Genetically engineered bacterial biofilm materials enhances portable whole cell sensing.

In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions-temperature, salinity, and pH-to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 µM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes.

Applications of cell free protein synthesis in protein design.

In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs. Compared to in vivo protein screening, a wider range of different activities can be evaluated using CFPS, and the scale on which it can easily be used-screening tens to hundreds of designed proteins-is ideally suited to current needs. Protein design using physics-based strategies tended to have a relatively low success rate, compared with current machine-learning based methods. Screening steps (such as yeast display) were often used to identify proteins that displayed the desired activity from many designs that were highly ranked computationally. We also describe how CFPS is well-suited to identify the reasons designs fail, which may include problems with transcription, translation, and solubility, in addition to not achieving the desired structure and function.

Surface-Immobilized ZnNx Sites as High-Performance Catalysts for Continuous Flow Knoevenagel Condensation in Water.

By immobilizing the metal complex on the substrate surface, our previous results have demonstrated that heterogeneous catalysts with well-dispersed active MNC (metal-nitrogen-carbon) sites can be prepared in a rational and efficient manner. In this study, we employed agarose aerogel (AA) as the substrate to illustrate a straightforward strategy for immobilizing ZnNx sites on the surface. Under relatively low temperatures, the amine group of the ligand condenses with the surface carbonyl group generated in situ, resulting in the surface immobilized Zn sites. This can be supported by the IR, PXRD, and XPS data. Comprehensive characterization methods, including synchrotron powder XRD and spherical aberration-corrected TEM, confirmed the absence of ZnNx site aggregation in the surface immobilization process, even with a high Zn content (up to 8 wt %). The immobilized ZnNx sites exhibited high catalytic performance in Knoevenagel condensation, and α,ß-unsaturated compounds were obtained with high yield in both batch and continuous flow reactions. AA-ZnNx-200 showed the best catalytic activity, which was processed under 200 °C with a Zn content of 4.62 wt %. The immobilized ZnNx sites activated both the aldehyde and nitrile substrates, which were quantitatively converted into the corresponding α,ß-unsaturated compounds, with water as the solvent at room temperature. In continuous flow reaction conditions, a conversion rate up to 99% can be achieved with malononitrile. This heterogeneous catalyst can be facilely produced with quantitative yield in a large scale from cheap starting material under mild conditions. No catalyst deactivation was observed after seven batch reaction cycles or 80 h of continuous flow reaction, indicating its high robustness under catalytic reaction conditions. This catalyst enables a separation-free, energy-saving, and environment-friendly production process, offering a practical way for the industrial production.

Electrospun Polymeric Fibers Decorated with Silk Microcapsules via Encapsulation and Surface Immobilization for Drug Delivery.

Hollow polymer microcapsules as drug carriers have the advantages of drug protection, storage, and controlled release. Microcapsules combined with tissue engineering scaffolds such as electrospun microfibers can enhance long-term local drug retention. However, the combination methods of microcapsules and fibers still need to be further explored. Here, different technical approaches to functionalize electrospun polycaprolactone (PCL) microfibers with silk fibroin (SF) microcapsules through encapsulation and surface immobilization are developed, including direct blending and emulsion electrospinning for encapsulation, as well as covalent and cleavable disulfide-linkage for surface immobilization. The results of "blending" approach show that silk microcapsules with different sizes could be uniformly encapsulated inside electrospun fibers without aggregation. To further reduce the use of organic solvents, the microcapsules in the aqueous phase can be uniformly distributed in the PCL organic phase and successfully electrospun into fibers using surfactant span-80. For surface immobilization, silk microcapsules are efficiently covalent binding to the surface of electrospun PCL fibers via click chemistry and exhibited noncytotoxic. Based on this method, with the incorporation of a disulfide bond, the linkages between microcapsule and fiber could be cleaved under reducing conditions. These microcapsule-electrospun fiber combination methods provide sufficient options for different drug delivery requirements.

