Digital Surface-Enhanced Raman Spectroscopy-Lateral Flow Test Dipstick: Ultrasensitive, Rapid Virus Quantification in Environmental Dust.

This study introduces a novel surface-enhanced Raman spectroscopy (SERS)-based lateral flow test (LFT) dipstick that integrates digital analysis for highly sensitive and rapid viral quantification. The SERS-LFT dipsticks, incorporating gold-silver core-shell nanoparticle probes, enable pixel-based digital analysis of large-area SERS scans. Such an approach enables ultralow-level detection of viruses that readily distinguishes positive signals from background noise at the pixel level. The developed digital SERS-LFTs demonstrate limits of detection (LODs) of 180 fg for SARS-CoV-2 spike protein, 120 fg for nucleocapsid protein, and 7 plaque forming units for intact virus, all within <30 min. Importantly, digital SERS-LFT methods maintain their robustness and their LODs in the presence of indoor dust, thus underscoring their potential for accurate and reliable virus diagnosis and quantification in real-world environmental settings.

Machine learning-driven SERS fingerprinting of disintegrated viral components for rapid detection of SARS-CoV-2 in environmental dust.

Surveillance of airborne viruses in crowded indoor spaces is crucial for managing outbreaks, as highlighted by the SARS-CoV-2 pandemic. However, the rapid and on-site detection of fast-mutating viruses, such as SARS-CoV-2, in complex environmental backgrounds remains challenging. Our study introduces a machine learning (ML)-driven surface-enhanced Raman spectroscopy (SERS) approach for detecting viruses within environmental dust matrices. By decomposing intact virions into individual structural components via a Raman-background-free lysis protocol and concentrating them into nanogap SERS hotspots, we significantly enhance the SERS signal intensity and fingerprint information density from viral structural components. Utilizing Principal Component Analysis (PCA), we establish a robust connection between the SERS data of these structural components and their biological sequences, laying a solid foundation for virus detection through SERS. Furthermore, we demonstrate reliable quantitative detection of SARS-CoV-2 using identified SARS-CoV-2 peaks at concentrations down to 102 pfu/ml through Gaussian Process Regression (GPR) and a digital SERS methodology. Finally, applying a Principal Component Analysis-Linear Discriminant Analysis (PCA-LDA) algorithm, we identify SARS-CoV-2, influenza A virus, and Zika virus within an environmental dust background with over 86% accuracy. Therefore, our ML-driven SERS approach holds promise for rapid environmental virus monitoring to manage future outbreaks.

Voltage Modulation of Nanoplasmonic Metal Luminescence from Nano-Optoelectrodes in Electrolytes.

Metallic nanostructures supporting surface plasmon modes can concentrate optical fields and enhance luminescence processes from the metal surface at plasmonic hotspots. Such nanoplasmonic metal luminescence contributes to the spectral background in surface-enhanced Raman spectroscopy (SERS) measurements and is helpful in bioimaging, nanothermometry and chemical reaction monitoring applications. Although there is growing interest in nanoplasmonic metal luminescence, its dependence on voltage modulation has received limited attention in research investigations. Also, the hyphenated electrochemical surface-enhanced Raman spectroscopy (EC-SERS) technique typically ignores voltage-dependent spectral background information associated with nanoplasmonic metal luminescence due to limited mechanistic understanding and poor measurement reproducibility. Here, we report a combined experiment and theory study on dynamic voltage-modulated nanoplasmonic metal luminescence from hotspots at the electrode-electrolyte interface using multiresonant nanolaminate nano-optoelectrode arrays. Our EC-SERS measurements under 785 nm continuous wavelength laser excitation demonstrate that short-wavenumber nanoplasmonic metal luminescence associated with plasmon-enhanced electronic Raman scattering (PE-ERS) exhibits a negative voltage modulation slope (up to ≈30% V-1) in physiological ionic solutions. Furthermore, we have developed a phenomenological model to intuitively capture the plasmonic, electronic, and ionic characteristics at the metal-electrolyte interface to understand the observed dependence of the PE-ERS voltage modulation slope on voltage polarization and ionic strength. The current work represents a critical step toward the general application of nanoplasmonic metal luminescence signals in optical voltage biosensing, hybrid optical-electrical signal transduction, and interfacial electrochemical monitoring.

