A flap endonuclease 1-assisted universal viral nucleic acid sensing system using surface-enhanced Raman scattering.

The continued uncertainty of emerging infectious viral diseases has led to an extraordinary urgency to develop advanced molecular diagnostic tests that are faster, more reliable, simpler to use, and readily available than traditional methods. This study presents a system that can accurately and rapidly trace viral nucleic acids by employing flap endonuclease 1 (FEN1)-assisted specific DNA cleavage reactions and surface-enhanced Raman scattering (SERS)-based analysis. The designed Raman tag-labeled 5'- and 3'-flap provider DNA yielded structurally defined DNA substrates on magnetic nanoparticle surfaces when a target was present. The FEN1 enzyme subsequently processes the substrates formed via an invasive cleavage reaction, producing 5'-flap DNA products. Magnetic separation allows efficient purification of flap products from reaction mixtures. The isolated solution was directly applied onto high aspect-ratio plasmonic silver nanopillars serving as SERS-active substrates to induce amplified SERS signals. We verified the developed SERS-based sensing system using a synthetic target complementary to an avian influenza A (H9N2) virus gene and examined the detection performance of the system using complementary DNA (cDNA) derived from H9N2 viral RNA. As a result, we could detect a synthetic target with a detection limit of 41.1 fM with a single base-pair discrimination ability and achieved multiplexed detection capability for two targets. Using cDNA samples from H9N2 viruses, we observed a high concordance of R2 = 0.917 between the data obtained from SERS and the quantitative polymerase chain reaction. We anticipate that this enzyme-assisted SERS sensor may provide insights into the development of high-performance molecular diagnostic tools that can respond rapidly to viral pathogens.

Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes.

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

Application of Nanomaterials as an Advanced Strategy for the Diagnosis, Prevention, and Treatment of Viral Diseases.

The coronavirus disease (COVID-19) pandemic poses serious global health concerns with the continued emergence of new variants. The periodic outbreak of novel emerging and re-emerging infectious pathogens has elevated concerns and challenges for the future. To develop mitigation strategies against infectious diseases, nano-based approaches are being increasingly applied in diagnostic systems, prophylactic vaccines, and therapeutics. This review presents the properties of various nanoplatforms and discusses their role in the development of sensors, vectors, delivery agents, intrinsic immunostimulants, and viral inhibitors. Advanced nanomedical applications for infectious diseases have been highlighted. Moreover, physicochemical properties that confer physiological advantages and contribute to the control and inhibition of infectious diseases have been discussed. Safety concerns limit the commercial production and clinical use of these technologies in humans; however, overcoming these limitations may enable the use of nanomaterials to resolve current infection control issues via application of nanomaterials as a platform for the diagnosis, prevention, and treatment of viral diseases.

Advanced Nanomaterials for Preparedness Against (Re-)Emerging Viral Diseases.

While the coronavirus disease (COVID-19) accounts for the current global pandemic, the emergence of other unknown pathogens, named "Disease X," remains a serious concern in the future. Emerging or re-emerging pathogens continue to pose significant challenges to global public health. In response, the scientific community has been urged to create advanced platform technologies to meet the ever-increasing needs presented by these devastating diseases with pandemic potential. This review aims to bring new insights to allow for the application of advanced nanomaterials in future diagnostics, vaccines, and antiviral therapies, thereby addressing the challenges associated with the current preparedness strategies in clinical settings against viruses. The application of nanomaterials has advanced medicine and provided cutting-edge solutions for unmet needs. Herein, an overview of the currently available nanotechnologies is presented, highlighting the significant features that enable them to control infectious diseases, and identifying the challenges that remain to be addressed for the commercial production of nano-based products is presented. Finally, to conclude, the development of a nanomaterial-based system using a "One Health" approach is suggested. This strategy would require a transdisciplinary collaboration and communication between all stakeholders throughout the entire process spanning across research and development, as well as the preclinical, clinical, and manufacturing phases.