Using Single-Molecule FRET to Evaluate DNA Nanodevices at Work.

The observation of DNA nanodevices at a single molecule (i.e., device) level and in real time provides rich information that is typically masked in ensemble measurements. Single-molecule fluorescence resonance energy transfer (smFRET) offers a means to directly follow dynamic conformational or compositional changes that DNA nanodevices undergo while operating, thereby retrieving insights critical for refining them toward optimal function. To be successful, smFRET measurements require careful execution and meticulous data analysis for robust statistics. Here we outline the elemental steps for smFRET experiments on DNA nanodevices, starting from microscope slide preparation for single-molecule observation to data acquisition and analysis.

Atomically Dispersed NiNx Site with High Oxygen Electrocatalysis Performance Facilely Produced via a Surface Immobilization Strategy.

Nonprecious-metal heterogeneous catalysts with atomically dispersed active sites demonstrated high activity and selectivity in different reactions, and the rational design and large-scale preparation of such catalysts are of great interest but remain a huge challenge. Current approaches usually involve extremely high-temperature and tedious procedures. Here, we demonstrated a straightforward and scalable preparation strategy. In two simple steps, the atomically dispersed Ni electrocatalyst can be synthesized in a tens grams scale with quantitative yield under mild conditions, and the active Ni sites were produced by immobilizing preorganized NiNx complex on the substrate surface via organic thermal reactions. This catalyst exhibits excellent catalysis performances in both oxygen evolution and reduction reactions. It also exhibited tunable catalysis activity, high catalysis reproducibility, and high stability. The atomically dispersed NiNx sites are tolerant at high Ni concentration, as the random reactions and metal nanoparticle formation that generally occurred at high temperatures were avoided. This strategy illustrated a practical and green method for the industrial manufacture of nonprecious-metal single-site catalysts with a predictable structure.

Generation of Functional-RNA Arrays by In Vitro Transcription and In Situ RNA Capture for the Detection of RNA-RNA Interactions.

RNA performs a wide variety of vital cellular functions. These functions typically require interactions with other biological macromolecules, often as part of an intricate communication network. High-throughput techniques capable of analyzing RNA-based interactions are therefore essential. Functional-RNA arrays address this need, providing the capability of performing hundreds of miniature assays in parallel. Here we describe a method to generate functional-RNA arrays using in vitro transcription of a DNA template array and in situ RNA capture. We also suggest how functional-RNA arrays could be applied to investigating RNA-RNA interactions.

Layer-by-Layer Surface Modification of Alendronate-Loaded Polyester Microparticles-Enabling Protein Immobilization.

The highly inert surface of polyester micro- and nano- drug carriers is a challenging substrate for further modification. The presence of surface moieties suitable for macromolecule coupling is crucial in the development of targeted drug delivery systems. Among available methods of surface activation, those based on adsorption of charged macromolecules may be carried out in mild conditions. In this work, alendronate-loaded microcores of three polyesters: poly-ε-caprolactone (PCL), poly(l-lactide-co-ε-caprolactone) (PLA-co-PCL) and poly(lactic-co-glycolic acid) (PLGA) were coated with three polyelectrolyte shells composed of chitosan/heparin (CHIT/HEP), polyallylamine/heparin (PAH/HEP), and polyethyleneimine/heparin (PEI/HEP) via the layer-by-layer method. Subsequently, the feasibility of model protein immobilization on obtained shells was assessed. Electrokinetic potential measurements confirmed the possibility of deposition of all investigated coating variants, and a positive correlation between initial core ζ potential and intensity of charge alterations after deposition of subsequent layers was identified. PEI/HEP assembly was stable in physiological-like conditions, while PAH/HEP multilayers disassembled in presence of phosphate ions, and CHIT/HEP shell showed limited stability in pH 7.4. Fluorescence assays of fluorescein tagged lysozyme surface coupled via ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) click reaction with all shell variants indicated satisfying reaction efficiency. Poly-ε-caprolactone cores coated with CHIT/HEP tetralayer were selected as suitable for model IgG surface immobilization. Antibodies immobilized on the shell surface exhibited a moderate degree of affinity to fluorescent IgG binding protein.

