Lateral flow immunoassay for rapid and sensitive detection of dsRNA contaminants in in vitro-transcribed mRNA products.

High purity is essential in mRNA-based therapeutic applications. A major contaminant of in vitro-transcribed (IVT) mRNA manufacturing is double-stranded RNA (dsRNA), which can induce severe anti-viral immune responses. Detection methods, such as agarose gel electrophoresis, ELISA, and dot-blot assay, are used to detect the existence of dsRNA in IVT mRNA products. However, these methods are either not sensitive enough or time-consuming. To overcome these challenges, we develop a rapid, sensitive, and easy-to-implement colloidal gold nanoparticle-based lateral flow strip assay (LFSA) with sandwich format for the detection of dsRNA from IVT process. dsRNA contaminant can be determined visually on the test strip or quantitatively with a portable optical detector. This method allows for a 15 min detection of N1-methyl-pseudouridine (m1Ψ)-containing dsRNA with a detection limit of 69.32 ng/mL. Furthermore, we establish the correlation between the LFSA test results and the immune response caused by dsRNA in mice. The LFSA platform allows the rapid, sensitive, and quantitative monitoring of purity in massive IVT mRNA products and aids for the prevention of immunogenicity by dsRNA impurities.

Photosynthetic biohybrid coculture for tandem and tunable CO2 and N2 fixation.

Solar-driven bioelectrosynthesis represents a promising approach for converting abundant resources into value-added chemicals with renewable energy. Microorganisms powered by electrochemical reducing equivalents assimilate CO2, H2O, and N2 building blocks. However, products from autotrophic whole-cell biocatalysts are limited. Furthermore, biocatalysts tasked with N2 reduction are constrained by simultaneous energy-intensive autotrophy. To overcome these challenges, we designed a biohybrid coculture for tandem and tunable CO2 and N2 fixation to value-added products, allowing the different species to distribute bioconversion steps and reduce the individual metabolic burden. This consortium involves acetogen Sporomusa ovata, which reduces CO2 to acetate, and diazotrophic Rhodopseudomonas palustris, which uses the acetate both to fuel N2 fixation and for the generation of a biopolyester. We demonstrate that the coculture platform provides a robust ecosystem for continuous CO2 and N2 fixation, and its outputs are directed by substrate gas composition. Moreover, we show the ability to support the coculture on a high-surface area silicon nanowire cathodic platform. The biohybrid coculture achieved peak faradaic efficiencies of 100, 19.1, and 6.3% for acetate, nitrogen in biomass, and ammonia, respectively, while maintaining product tunability. Finally, we established full solar to chemical conversion driven by a photovoltaic device, resulting in solar to chemical efficiencies of 1.78, 0.51, and 0.08% for acetate, nitrogenous biomass, and ammonia, correspondingly. Ultimately, our work demonstrates the ability to employ and electrochemically manipulate bacterial communities on demand to expand the suite of CO2 and N2 bioelectrosynthesis products.

Enhancing Biohybrid CO2 to Multicarbon Reduction via Adapted Whole-Cell Catalysts.

Catalytic CO2 conversion to renewable fuel is of utmost importance to establish a carbon-neutral society. Bioelectrochemical CO2 reduction, in which a solid cathode interfaces with CO2-reducing bacteria, represents a promising approach for renewable and sustainable fuel production. The rational design of biocatalysts in the biohybrid system is imperative to effectively reduce CO2 into valuable chemicals. Here, we introduce methanol adapted Sporomusa ovata (S. ovata) to enhance the slow metabolic activity of wild-type microorganisms to our semiconductive silicon nanowires (Si NWs) array for efficient CO2 reduction. The adapted whole-cell catalysts enable an enhancement of CO2 fixation with a superior faradaic efficiency on the poised Si NWs cathode. The synergy of the high-surface-area cathode and the adapted strain achieves a CO2-reducing current density of 0.88 ± 0.11 mA/cm2, which is 2.4-fold higher than the wild-type strain. This new generation of biohybrids using adapted S. ovata also decreases the charge transfer resistance at the cathodic interface and facilitates the faster charge transfer from the solid electrode to bacteria.