In Situ Spatiotemporal SERS Measurements and Multivariate Analysis of Virally Infected Bacterial Biofilms Using Nanolaminated Plasmonic Crystals.

In situ spatiotemporal biochemical characterization of the activity of living multicellular biofilms under external stimuli remains a significant challenge. Surface-enhanced Raman spectroscopy (SERS), combining the molecular fingerprint specificity of vibrational spectroscopy with the hotspot sensitivity of plasmonic nanostructures, has emerged as a promising noninvasive bioanalysis technique for living systems. However, most SERS devices do not allow reliable long-term spatiotemporal SERS measurements of multicellular systems because of challenges in producing spatially uniform and mechanically stable SERS hotspot arrays to interface with large cellular networks. Furthermore, very few studies have been conducted for multivariable analysis of spatiotemporal SERS datasets to extract spatially and temporally correlated biological information from multicellular systems. Here, we demonstrate in situ label-free spatiotemporal SERS measurements and multivariate analysis of Pseudomonas syringae biofilms during development and upon infection by bacteriophage virus Phi6 by employing nanolaminate plasmonic crystal SERS devices to interface mechanically stable, uniform, and spatially dense hotspot arrays with the P. syringae biofilms. We exploited unsupervised multivariate machine learning methods, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), to resolve the spatiotemporal evolution and Phi6 dose-dependent changes of major Raman peaks originating from biochemical components in P. syringae biofilms, including cellular components, extracellular polymeric substances (EPS), metabolite molecules, and cell lysate-enriched extracellular media. We then employed supervised multivariate analysis using linear discriminant analysis (LDA) for the multiclass classification of Phi6 dose-dependent biofilm responses, demonstrating the potential for viral infection diagnosis. We envision extending the in situ spatiotemporal SERS method to monitor dynamic, heterogeneous interactions between viruses and bacterial networks for applications such as phage-based anti-biofilm therapy development and continuous pathogenic virus detection.

Biomimetic Transparent Nanoplasmonic Meshes by Reverse-Nanoimprinting for Bio-Interfaced Spatiotemporal Multimodal SERS Bioanalysis.

Multicellular systems, such as microbial biofilms and cancerous tumors, feature complex biological activities coordinated by cellular interactions mediated via different signaling and regulatory pathways, which are intrinsically heterogeneous, dynamic, and adaptive. However, due to their invasiveness or their inability to interface with native cellular networks, standard bioanalysis methods do not allow in situ spatiotemporal biochemical monitoring of multicellular systems to capture holistic spatiotemporal pictures of systems-level biology. Here, a high-throughput reverse nanoimprint lithography approach is reported to create biomimetic transparent nanoplasmonic microporous mesh (BTNMM) devices with ultrathin flexible microporous structures for spatiotemporal multimodal surface-enhanced Raman spectroscopy (SERS) measurements at the bio-interface. It is demonstrated that BTNMMs, supporting uniform and ultrasensitive SERS hotspots, can simultaneously enable spatiotemporal multimodal SERS measurements for targeted pH sensing and non-targeted molecular detection to resolve the diffusion dynamics for pH, adenine, and Rhodamine 6G molecules in agarose gel. Moreover, it is demonstrated that BTNMMs can act as multifunctional bio-interfaced SERS sensors to conduct in situ spatiotemporal pH mapping and molecular profiling of Escherichia coli biofilms. It is envisioned that the ultrasensitive multimodal SERS capability, transport permeability, and biomechanical compatibility of the BTNMMs can open exciting avenues for bio-interfaced multifunctional sensing applications both in vitro and in vivo.

Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein spike for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live, attenuated, recombinant human respiratory syncytial virus expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200-fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunoglobulin A in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against virus variants of concern Alpha, Beta, and Delta. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in Phase 1 clinical trials as an intranasal COVID-19 vaccine.

Microporous Multiresonant Plasmonic Meshes by Hierarchical Micro-Nanoimprinting for Bio-Interfaced SERS Imaging and Nonlinear Nano-Optics.

