Biohybrid Systems Research Articles

Embedding biocatalysts in a redox polymer enhances the performance of dye-sensitized photocathodes in bias-free photoelectrochemical water splitting

Dye-sensitized photoelectrodes consisting of photosensitizers and molecular catalysts with tunable structures and adjustable energy levels are attractive for low-cost and eco-friendly solar-assisted synthesis of energy rich products. Despite these advantages, dye-sensitized NiO photocathodes suffer from severe electron-hole recombination and facile molecule detachment, limiting photocurrent and stability in photoelectrochemical water-splitting devices. In this work, we develop an efficient and robust biohybrid dye-sensitized NiO photocathode, in which the intermolecular charge transfer is enhanced by a redox polymer. Owing to efficient assisted electron transfer from the dye to the catalyst, the biohybrid NiO photocathode showed a satisfactory photocurrent of 141±17 μA·cm−2 at neutral pH at 0 V versus reversible hydrogen electrode and a stable continuous output within 5 h. This photocathode is capable of driving overall water splitting in combination with a bismuth vanadate photoanode, showing distinguished solar-to-hydrogen efficiency among all reported water-splitting devices based on dye-sensitized photocathodes. These findings demonstrate the opportunity of building green biohybrid systems for artificial synthesis of solar fuels.

Interspecies cooperation-driven photogenerated electron transfer processes and efficient multi-pathway nitrogen removal in the g-C3N4-anammox consortia biohybrid system

Photocatalytic materials-microbial biohybrid systems pave the way for solar-driven wastewater nitrogen removal. In this study, interspecies cooperation in photogenerated electron transfer and efficient nitrogen removal mechanism in the g-C3N4-anammox consortia biohybrid system were first deciphered. The results indicated that the essential extracellular electron carriers (cytochrome c and flavin) for anammox genomes were provided by associated bacteria (BACT3 and CHLO2). This cooperation, regulated by the ArcAB system and electron transfer flavoprotein, made anammox bacteria the primary photogenerated electron sink. Furthermore, an efficient photogenerated electron harness was used to construct a reductive glycine pathway (rGlyP) in anammox bacteria inventively, which coexisted with the Wood–Ljungdahl pathway (WLP), constituting a dual-pathway carbon fixation model, rGlyP-WLP. Carbon fixation products efficiently contributed to the tricarboxylic acid cycle, while inhibiting electron diversion in anabolism. Photogenerated electrons were targeted channeled into nitrogen metabolism-available electron carriers, enhancing anammox and dissimilatory nitrate reduction to ammonium (DNRA) processes. Moreover, ammonia assimilation by the glycine cleavage system in rGlyP established an alternative ammonia removal route. Ultimately, multi-pathway nitrogen removal involving anammox, DNRA, and rGlyP achieved 100 % ammonia removal and 94.25 % total nitrogen removal efficiency. This study has expanded understanding of anammox metabolic diversity, enhancing its potential application in carbon-neutral wastewater treatment.

Electroacoustic Responsive Cochlea-on-a-Chip.

Organ-on-chips can highly simulate the complex physiological functions of organs, exhibiting broad application prospects in developmental research, disease simulation, as well as new drug research and development. However, there is still less concern about effectively constructing cochlea-on-chips. Here, a novel cochlear organoids-integrated conductive hydrogel biohybrid system with cochlear implant electroacoustic stimulation (EAS) for cochlea-on-a-chip construction and high-throughput drug screening, is presented. Benefiting from the superior biocompatibility and electrical property of conductive hydrogel, together with cochlear implant EAS, the inner ear progenitor cells can proliferate and spontaneously shape into spheres, finally forming cochlear organoids with good cell viability and structurally mature hair cells. By incorporating these progenitor cells-encapsulated hydrogels into a microfluidic-based cochlea-on-a-chip with culture chambers and a concentration gradient generator, a dynamic and high-throughput evaluation of inner ear disease-related drugs is demonstrated. These results indicate that the proposed cochlea-on-a-chip platform has great application potential in organoid cultivation and deafness drug evaluation.