Photoelectrochemical CO2 Reduction toward Multicarbon Products with Silicon Nanowire Photocathodes Interfaced with Copper Nanoparticles.

The development of photoelectrochemical systems for converting CO2 into chemical feedstocks offers an attractive strategy for clean energy storage by directly utilizing solar energy, but selectivity and stability for these systems have thus been limited. Here, we interface silicon nanowire (SiNW) photocathodes with a copper nanoparticle (CuNP) ensemble to drive efficient photoelectrochemical CO2 conversion to multicarbon products. This integrated system enables CO2-to-C2H4 conversion with faradaic efficiency approaching 25% and partial current densities above 2.5 mA/cm2 at -0.50 V vs RHE, while the nanowire photocathodes deliver 350 mV of photovoltage under 1 sun illumination. Under 50 h of continual bias and illumination, CuNP/SiNW can sustain stable photoelectrochemical CO2 reduction. These results demonstrate the nanowire/catalyst system as a powerful modular platform to achieve stable photoelectrochemical CO2 reduction and the feasibility to facilitate complex reactions toward multicarbons using generated photocarriers.

Production of PHB From CO2-Derived Acetate With Minimal Processing Assessed for Space Biomanufacturing.

Providing life-support materials to crewed space exploration missions is pivotal for mission success. However, as missions become more distant and extensive, obtaining these materials from in situ resource utilization is paramount. The combination of microorganisms with electrochemical technologies offers a platform for the production of critical chemicals and materials from CO2 and H2O, two compounds accessible on a target destination like Mars. One such potential commodity is poly(3-hydroxybutyrate) (PHB), a common biopolyester targeted for additive manufacturing of durable goods. Here, we present an integrated two-module process for the production of PHB from CO2. An autotrophic Sporomusa ovata (S. ovata) process converts CO2 to acetate which is then directly used as the primary carbon source for aerobic PHB production by Cupriavidus basilensis (C. basilensis). The S. ovata uses H2 as a reducing equivalent to be generated through electrocatalytic solar-driven H2O reduction. Conserving and recycling media components is critical, therefore we have designed and optimized our process to require no purification or filtering of the cell culture media between microbial production steps which could result in up to 98% weight savings. By inspecting cell population dynamics during culturing we determined that C. basilensis suitably proliferates in the presence of inactive S. ovata. During the bioprocess 10.4 mmol acetate L -1 day-1 were generated from CO2 by S. ovata in the optimized media. Subsequently, 12.54 mg PHB L-1 hour-1 were produced by C. basilensis in the unprocessed media with an overall carbon yield of 11.06% from acetate. In order to illustrate a pathway to increase overall productivity and enable scaling of our bench-top process, we developed a model indicating key process parameters to optimize.

Molecular insights and future frontiers in cell photosensitization for solar-driven CO2 conversion.

The conversion of CO2 to value-added products powered with solar energy is an ideal solution to establishing a closed carbon cycle. Combining microorganisms with light-harvesting nanomaterials into photosynthetic biohybrid systems (PBSs) presents an approach to reaching this solution. Metabolic pathways precisely evolved for CO2 fixation selectively and reliably generate products. Nanomaterials harvest solar light and biocompatibly associate with microorganisms owing to similar lengths scales. Although this is a nascent field, a variety of approaches have been implemented encompassing different microorganisms and nanomaterials. To advance the field in an impactful manner, it is paramount to understand the molecular underpinnings of PBSs. In this perspective, we highlight studies inspecting charge uptake pathways and singularities in photosensitized cells. We discuss further analyses to more completely elucidate these constructs, and we focus on criteria to be met for designing photosensitizing nanomaterials. As a result, we advocate for the pairing of microorganisms with naturally occurring and highly biocompatible mineral-based semiconductor nanomaterials.

Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown.

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

Two-Step Patterning of Scalable All-Inorganic Halide Perovskite Arrays.

