Secondary Structure in Enzyme-Inspired Polymer Catalysts Impacts Water Oxidation Efficiency.

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.

Highly Efficient Carbon Dioxide Electroreduction via DNA-Directed Catalyst Immobilization.

Electrochemical reduction of carbon dioxide (CO2) is a promising route to up-convert this industrial byproduct. However, to perform this reaction with a small-molecule catalyst, the catalyst must be proximal to an electrode surface. Efforts to immobilize molecular catalysts on electrodes have been stymied by the need to optimize the immobilization chemistries on a case-by-case basis. Taking inspiration from nature, we applied DNA as a molecular-scale "Velcro" to investigate the tethering of three porphyrin-based catalysts to electrodes. This tethering strategy improved both the stability of the catalysts and their Faradaic efficiencies (FEs). DNA-catalyst conjugates were immobilized on screen-printed carbon and carbon paper electrodes via DNA hybridization with nearly 100% efficiency. Following immobilization, a higher catalyst stability at relevant potentials is observed. Additionally, lower overpotentials are required for the generation of carbon monoxide (CO). Finally, high FE for CO generation was observed with the DNA-immobilized catalysts as compared to the unmodified small-molecule systems, as high as 79.1% FE for CO at -0.95 V vs SHE using a DNA-tethered catalyst. This work demonstrates the potential of DNA "Velcro" as a powerful strategy for catalyst immobilization. Here, we demonstrated improved catalytic characteristics of molecular catalysts for CO2 valorization, but this strategy is anticipated to be generalizable to any reaction that proceeds in aqueous solutions.

Novel delivery systems for controlled release of bacterial therapeutics.

As more is learned about the benefits of microbes, their potential to prevent and treat disease is expanding. Microbial therapeutics are less burdensome and costly to produce than traditional molecular drugs, often with superior efficacy. Yet, as with most medicines, controlled dosing and delivery to the area of need remain key challenges for microbes. Advances in materials to control small-molecule delivery are expected to translate to microbes, enabling similar control with equivalent benefits. In this perspective, recent advances in living biotherapeutics are discussed within the context of new methods for their controlled release. The integration of these advances provides a roadmap for the design, synthesis, and analysis of controlled microbial therapeutic delivery systems.

Improving electrochemical hybridization assays with restriction enzymes.

Nucleic acids in blood are early indicators of disease that could be detected by point-of-care biosensors if sufficiently sensitive and facile sensors existed. Electrochemical hybridization assays are sensitive and specific but are limited to very short nucleic acids. We have developed a restriction enzyme-assisted electrochemical hybridization (REH) assay for improved nucleic acid detection. By incorporating target-specific restriction enzymes, we detect long nucleic acids, with performance dependent on the location of the cut site relative to the electrode surface. Thus, we have further established guidelines for REH design to serve as a generalizable platform for robust electrochemical detection of long nucleic acids.

Zeta potential characterization using commercial microfluidic chips.

Surface charge is a critical feature of microbes that affects their interactions with other cells and their environment. Because bacterial surface charge is difficult to measure directly, it is typically indirectly inferred through zeta potential measurements. Existing tools to perform such characterization are either costly and ill-suited for non-spherical samples or rely on microfluidic techniques requiring expensive fabrication equipment or specialized facilities. Here, we report the application of commercially available PMMA microfluidic chips and open-source data analysis workflows for facile electrokinetic characterization of particles and cells after prior zeta potential measurement with a Zetasizer for calibration. Our workflows eliminate the need for microchannel fabrication, increase measurement reproducibility, and make zeta potential measurements more accessible. This novel methodology was tested with functionalized 1 µm and 2 µm polystyrene beads as well as Escherichia coli MG1655 strain. Measured zeta potentials for these samples were in agreement with literature values obtained by conventional measurement methods. Taken together, our data demonstrate the power of this workflow to broadly enable critical measurements of particle and bacterial zeta potential for numerous applications.

Self-Assembled Nanocoatings Protect Microbial Fertilizers for Climate-Resilient Agriculture.

Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their use is unsustainable. Pseudomonas chlororaphis is a nitrogen-fixing bacterium that could be used as a fertilizer replacement, but this microbe is delicate. It is sensitive to stressors, such as freeze-drying and high temperatures. Here, we demonstrate protection of P. chlororaphis from freeze-drying, high temperatures (50 oC), and high humidity using self-assembling metal-phenolic network (MPN) coatings. The composition of the MPN is found to significantly impact its protective efficacy, and with optimized compositions, no viability loss is observed for MPN-coated microbes under conditions where uncoated cells do not survive. Further, we demonstrate that MPN-coated microbes improve germination of seeds by 150% as compared to those treated with fresh P. chlororaphis. Taken together, these results demonstrate the protective capabilities of MPNs against environmental stressors and represent a critical step towards enabling the production and storage of delicate microbes under nonideal conditions.

Carbon Electrode-Based Biosensing Enabled by Biocompatible Surface Modification with DNA and Proteins.

Modification of electrodes with biomolecules is an essential first step for the development of bioelectrochemical systems, which are used in a variety of applications ranging from sensors to fuel cells. Gold is often used because of its ease of modification with thiolated biomolecules, but carbon screen-printed electrodes (SPEs) are gaining popularity due to their low cost and fabrication from abundant resources. However, their effective modification with biomolecules remains a challenge; the majority of work to-date relies on nonspecific adhesion or broad amide bond formation to chemical handles on the electrode surface. By combining facile electrochemical modification to add an aniline handle to electrodes with a specific and biocompatible oxidative coupling reaction, we can readily modify carbon electrodes with a variety of biomolecules. Importantly, both proteins and DNA maintain bioactive conformations following coupling. We have then used biomolecule-modified electrodes to generate microbial monolayers through DNA-directed immobilization. This work provides an easy, general strategy to modify inexpensive carbon electrodes, significantly expanding their potential as bioelectrochemical systems.

An Improved Spectrophotometric Method for Toluene-4-Monooxygenase Activity.

Monooxygenases, an important class of enzymes, have been the subject of enzyme engineering due to their high activity and versatile substrate scope. Reactions performed by these biocatalysts have long been monitored by a colorimetric method involving the coupling of a dye precursor to naphthalene hydroxylation products generated by the enzyme. Despite the popularity of this method, we found the dye product to be unstable, preventing quantitative readout. By incorporating an extraction step to solubilize the dye produced, we have improved this assay to the point where quantitation of enzyme activity is possible. Further, by incorporating spectral deconvolution, we have, for the first time, enabled independent quantification of the two possible regioisomeric products: 1-naphthol and 2-naphthol. Previously, such analysis was only possible with chromatographic separation, increasing the cost and complexity of analysis. The efficacy of our improved workflow was evaluated by monitoring the activity of a toluene-4-monooxygenase enzyme from Pseudomonas mendocina KR-1. Our colorimetric regioisomer quantification was found to be consistent with chromatographic analysis by HPLC. The development and validation of a quantitative colorimetric assay for monooxygenase activity that enables regioisomeric distinction and quantification represents a significant advance in analytical methods to monitor enzyme activity. By maintaining facile, low-cost, high-throughput readout while incorporating quantification, this assay represents an important alternative to more expensive chromatographic quantification techniques.

Recent advances in gold electrode fabrication for low-resource setting biosensing.

Gold electrodes are some of the most prevalent electrochemical biosensor substrate materials because they are readily functionalized with thiolated biomolecules. Yet, conventional methods to fabricate gold electrodes are costly and require onerous equipment, precluding them from implementation in low-resource settings (LRS). Recently, a number of alternative gold electrode fabrication methods have been developed to simplify and lower the cost of manufacturing. These methods include screen and inkjet printing as well as physical fabrication with common materials such as wire or gold leaf. All electrodes generated with these methods have successfully been functionalized with thiolated molecules, demonstrating their suitability for use in biosensors. Here, we detail recent advances in the fabrication, characterization and functionalization of these next-generation gold electrodes, with an emphasis on comparisons between cost and complexity with traditional cleanroom fabrication. We highlight gold leaf electrodes for their potential in LRS. This class of electrodes is anticipated to be broadly applicable beyond LRS due to their numerous inherent advantages.

Metal-phenolic networks as tuneable spore coat mimetics.