Synergistic Approaches for Enhanced Light-Driven Hydrogen Production: A Membrane-Anchoring Protein-Engineered Biohybrid System with Dual Photosensitizers Strategy

Synergistic Approaches for Enhanced Light-Driven Hydrogen Production: A Membrane-Anchoring Protein-Engineered Biohybrid System with Dual Photosensitizers Strategy

Biohybrid Energy Storage Circuits Based on Electronically Functionalized Plant Roots.

Biohybrid systems based on plants integrate plant structures and processes into technological components targeting more sustainable solutions. Plants' biocatalytic machinery, for example, has been leveraged for the organization of electronic materials directly in the vasculature and roots of living plants, resulting in biohybrid electrochemical devices. Among other applications, energy storage devices were demonstrated where the charge storage electrodes were seamlessly integrated into the plant tissue. However, the capacitance and the voltage output of a single biohybrid supercapacitor are limited. Here, we developed biohybrid circuits based on functionalized conducting roots, extending the performance of plant based biohybrid energy storage systems. We show that root-supercapacitors can be combined in series and in parallel configuration, achieving up to 1.5 V voltage output or up to 11 mF capacitance, respectively. We further demonstrate that the supercapacitors circuit can be charged with an organic photovoltaic cell, and that the stored charge can be used to power an electrochromic display or a bioelectronic device. Furthermore, the functionalized roots degrade in composting similarly to native roots. The proof-of-concept demonstrations illustrate the potential of this technology to achieve more sustainable solutions for powering low consumption devices such as bioelectronics for agriculture or IoT applications.

Biohybrid CO2 electrolysis under external mode: using pure formic acid extracted from CO2 electroreduction for diverse microbial conversion

The demand for converting CO2 into fuels or chemicals is on the rise to achieve a carbon-efficient circular economy. Biohybrid CO2 electrolysis shows potential for increasing production rates and diversifying product spectra by combining electrocatalysts and microbial catalysts. However, it is important to note that utilizing a shared catholyte for biohybrid CO2 electrolysis has not demonstrated significant performance improvements to date. In this study, we developed a biohybrid CO2 electrolysis system utilizing a solid electrolyte operating in an external mode. The produced formic acid was extracted and used as an intermediate for microbial conversion. Impressively, the solid-electrolyte CO2 electrolysers obtained a remarkable total Faraday efficiency of 81.4% for formic acid production. In-situ mechanism studies unveiled metallic tin as the probable real active site, prompting further exploration of strategies to boost the activity and stability of electrocatalysts. In the bioconversion step, we achieved a noteworthy 8-day duration for generating bioelectricity, nearly 100% electron recovery for biomethane production, and 90.8% for acetate generation. Additionally, when ethanol was co-fed, a C6 specificity of 41.1% was observed for the generation of medium-chain fatty acids (MCFAs). This study presents groundbreaking experimental data that demonstrates the numerous advantages of utilizing hybrid systems as advanced synthesis techniques.

A method for detecting spatiotemporal patterns of cancer biomarkers-evoked activity using radial basis function network extracted time-domain features from calcium imaging data

BackgroundTwo-photon calcium imaging is widely used to study the odor-evoked glomerular activity in the dorsal olfactory bulb of macrosmatic animals. The nonstationary character of activated patterns sets a limit on the use of a traditional image processing approaches. New methodThe developed method makes it possible to automatically map cancer biomarkers-activated glomeruli in the rat dorsal olfactory bulb. We interpolated fluorescence intensity of calcium dynamics based on the Gaussian RBF network and synthesized the physiological fluorescence model of the receptive glomerular field. ResultsThe experiments on 5 rats confirmed the correctness of the developed approach. Patterns evoked by the 6-methyl-5-hepten-2-one (stomach cancer biomarker) and benzene (lung cancer biomarker) were correctly identified. Comparison with existing methodsThe proposed method was compared with the nonnegative matrix factorization method and with the method based on computer vision algorithms. The developed approach showed better accuracy in experiments and provided the mathematical models of the odor-evoked patterns synthesis. These models can be used to generate synthetic images of odor-evoked glomerular activity and thus to overcome the problem of small experimental data collected in calcium imaging. ConclusionsThe proposed method should be considered part of the toolkit for fully automatic analysis of calcium imaging-based studies. Currently available methodology is not able to use breath biomarkers to reliably discriminate between cancer patients and healthy controls. Nevertheless, the effective identification of the spatial patterns of cancer biomarkers-evoked glomerular activity can serve as the foundation for highly sensitive biohybrid systems for cancer screening.