A Heterogenized Copper Phenanthroline System to Catalyze the Oxygen Reduction Reaction.

Upon the electrochemical reduction of an in situ generated 5-diazo-1,10-phenanthroline ion, phenanthroline was covalently attached to a gold electrode. The grafted molecules act as a ligand when brought in contact with a copper-containing electrolyte solution. As the ligands are limited in spatial movement, the exclusive formation of the active species with only one phenanthroline ligand coordinated was expected. The in situ generated complexes have been investigated for activity in the oxygen reduction reaction, for which an overpotential of 800 mV is observed. During catalysis, initially a thick copper layer is formed on top of an organic layer that is still present on the gold surface. Upon deterioration of the organic layer underneath the copper over time, the amount of copper on the electrode and thereby the electrocatalytic activity decreases.

Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins.

We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using "Catcher/Tag" technology. Importantly, proteins of interest can be captured either when synthesised in situ by CFPS above the BslA surfaces, or when added as pure protein. The simplicity and cost efficiency of this method suggest that it will find many applications in cell-free-based methods.

Tiger 17 and pexiganan as antimicrobial and hemostatic boosters of cellulose acetate-containing poly(vinyl alcohol) electrospun mats for potential wound care purposes.

In this research, we propose to engineer a nanostructured mat that can simultaneously kill bacteria and promote an environment conducive to healing for prospective wound care. Polyvinyl alcohol (PVA) and cellulose acetate (CA) were combined at different polymer ratios (100/0, 90/10, 80/20% v/v), electrospun and crosslinked with glutaraldehyde vapor. Crosslinked fibers increased in diameter (from 194 to 278 nm), retaining their uniform structure. Fourier-transform infrared spectroscopy and thermal analyses proved the excellent miscibility between polymers. CA incorporation incremented the fibers swelling capacity and reduced the water vapor and air permeabilities of the mats, preventing the excessive drying of wounds. The antimicrobial peptide cys-pexiganan and the immunoregulatory peptide Tiger 17 were incorporated onto the mats via polyethylene glycol spacer (hydroxyl-PEG2-maleimide) and physisorbed, respectively. Time-kill kinetics evaluations revealed the mats effectiveness against Staphylococcus aureus and Pseudomonas aeruginosa. Tiger 17 played a major role in accelerating clotting of re-calcified plasma. Data reports for the first time the collaborative effect of pexiganan and Tiger 17 against bacterial infections and in boosting hemostasis. Cytocompatibility data verified the peptide-modified mats safety. Croslinked 90/10 PVA/CA mats were deemed the most promising combination due to their moderate hydrophilicity and permeabilities, swelling capacity, and high yields of peptide loading.

Nanoarchitectured prototypes of mesoporous silica nanoparticles for innovative biomedical applications.

Despite exceptional morphological and physicochemical attributes, mesoporous silica nanoparticles (MSNs) are often employed as carriers or vectors. Moreover, these conventional MSNs often suffer from various limitations in biomedicine, such as reduced drug encapsulation efficacy, deprived compatibility, and poor degradability, resulting in poor therapeutic outcomes. To address these limitations, several modifications have been corroborated to fabricating hierarchically-engineered MSNs in terms of tuning the pore sizes, modifying the surfaces, and engineering of siliceous networks. Interestingly, the further advancements of engineered MSNs lead to the generation of highly complex and nature-mimicking structures, such as Janus-type, multi-podal, and flower-like architectures, as well as streamlined tadpole-like nanomotors. In this review, we present explicit discussions relevant to these advanced hierarchical architectures in different fields of biomedicine, including drug delivery, bioimaging, tissue engineering, and miscellaneous applications, such as photoluminescence, artificial enzymes, peptide enrichment, DNA detection, and biosensing, among others. Initially, we give a brief overview of diverse, innovative stimuli-responsive (pH, light, ultrasound, and thermos)- and targeted drug delivery strategies, along with discussions on recent advancements in cancer immune therapy and applicability of advanced MSNs in other ailments related to cardiac, vascular, and nervous systems, as well as diabetes. Then, we provide initiatives taken so far in clinical translation of various silica-based materials and their scope towards clinical translation. Finally, we summarize the review with interesting perspectives on lessons learned in exploring the biomedical applications of advanced MSNs and further requirements to be explored.