Bacillus subtilis are important probiotic microbes currently formulated for delivery as spores, but their ability to germinate in the gut remains debatable. To optimize their application, cells should be delivered in their vegetative state, but the sensitivity of B. subtilis prevents this. Through the application of self-assembled metal-phenolic network (MPN) cellular coatings, B. subtilis are protected from lyophilization stresses. These MPNs are an important class of self-assembled materials comprised of polyphenols and metal ions, and the efficacy of MPN protection was found to be dependent on the MPN components used for assembly. Both the size of the polyphenol and stability of the metal-phenol coordination were important factors that influenced their cellular protection; the smallest polyphenol, gallic acid, and the most stable chelated ion, FeIII, were found to provide the highest level of protection. Further, delivery to the gut involves exposure to acidic conditions in the form of stomach acid and intestinal fluid. MPN coatings rapidly disassemble upon mild acid treatment but were found to protect B. subtilis from the negative impacts of the acid. Overall, optimized MPNs were found to protect vegetative B. subtilis cells from lyophilization stress and enable a more complete understanding of the role of each component in MPNs.

Recent Advances in Signal Amplification to Improve Electrochemical Biosensing for Infectious Diseases.

The field of infectious disease diagnostics is burdened by inequality in access to healthcare resources. In particular, "point-of-care" (POC) diagnostics that can be utilized in non-laboratory, sub-optimal environments are appealing for disease control with limited resources. Electrochemical biosensors, which combine biorecognition elements with electrochemical readout to enable sensitive and specific sensing using inexpensive, simple equipment, are a major area of research for the development of POC diagnostics. To improve the limit of detection (LOD) and selectivity, signal amplification strategies have been applied towards these sensors. In this perspective, we review recent advances in electrochemical biosensor signal amplification strategies for infectious disease diagnostics, specifically biosensors for nucleic acids and pathogenic microbes. We classify these strategies into target-based amplification and signal-based amplification. Target-based amplification strategies improve the LOD by increasing the number of detectable analytes, while signal-based amplification strategies increase the detectable signal by modifying the transducer system and keep the number of targets static. Finally, we argue that signal amplification strategies should be designed with application location and disease target in mind, and that the resources required to produce and operate the sensor should reflect its proposed application, especially when the platform is designed to be utilized in low-resource settings. We anticipate that, based on current technologies to diagnose infectious diseases, incorporating signal-based amplification strategies will enable electrochemical POC devices to be deployed for illnesses in a wide variety of settings.

Metal-Phenolic Networks as Versatile Coating Materials for Biomedical Applications.

Polyphenols are naturally derived organic compounds that have long been used as food additives, antioxidants, and adhesives owing to their intrinsic physicochemical properties. Recently, there has been growing interest in the fabrication of coordination networks based on the self-assembly of polyphenols and metal ions, termed metal-phenolic networks (MPNs), for multiple biological applications including bioimaging, drug delivery, and cell encapsulation. The as-synthesized MPN complexes feature pH responsiveness, controllable size and rigidity, and tunable permeability based on the choice of polyphenol-metal ion pairs. The aim of this Review is to introduce the physicochemical properties of MPNs, highlight their recent biological applications in cancer theranostics and single-cell encapsulation, and discuss the future utility of MPNs for biomedical applications.

Surface Requirements for Optimal Biosensing with Disposable Gold Electrodes.

Electrochemical biosensors are promising technologies for detection and monitoring in low-resource settings due to their potential for easy use and low-cost instrumentation. Disposable gold screen-printed electrodes (SPEs) are popular substrates for these biosensors, but necessary dopants in the ink used for their production can interfere with biosensor function and contribute to the heterogeneity of these electrodes. We recently reported an alternative disposable gold electrode made from gold leaf generated using low-cost, equipment-free fabrication. We have directly compared the surface topology, biorecognition element deposition, and functional performance of three disposable gold electrodes: our gold leaf electrodes and two commercial SPEs. Our leaf electrodes significantly outperformed the SPEs for reproducible and effective biosensing in a DNase I assay and are nearly an order of magnitude less expensive than the SPEs. Therefore, these electrodes are promising for further development as point-of-care diagnostics, especially in low-resource settings.

Electricity, chemistry and biomarkers: an elegant and simple package: The potential of electrochemical biosensors for developing novel point-of-care diagnostics: The potential of electrochemical biosensors for developing novel point-of-care diagnostics.

Electrochemical sensors to measure biomarkers from complex samples are a tried and tested technology with large untapped potential for addressing important public health needs.

Perspective-Electrochemical Sensors for Neurotransmitters and Psychiatrics: Steps toward Physiological Mental Health Monitoring.

Therapeutic monitoring of neurotransmitters (NTs) and psychiatric medications is essential for the diagnosis and treatment of mental illness. However, in-vivo monitoring of NTs in humans as well as continuous physiological monitoring of psychiatrics have yet to be realized. In pursuit of this goal, there has been a plethora of work to develop electrochemical sensors for both in-vivo NT monitoring as well as in-vitro detection of psychiatric medications. We review these sensors here while discussing next steps needed to achieve concurrent, continuous physiological monitoring of NTs and psychiatric medications as part of a closed-loop feedback system that guides medication administration.