A proposed metabolic pathway mechanism along with Lactobacillus acidophilus growth modeling in a novel semi-artificial biohybrid system based on CdS-NPs

A metabolic pathway mechanism in a newly developed semi-artificial biohybrid system (SABS) constructed from Lactobacillus acidophilus and cadmium sulfide nanoparticles (CdS-NPs) was proposed. Also, the bacterium growth in the biohybrid system was modeled via historical data design (HDD). The findings showed that the bacterial biomass production in the biosystem occurs in several steps, including Light harvesting, Electron generation, Transduction, Glycolysis pathway, Electro-fermentation, Substrate for biomass accumulation, Gluconeogenesis pathway and anabolism, and Biomass production. In general, electron transfer (ET) increases the nicotinamide adenine dinucleotide (NADH/NAD+) ratio, accumulates pyruvate, generates more adenosine triphosphate (ATP), and incites the bacterium toward more growth. The modeling with HDD indicated that the correlation between time and absorbance in the biohybrid system corresponded with a cubic model with R2 > 0.99 and the optimum point of 1.93 at the time of 64.14 h.

In Vitro and In Vivo Evaluation of Chitosan/HPMC/Insulin Hydrogel for Wound Healing Applications.

Treatment of chronic wounds is challenging, and the development of different formulations based on insulin has shown efficacy due to their ability to regulate oxidative stress and inflammatory reactions. The formulation of insulin with polysaccharides in biohybrid hydrogel systems has the advantage of synergistically combining the bioactivity of the protein with the biocompatibility and hydrogel properties of polysaccharides. In this study, a hydrogel formulation containing insulin, chitosan, and hydroxypropyl methyl cellulose (Chi/HPMC/Ins) was prepared and characterized by FTIR, thermogravimetric, and gel point analyses. The in vitro cell viability and cell migration potential of the Chi/HPMC/Ins hydrogel were evaluated in human keratinocyte cells (HaCat) by MTT and wound scratch assay. The hydrogel was applied to excisional full-thickness wounds in diabetic mice for twenty days for in vivo studies. Cell viability studies indicated no cytotoxicity of the Chi/HPMC/Ins hydrogel. Moreover, the Chi/HPMC/Ins hydrogel promoted faster gap closure in the scratch assay. In vivo, the wounds treated with the Chi/HPMC/Ins hydrogel resulted in faster wound closure, formation of a more organized granulation tissue, and hair follicle regeneration. These results suggest that Chi/HPMC/Ins hydrogels might promote wound healing in vitro and in vivo and could be a new potential dressing for wound healing.

Polydopamine-immobilized yeast cells for portable electrochemical biosensors applied in environmental copper sensing

The coupling of biological organisms with electrodes enables the development of sustainable, low cost, and potentially self-sustained biosensors. A critical aspect is to obtain portable bioelectrodes where the biological material is immobilized on the electrode surface to be utilized on demand. Herein, we developed an approach for the rapid entrapment and immobilization of metabolically active yeast cells in a biocompatible polydopamine layer, which does not require a separate and time-consuming synthesis. The reported approach allows obtaining the “electrical wire” of intact and active yeast cells with resulting current generation from glucose oxidation. Additionally, the electrochemical performance of the biohybrid yeast-based system has been characterized in the presence of CuSO4, a widely used pesticide, in the environmentally relevant concentration range of 20–100 μM. The system enabled the rapid preliminary monitoring of the contaminant based on variations in current generation, with a limit of detection of 12.5 μM CuSO4. The present approach for the facile preparation of portable yeast-based electrochemical biosensors paves the way for the future development of sustainable systems for environmental monitoring.