Halide perovskites have many important optoelectronic properties, including high emission efficiency, high absorption coefficients, color purity, and tunable emission wavelength, which makes these materials promising for optoelectronic applications. However, the inability to precisely control large-scale patterned growth of halide perovskites limits their potential toward various device applications. Here, we report a patterning method for the growth of a cesium lead halide perovskite single crystal array. Our approach consists of two steps: (1) cesium halide salt arrays patterning and (2) chemical vapor transport process to convert salt arrays into single crystal perovskite arrays. Characterizations including energy-dispersive X-ray spectroscopy and photoluminescence have been employed to confirm the chemical compositions and the optical properties of the as-synthesized perovskite arrays. This patterning method enables the patterning of single crystal cesium lead halide perovskite arrays with tunable spacing (from 2 to 20 µm) and crystal size (from 200 nm to 1.2 µm) in high production yield (almost every pixel in the array is successfully grown with converted perovskite crystals). Our large-scale patterning method renders a platform for the study of fundamental properties and opportunities for perovskite-based optoelectronic applications.

Solar-driven carbon dioxide fixation using photosynthetic semiconductor bio-hybrids.

Solar-driven conversion of carbon dioxide to value-added carbon products is an ambitious objective of ongoing research efforts. However, high overpotential, low selectivity and poor CO2 mass transfer plague purely inorganic electrocatalysts. In this instance, we can consider a class of biological organisms that have evolved to achieve CO2 fixation. We can harness and combine the streamlined CO2 fixation pathways of these whole organisms with the exceptional ability of semiconducting nanomaterials to harvest solar energy. A novel nanomaterial-biological interface has been pioneered in which light-capturing cadmium sulfide nanoparticles reside within individual organisms essentially powering biological CO2 fixation by solar energy. In order to further develop the photosensitized organism platform, more biocompatible photosensitizers and cytoprotective strategies are required as well as elucidation of charge transfer mechanisms. Here, we discuss the ability of gold nanoclusters to photosensitize a model acetogen effectively and biocompatibly. Additionally, we present innovative materials including two-dimensional metal organic framework sheets and alginate hydrogels to shield photosensitized cells. Finally, we delve into original work using transient absorption spectroscopy to inform on charge transfer mechanisms.

Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules.

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.

Bacteria photosensitized by intracellular gold nanoclusters for solar fuel production.

The demand for renewable and sustainable fuel has prompted the rapid development of advanced nanotechnologies to effectively harness solar power. The construction of photosynthetic biohybrid systems (PBSs) aims to link preassembled biosynthetic pathways with inorganic light absorbers. This strategy inherits both the high light-harvesting efficiency of solid-state semiconductors and the superior catalytic performance of whole-cell microorganisms. Here, we introduce an intracellular, biocompatible light absorber, in the form of gold nanoclusters (AuNCs), to circumvent the sluggish kinetics of electron transfer for existing PBSs. Translocation of these AuNCs into non-photosynthetic bacteria enables photosynthesis of acetic acid from CO2. The AuNCs also serve as inhibitors of reactive oxygen species (ROS) to maintain high bacterium viability. With the dual advantages of light absorption and biocompatibility, this new generation of PBS can efficiently harvest sunlight and transfer photogenerated electrons to cellular metabolism, realizing CO2 fixation continuously over several days.

Physical Biology of the Materials-Microorganism Interface.

Future solar-to-chemical production will rely upon a deep understanding of the material-microorganism interface. Hybrid technologies, which combine inorganic semiconductor light harvesters with biological catalysis to transform light, air, and water into chemicals, already demonstrate a wide product scope and energy efficiencies surpassing that of natural photosynthesis. But optimization to economic competitiveness and fundamental curiosity beg for answers to two basic questions: (1) how do materials transfer energy and charge to microorganisms, and (2) how do we design for bio- and chemocompatibility between these seemingly unnatural partners? This Perspective highlights the state-of-the-art and outlines future research paths to inform the cadre of spectroscopists, electrochemists, bioinorganic chemists, material scientists, and biologists who will ultimately solve these mysteries.

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world.

Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing.

Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST) is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm) that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 µL samples of a model bacterial strain (E. coli ATCC 25922) treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.