Immobilization of Peroxo-Heteropoly Compound and Palladium on Hydroxyapatite for the Epoxidation of Propylene by Molecular Oxygen in Methanol.

Peroxo-heteropoly compound PO4[W(O)(O2)2] was synthesized on calcium-deficient hydroxyapatite using a reaction of surface [HPO4]2- groups on hydroxyapatite with a Na2[W2O3(O2)4] aqueous solution. The vibration of [HPO4]2- at 875 cm-1 became very weak, and the vibration of the peroxo-oxygen bond [O-O]2- at 845 cm-1 appeared in the FT-IR spectrum of the solid product, indicating that PO4[W(O)(O2)2] was formed on the surface of hydroxyapatite. The formed solid sample was further reacted with PdCl2(PhCN)2 in an acetone solution to fix PdCl2 between the O sites on the hydroxyapatite. Elemental analyses proved that the resultant solid contained 1.2 wt.% Pd, implying that PdCl2 molecules were immobilized on the surface of hydroxyapatite. The hydroxyapatite-based hybrid compound containing Pd and PO4[W(O)(O2)2] was used as a heterogeneous catalyst in a methanol solvent for propylene epoxidation by molecular oxygen in an autoclave batch reaction system. A propylene conversion of 53.4% and a selectivity for propylene oxide of 88.7% were obtained over the solid catalyst after reaction at 363 K for 8 h. The novel catalyst could be reused by a simple centrifugal separation, and the yield of propylene oxide did not decrease after the reaction for five runs. By prolonging the reaction time to 13 h, the highest yield of propylene oxide at 363 K over the solid catalyst was obtained as 53.8%, which was almost the same as that of the homogeneous catalyst containing PdCl2(PhCN)2 and [(C6H13)4N]2{HPO4[W(O)(O2)2]2} for the propylene epoxidation. Methanol was used as a solvent as well as a reducing agent in the propylene epoxidation by molecular oxygen. Small particles of Pd metal were formed on the surface of the hybrid solid catalyst during the reaction, and acted as active species to achieve the catalytic turnover of PO4[W(O)(O2)2] in the propylene epoxidation by molecular oxygen in methanol.

Rewritable Surface on a Plastic Substrate Using Fluorous Affinity.

Fluorous chemistry has unique features and high potential applicability, which are distinct from those of nonfluorinated organic compounds. However, there are limited reports detailing the applications of fluorous-fluorous interactions (fluorophilicity or fluorous affinity), likely because these interactions are not found in nature. In the present study, we describe the rewritable surface functionalization of a plastic substrate based on fluorous affinity. Plastic substrates were dip-coated with a series of methacrylate-based fluoropolymers to generate fluorous surfaces. Fluorous-tagged small molecules [perfluoroalkyl (Rf) amines] were immobilized on the fluorous surfaces via fluorous-fluorous interactions, thereby introducing reactive functional groups (amino moieties) on the surface. The amino groups displayed on the surface (accessible by a reactant) were successfully quantified using a reactive fluorophore, which enabled quantitative analysis of the Rf-amines immobilized on the fluorous surface that were available for the subsequent reaction. The effects of the molecular structures of the fluoropolymers and Rf-amines on the surface immobilization of Rf-amines were also investigated quantitatively. The surface coated with a fluoropolymer containing -C8F17 most effectively immobilized an Rf-amine comprising two -C6F13 chains. The adhered Rf-amines were easily removed by washing the surface with methanol, and then, they could successfully be re-immobilized on the surface. Finally, the presented approach enabled the rewritable micropatterning of an Rf-tagged biomolecule on a plastic surface through microcontact printing.