A hybrid photocatalytic system enables direct glucose utilization for methanogenesis

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic-abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals.

Kinetic analysis of in vitro release profiles of salicylic acid and fluocinolone acetonide from dual delivery systems composed of polymeric nanocarriers and a hydrogel matrix

Many research studies have focused on the design of dressing materials that ensure fast and healthy regeneration of the skin while maintaining sterility and delivering active substances at the required time for treatment. In this study, a method for synthesizing a pH/thermosensitive polymeric nanocarrier and its encapsulation with salicylic acid (SA) and fluocinolone acetonide (FA) is reported for the first time. Dual-drug delivery systems comprising a sodium alginate/poly (vinyl alcohol)/aloe vera-based hydrogel matrix and drug-loaded polymeric nanocarriers (T-SA + FA or pH-SA + FA) were obtained and characterized by scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) spectroscopy as well as degradation, mechanical, microbiological, and cytotoxicity tests. The swelling degree in water of the base matrix (M) and those of the bio-hybrids M-pH-SA + FA and M-T-SA + FA were in the range of 87–262%, 158–203%, and 87–156%, respectively. Degradation rates were comparable for all the analyzed samples, with values of 40.3%, 44.7%, and 42.5% for the base matrix (M) and bio-hybrid hydrogels M-pH-SA + FA and M-T-SA + FA, respectively. Both bio-hybrids inhibited the growth of different bacteria, such as Staphylococcus aureus and Escherichia coli. Cytotoxicity tests proved that the materials obtained do not show cytotoxic effects and are safe for skin cells. The release of dual drugs from the bio-hybrid system occurred in a prolonged and controlled way, according to the Korsmeyer–Peppas model.

Hole-scavenging in photo-driven N2 reduction catalyzed by a CdS-nitrogenase MoFe protein biohybrid system

The light-driven reduction of dinitrogen (N2) to ammonia (NH3) catalyzed by a cadmium sulfide (CdS) nanocrystal‑nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N2 reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N2 reduction to NH3 in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N2 reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N2 reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N2 reduction by the biohybrid complex to the standard reduction potential (Eo) of the SEDs reveals that Eo is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N2 reduction reaction catalyzed by a CdS QD–MoFe protein hybrid.

Biophysical investigation of liposome systems decorated with bioconjugated copolymers in the presence of amantadine.

Liposome-based technologies derived from lipids and polymers (e.g., PEGylated liposomes) have been recognized because of their applications in nanomedicine. However, since such systems represent myriad challenges and may promote immune responses, investigation of new biomaterials is mandatory. Here, we report on a biophysical investigation of liposomes decorated with bioconjugated copolymers in the presence (or absence) of amantadine (an antiviral medication). First, copolymers of poly(N,N-dimethylacrylamide-co-fluoresceinacrylate-co-acrylic acid-N-succinimide ester)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM) containing a fluorescence label were biofunctionalized with short peptides that resemble the sequence of the loops 220 and 130 of the binding receptor of the hemagglutinin (HA) protein of the influenza A virus. Then, the bioconjugated copolymers were self-assembled along with liposomes composed of 1,2 dimyristoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol (MSC). These biohybrid systems, with and without amantadine, were systematically characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryoTEM). Finally, the systems were tested in an in vitro study to evaluate cytotoxicity and direct immunofluorescence in Madin Darbin Canine Kidney (MDCK) cells. The biohybrid systems displayed long-term stability, thermo-responsiveness, hydrophilic-hydrophobic features, and fluorescence properties and were presumable endowed with cell targeting properties intrinsically integrated into the amino acid sequences of the utilized peptides, which indeed turn them into promising nanodevices for biomedical applications.