Protection of Anaerobic Microbes from Processing Stressors Using Metal-Phenolic Networks.

The gut microbiome is essential to maintain overall health and prevent disease, which can occur when these microbes are not in homeostasis. Microbial biotherapeutics are important to combat these issues, but they must be alive at the time of delivery for efficacy. Many potentially therapeutic species are anaerobes and thus are difficult to manufacture because of the limited efficacy of existing protective methods, making their production nearly impossible. We have developed a self-assembling cellular coating to improve the viability and stability of the next-generation biotherapeutic Bacteroides thetaiotaomicron. We show protection from both harsh processing conditions and oxygen exposure, even in the absence of canonical cryoprotectants. This advance will increase the range of microbes that can be stably manufactured and facilitate the development of emerging strains of interest by ensuring their postproduction viability.

A Microbial Electrochemical Technology to Detect and Degrade Organophosphate Pesticides.

Organophosphate (OP) pesticides cause hundreds of illnesses and deaths annually. Unfortunately, exposures are often detected by monitoring degradation products in blood and urine, with few effective methods for detection and remediation at the point of dispersal. We have developed an innovative strategy to remediate these compounds: an engineered microbial technology for the targeted detection and destruction of OP pesticides. This system is based upon microbial electrochemistry using two engineered strains. The strains are combined such that the first microbe (E. coli) degrades the pesticide, while the second (S. oneidensis) generates current in response to the degradation product without requiring external electrochemical stimulus or labels. This cellular technology is unique in that the E. coli serves only as an inert scaffold for enzymes to degrade OPs, circumventing a fundamental requirement of coculture design: maintaining the viability of two microbial strains simultaneously. With this platform, we can detect OP degradation products at submicromolar levels, outperforming reported colorimetric and fluorescence sensors. Importantly, this approach affords a modular, adaptable strategy that can be expanded to additional environmental contaminants.

Strategies for Engineering Affordable Technologies for Point-of-Care Diagnostics of Infectious Diseases.

Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS.The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold-thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for a fraction of the cost of commercially available gold electrodes.

Bioelectrochemical platforms to study and detect emerging pathogens.

The ongoing SARS-CoV-2 pandemic has emphasized the importance of technologies to rapidly detect emerging pathogens and understand their interactions with hosts. Platforms based on the combination of biological recognition and electrochemical signal transduction, generally termed bioelectrochemical platforms, offer unique opportunities to both sense and study pathogens. Improved bio-based materials have enabled enhanced control over the biotic-abiotic interface in these systems. These improvements have generated platforms with the capability to elucidate biological function rather than simply detect targets. This advantage is a key feature of recent bioelectrochemical platforms applied to infectious disease. Here, we describe developments in materials for bioelectrochemical platforms to study and detect emerging pathogens. The incorporation of host membrane material into electrochemical devices has provided unparalleled insights into the interaction between viruses and host cells, and new capture methods have enabled the specific detection of bacterial pathogens, such as those that cause secondary infections with SARS-CoV-2. As these devices continue to improve through the merging of hi-tech materials and biomaterials, the scalability and commercial viability of these devices will similarly improve.

Electrochemical Strategy for Low-Cost Viral Detection.

Sexually transmitted infections, including the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), disproportionally impact those in low-resource settings. Early diagnosis is essential for managing HIV. Similarly, HPV causes nearly all cases of cervical cancer, the majority (90%) of which occur in low-resource settings. Importantly, infection with HPV is six times more likely to progress to cervical cancer in women who are HIV-positive. An inexpensive, adaptable point-of-care test for viral infections would make screening for these viruses more accessible to a broader set of the population. Here, we report a novel, cost-effective electrochemical platform using gold leaf electrodes to detect clinically relevant viral loads. We have combined this platform with loop-mediated isothermal amplification and a CRISPR-based recognition assay to detect HPV. Lower limits of detection were demonstrated down to 104 total copies of input nucleic acids, which is a clinically relevant viral load for HPV DNA. Further, proof-of-concept experiments with cervical swab samples, extracted using standard extraction protocols, demonstrated that the strategy is extendable to complex human samples. This adaptable technology could be applied to detect any viral infection rapidly and cost-effectively.