Overview and emerging trends in optical fiber aptasensing.

Optical fiber biosensors have attracted growing interest over the last decade and quickly became a key enabling technology, especially for the detection of biomarkers at extremely low concentrations and in small volumes. Among the many and recent fiber-optic sensing amenities, aptamers-based sensors have shown unequalled performances in terms of ease of production, specificity, and sensitivity. The immobilization of small and highly stable bioreceptors such as DNA has bolstered their use for the most varied applications e.g., medical diagnosis, food safety and environmental monitoring. This review highlights the recent advances in aptamer-based optical fiber biosensors. An in-depth analysis of the literature summarizes different fiber-optic structures and biochemical strategies for molecular detection and immobilization of receptors over diverse surfaces. In this review, we analyze the features offered by those sensors and discuss about the next challenges to be addressed. This overview investigates both biochemical and optical parameters, drawing the guiding lines for forthcoming innovations and prospects in this ever-growing field of research.

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals.

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv-GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv-pH-Lemon and TAv-GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing.

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.

Aggregation-related quenching of LHCII fluorescence in liposomes revealed by single-molecule spectroscopy.

Incorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted major light-harvesting complexes of plants (LHCII). By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates bound to the surface. Our results showed that fluorescence lifetime obtained in bulk and in single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII - LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Evaluation of various waste substrates for biofilm formation and subsequent use in aerobic packed-bed reactor for secondary treatment of domestic wastewater.

Immobilization of bacterial cells on suitable substrates is of utmost importance in the secondary treatment of wastewater using fixed-film reactors. Therefore, screening of efficient and cheaper materials for bacterial surface immobilization was carried out. Eleven waste materials were used as substrates, packed in a column, and bacterial surface immobilization was carried out using cow dung slurry/MLSS mixture. All the chosen substrates were screened for bacterial immobilization/biofilm formation by standard bacterial enumeration technique. The substrate with the highest biofilm-forming ability was used for secondary treatment of raw domestic wastewater. The results showed that high-density polyethylene and aluminium foil sheets have poor immobilizing characteristics with 2.2 × 108 and 2.4 × 108 CFU/cm2 respectively, whereas jute fibres were observed to be the most efficient among the substrates with 5.1 × 1023 CFU/cm2. The column packed with jute fibres was used for wastewater treatment. Various physico-chemical parameters were analyzed before and after treatment and there was a significant reduction in major parameters after treatment. The bacteria-immobilized jute fibres showed maximum immobilization potential and were highly efficient in wastewater treatment, and therefore these findings offer immense promise in the synthesis of composite polymers for bacterial immobilization and subsequent secondary treatment.

Microwave Atmospheric Plasma: A Versatile and Fast Way to Confer Antimicrobial Activity toward Direct Chitosan Immobilization onto Poly(lactic acid) Substrate.

In this study, a simple method to immobilize chitosan on a poly(lactic acid) (PLA) surface was developed in a fast manner. The immobilization was realized in two steps. First, an atmospheric plasma (MWAP) torch was used to modify the PLA surface in less than 5 min in order to create enough activated sites toward the chitosan adhesion, followed by a direct dip coating to spread and immobilize chitosan on this MWAP-modified PLA surface. The modification of the PLA surface properties was confirmed by X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy. It resulted that the activated species derived from the plasma torch, i.e., hydroxyl and carboxylic acid moieties, enabled an increase of the hydrophilicity of the PLA surface. Interestingly, this activated surface allows a good spreading of chitosan solution from dip coating and leads to a homogeneous stable coating. Our XPS results bring us the hypothesis that the stabilization of the chitosan layer is mainly induced by noncovalent interactions such as hydrogen bonding and electrostatic interactions. A first insight into the biological properties of theses surfaces was assessed in terms of the antimicrobial activity of the here-designed surfaces.