Engineering of bespoke photosensitiser-microbe interfaces for enhanced semi-artificial photosynthesis.

Biohybrid systems for solar fuel production integrate artificial light-harvesting materials with biological catalysts such as microbes. In this perspective, we discuss the rational design of the abiotic-biotic interface in biohybrid systems by reviewing microbes and synthetic light-harvesting materials, as well as presenting various approaches to coupling these two components together. To maximise performance and scalability of such semi-artificial systems, we emphasise that the interfacial design requires consideration of two important aspects: attachment and electron transfer. It is our perspective that rational design of this photosensitiser-microbe interface is required for scalable solar fuel production. The design and assembly of a biohybrid with a well-defined electron transfer pathway allows mechanistic characterisation and optimisation for maximum efficiency. Introduction of additional catalysts to the system can close the redox cycle, omitting the need for sacrificial electron donors. Studies that electronically couple light-harvesters to well-defined biological entities, such as emerging photosensitiser-enzyme hybrids, provide valuable knowledge for the strategic design of whole-cell biohybrids. Exploring the interactions between light-harvesters and redox proteins can guide coupling strategies when translated into larger, more complex microbial systems.

Semiconductor biohybrids for enhanced bifunctional wastewater sulfur and heavy metal removal

The proposed mechanism of the light-driven microbial desulphurization by the CdS–H. neapolitanus biohybrid system.

(Invited) Organic Polymer Dots for Photocatalysis

Organic polymeric photocatalysts have been attracted scientists’ interests during past years due to their low-cost, tunable bandgaps and energy levels, and promising photocatalytic performance. Making the traditional hydrophobic polymeric photocatalyst into nano-particles with size less than 100 nm, so-called polymer dots (Pdots), has shown significantly enhanced photocatalytic activity. Rational design of organic polymers is one of strategies to improve photocatalytic performance for hydrogen production. Moreover, making heterojunction Pdots can efficiently separate photogenerated charges within Pdots as well as utilize energy transfer process to enhance the light harvesting efficiency. In this talk, I will discuss our research achievements in optimization of polymer structures on Pdots photocatalysis, study of photocatalytic mechanism in heterojunction Pdots systems and applications of Pdots in bio-hybrid photocatalytic systems.Relevant publications:[1] Wang L., et al., Angew. Chem., 2016, 128 (40), 12494-12498[2] Pati P., et al., Energy Environ. Sci., 2017 10 (6), 1372-1376[3] Liu A., et al., J. Am. Chem. Soc., 2021, 143 (7), 2875-2885[4] Pavliuk M., et al., J. Am. Chem. Soc. 2022, 144(30) 13600–13611[5] Wang S., et al., Angew. Chem. Int. Ed., 2022, 61(23), e202202733

Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation

AbstractSortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation.

Sortase-mediated ligation (SML) has emerged as a powerful and versatile methodology for site-specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side-products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state-of-the-art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

PenTag, a Versatile Platform for Synthesizing Protein‐Polymer Biohybrid Materials

AbstractThe site‐specific and covalent conjugation of proteins on solid supports and in hydrogels is the basis for the synthesis of biohybrid materials offering broad applications. Current methods for conjugating proteins to desired targets are often challenging due to unspecific binding, unstable (noncovalent) coupling, or expensive and difficult‐to‐synthesize ligand molecules. Here, is presented PenTag, an approach for the bioorthogonal, highly specific, and covalent conjugation of a protein to its ligand for various applications in materials sciences. Penicillin‐binding protein 3 (PBP3) is engineered and shows that this protein can be used for the stable and spontaneous conjugation of proteins to dyes, polymers, or solid supports. PenTag as a crosslinking tool is applied for synthesizing stimuli‐responsive hydrogels or for the development of a biohybrid material system performing computational operations emulating a 4:2 encoder. Based on this broad applicability and the use of a small, cheap, and easy‐to‐functionalize ligand and a stable, soluble recombinant protein, is seen PenTag as a versatile approach toward biohybrid material synthesis.