low strain (1%) rapidly changes to the sol-like behavior (G’<G’’) at high strain (500%), and quickly recovers after removing the strain. Shear thinning behavior is another important characteristic of the hydrogel used as 3D printing ink. The decrease in viscosity with the increase in shear rate enables the use of high-viscosity inks with better printability during printing, and lower shear stress can be used during the process to maintain the activity of biological components. Figure 6 Displays the ink viscosity-shear rate scan results, using the power law model η=k·ɤ (n−1) The power law exponents n were all less than 1 after fitting, which quantitatively indicated that the bio-ink was indeed a shear-thinning pseudoplastic non-Newtonian fluid, which may have higher fidelity and cell survival rate during printing.

[0094] Example 6

[0095] The process of 3D bioprinting working electrodes:

[0096] First, the engineered Shewanella oneidensis MR-1 bacterial culture solution was centrifuged at 25°C and 4000 rpm for 8 minutes to remove the LB medium used for culture, and the obtained bacteria were resuspended in PBS liquid culture medium; then 0.87 g of sodium alginate-polyacrylamide hydrogel was freeze-dried and mixed with a small amount of liquid culture medium to rehydrate it, and the mixture consisting of the engineered Shewanella oneidensis MR-1 culture solution was added thereto to make the final OD 600 The value was 2.0, and the mixture was stirred evenly to obtain 3D printed bio-ink. The bio-ink was transferred to a 10 mL sterile syringe and added to a 3D bio-printer (ALLEVI, USA) equipped with a 17G nozzle (spinneret diameter 1.04 mm) for 3D printing to obtain a semi-finished fractal structure material (the fractal structure was modeled based on the Hilbert curve, as shown in Figure 7 As shown, the mesh diameter is about 1 mm); the 3D printed block is placed in a 0.5 M calcium chloride solution for cross-linking.

[0097] Example 7

[0098] The electrode material prepared in Example 6 was recorded as PAMSA-25-bacteria, where 25 means the mass fraction of polyacrylamide. The hydrogels prepared by adding different mass fractions of polyacrylamide to sodium alginate were used as control groups, which were recorded as SA-bacteria, PAMSA-10-bacteria, and PAMSA-50-bacteria. After culturing for 1 day, the cross-linking of different electrodes was removed and the culture was continued in LB medium. The results are shown in FIG. Figure 8 As shown in Table 1, the biocompatibility of polyacrylamide-sodium alginate hydrogel PAMSA was slightly affected by the addition of non-natural polymer polyacrylamide, but the selected PAMSA-25-bacteria showed good biocompatibility and could be used for subsequent experiments.

[0099] Table 1

[0100]

[0101] Example 8

[0102] A bioelectrochemical reactor was constructed using the 3D printed bioelectrode material prepared in Example 6. The bioelectrochemical reactor consisted of a three-electrode single-chamber battery. The 3D printed bioelectrode described in the above technical solution was used as the main working electrode, the Ag / AgCl electrode was used as the reference electrode, and the Pt wire electrode was used as the counter electrode to construct the three-electrode single-chamber battery. 200 μL of 60% sodium lactate was pipetted into 60 mL of liquid culture medium using a pipette to obtain simulated sewage with a sodium lactate concentration of 20 mM. The simulated sewage was added to the electrode chamber of the bioelectrochemical reactor, nitrogen was aerated for 30 minutes to make the liquid environment an anaerobic environment, magnetic stirring was turned on at a speed of 400 r / min, and monitoring was started through the it mode of the electrochemical workstation or the current acquisition module. After the current signal was stable, 2, 4-dinitrotoluene solution of different concentrations was added thereto so that its final concentration was 200-700 μg / L, and the current was continued to be monitored after the addition. The parameters of the electrochemical workstation IT mode are: voltage 0.2 V, silence time 30 s, and sampling interval 10 s. Fig. 9 , Fig.10 The results of time-current acquisition by the electrochemical workstation show that after adding different concentrations of 2, 4-dinitrotoluene solution, the current variable of the bulk electrode has a linear relationship with the DNT concentration, but the credibility of the linear relationship is higher over 30 minutes. The bioelectrode regulated into a fractal configuration by 3D bioprinting can reach the maximum current in 20 minutes, and the credibility of the dose-effect relationship curve can reach 0.9987 at 5 minutes.

[0103] Verification Example 1

[0104] The present invention conducted a preliminary experiment when preparing the hydrogel, and the effect is shown in Table 2.

[0105] Table 2

[0106]

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that include: hydrogel and Shewanella oneidensis MR-1; The hydrogel is obtained by cross-linking polyacrylamide and sodium alginate.

2. The composition according to claim 1, characterized in that The Shewanella oneidensis MR-1 is obtained by transformation of a recombinant plasmid and / or transfection of a chassis strain; The recombinant plasmid comprises: promoter P yqjF , transcriptional activators YhaJ and cymA genes; The chassis strain is: the Shewanella oneidensis MR-1 in which the cymA gene is knocked out.

3. Bio-ink, characterized in that include: The composition and liquid culture medium as claimed in claim 1 or 2.

4. The bio-ink according to claim 4, characterized in that: The mass volume ratio of the polyacrylamide to the liquid culture medium is (0.01-0.035) g:1 mL.

5. The method for preparing the biological ink according to claim 3 or 4, characterized in that: The polyacrylamide, the alginic acid and the liquid culture medium are mixed, degassed, and mixed with the bacterial solution of Shewanella oneidensis MR-1 to obtain the biological ink.

6. The preparation method according to claim 5, characterized in that: The OD of the bacterial solution of Shewanella oneidensis MR-1 600 It is 2~4.

7. A bioelectrode material, characterized in that include: The bio-ink as described in claim 3 or 4 and / or the bio-ink obtained by the preparation method as described in claim 5 or 6.

8. The method for preparing a bioelectrode material according to claim 7, characterized in that: The bio-ink is 3D printed and cross-linked to obtain the bio-electrode material.

9. Use of the composition as described in claim 1 or 2, the bio-ink as described in claim 3 or 4, the bio-ink obtained by the preparation method as described in claim 5 or 6, the bio-electrode material as described in claim 7 and / or the bio-electrode material obtained by the preparation method as described in claim 8 in sewage monitoring.

10. A method for sewage monitoring, characterized in that: The sample to be tested is placed in the bioelectrode material as claimed in claim 7 and / or the bioelectrode material obtained by the preparation method as claimed in claim 8 for sensing detection; the sample to be tested contains organic pollutants; the organic pollutants include: sodium lactate and / or 2, 4-dinitrotoluene.

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