Microporous mesh plasmonic devices have the potential to combine the biocompatibility of microporous polymeric meshes with the capabilities of plasmonic nanostructures to enhance nanoscale light-matter interactions for bio-interfaced optical sensing and actuation. However, scalable integration of dense and uniformly structured plasmonic hotspot arrays with microporous polymeric meshes remains challenging due to the processing incompatibility of conventional nanofabrication methods with flexible microporous substrates. Here, scalable nanofabrication of microporous multiresonant plasmonic meshes (MMPMs) is achieved via a hierarchical micro-/nanoimprint lithography approach using dissolvable polymeric templates. It is demonstrated that MMPMs can serve as broadband nonlinear nanoplasmonic devices to generate second-harmonic generation, third-harmonic generation, and upconversion photoluminescence signals with multiresonant plasmonic enhancement under fs pulse excitation. Moreover, MMPMs are employed and explored as bio-interfaced surface-enhanced Raman spectroscopy mesh sensors to enable in situ spatiotemporal molecular profiling of bacterial biofilm activity. Microporous mesh plasmonic devices open exciting avenues for bio-interfaced optical sensing and actuation applications, such as inflammation-free epidermal sensors in conformal contact with skin, combined tissue-engineering and biosensing scaffolds for in vitro 3D cell culture models, and minimally invasive implantable probes for long-term disease diagnostics and therapeutics.

Nanobiotechnology enabled approaches for wastewater based epidemiology.

The impacts of the ongoing coronavirus pandemic highlight the importance of environmental monitoring to inform public health safety. Wastewater based epidemiology (WBE) has drawn interest as a tool for analysis of biomarkers in wastewater networks. Wide scale implementation of WBE requires a variety of field deployable analytical tools for real-time monitoring. Nanobiotechnology enabled sensing platforms offer potential as biosensors capable of highly efficient and sensitive detection of target analytes. This review provides an overview of the design and working principles of nanobiotechnology enabled biosensors and recent progress on the use of biosensors in detection of biomarkers. In addition, applications of biosensors for analysis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus are highlighted as they relate to the potential expanded use of biosensors for WBE-based monitoring. Finally, we discuss the opportunities and challenges in future applications of biosensors in WBE for effective monitoring and investigation of public health threats.

Reusable Surface-Enhanced Raman Spectroscopy Membranes and Textiles via Template-Assisted Self-Assembly and Micro/Nanoimprinting.

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool for ultrasensitive fingerprint recognition of molecules with considerable potential in wearable biochemical sensing. However, previous efforts to fabricate wearable SERS devices by directly treating fabrics with plasmonic nanoparticles have generated a nonuniform assembly of nanoparticles, weakly adsorbed on fabrics via van der Waals forces. Here, we report the creation of washing reusable SERS membranes and textiles via template-assisted self-assembly and micro/nanoimprinting approaches. Uniquely, we employ the capillary force driven self-assembly process to generate micropatch arrays of Au nanoparticle (NP) aggregates within hydrophobic microstructured templates, which are then robustly bonded onto semipermeable transparent membranes and stretchable textiles using the UV-resist based micro/nanoimprinting technique. A mild reactive ion etching (RIE) treatment of SERS membranes and textiles can physically expose the SERS hotspots of Au NP-aggregates embedded within the polymer UV resist for further improvement of their SERS performance. Also, we demonstrate that the semipermeable transparent SERS membranes can keep the moisture content of meat from evaporating to enable stable in situ SERS monitoring of biochemical environments at the fresh meat surface. By contrast, stretchable SERS textiles can allow the spreading, soaking, and evaporation of solution analyte samples on the fabric matrix for continuous enrichment of analyte molecules at the hotspots in biochemical SERS detection. Due to the mechanical robustness of the UV-resist immobilized Au NP aggregates, simple detergent-water washing with ultrasound sonication or mechanical stirring can noninvasively clean contaminated hot spots to reuse SERS textiles. Therefore, we envision that washing reusable SERS membranes and textiles by template-assisted self-assembly and micro/nanoimprinting fabrication are promising for wearable biochemical sensing applications, such as wound monitoring and body fluid monitoring.

Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 µM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

Pregnancy in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare disabling genetic disorder characterized by progressive postnatal heterotopic ossification leading to cumulative disability. Heterotopic bone formation in FOP usually begins in early childhood following a series of painful, post-traumatic, inflammatory soft-tissue swellings known as flare-ups, which later undergo ossification resulting in the progressive immobilization of the chest wall, limbs and jaw by early adulthood. Pregnancy in FOP has occurred infrequently and reproductive decisions are a dilemma for an individual or couple with FOP. We present the clinical course, medical management and potential concerns of four cases of pregnancy in FOP.