Plasmonic Optical Wells-Based Enhanced Rate PCR.

Rapid, sensitive, inexpensive point-of-care molecular diagnostics are crucial for the efficient control of spreading viral diseases and biosecurity of global health. However, the gold standard, polymerase chain reaction (PCR) is time-consuming and expensive and needs specialized testing laboratories. Here, we report a low-cost yet fast, selective, and sensitive Plasmonic Optical Wells-Based Enhanced Rate PCR: POWER-PCR. We optimized the efficient optofluidic design of 3D plasmonic optical wells via the computational simulation of light-to-heat conversion and thermophoretic convection in a self-created plasmonic cavity. The POWER-PCR chamber with a self-passivation layer can concentrate incident light to accumulate molecules, generate rapid heat transfer and thermophoretic flow, and minimize the quenching effect on the naked Au surface. Notably, we achieved swift photothermal cycling of nucleic acid amplification in POWER-PCR on-a-chip in 4 min 24 s. The POWER-PCR will provide an excellent solution for affordable and sensitive molecular diagnostics for precision medicine and preventive global healthcare.

Highly porous hydrogels for efficient solar water evaporation.

Solar energy is a plentiful renewable resource on Earth, with versatile applications in both domestic and industrial settings, particularly in solar steam generation (SSG). However, current SSG processes encounter challenges such as low efficiency and the requirement for extremely high concentrations of solar irradiation. Interfacial evaporation technology has emerged as a solution to these issues, offering improved solar performance compared to conventional SSG processes. Nonetheless, its implementation introduces additional complexities and costs to system construction. In this study, we present the development of hydrophilic, three-dimensional network-structured hydrogels with high porosity and swelling ratio using a facile fabrication technique. We systematically varied the mixing ratios of four key ingredients (polyethylene glycol diacrylate, PEGDA; polyethylene glycol methyl-ether acrylate, PEGMA; phosphate-buffered saline, PBS; and 2-hydroxy-2-methylpropiophenone, PI) to control the mean pore size and swelling ratio of the hydrogel. Additionally, plasmonic gold nanoparticles were incorporated into the hydrogel using a novel methodology to enhance solar light absorption and subsequent evaporation efficiency. The resulting material exhibited a remarkable solar efficiency of 77% and an evaporation rate of 1.6 kg m-2 h-1 under standard solar illumination (one sun), comparable to those of state-of-the-art SSG devices. This high efficiency can be attributed to the synergistic effects of the hydrogel's unique composition and nanoparticle concentration. These findings offer a promising avenue for the development of highly efficient solar-powered evaporation applications.

Fabry-Perot Cavity Control for Tunable Raman Scattering.

The Fabry-Perot (FP) resonator is an intuitive and versatile optical structure owing to its uniqueness in light-matter interactions, yielding resonance with a wide range of wavelengths as it couples with photonic materials encapsulated in a dielectric cavity. Leveraging the FP resonator for molecular detection, a simple geometry of the metal-dielectric-metal structure is demonstrated to allow tuning of the enhancement factors (EFs) of surface-enhanced Raman scattering (SERS). The optimum near-field EF from randomly dispersed gold nano-gaps and dynamic modulation of the far-field SERS EF by varying the optical resonance of the FP etalon are systematically investigated by performing computational and experimental analyses. The proposed strategy of combining plasmonic nanostructures with FP etalons clearly reveals wavelength matching of FP resonance to excitation and scattering wavelengths plays a key role in determining the magnitude of the SERS EF. Finally, the optimum near-field generating optical structure with controlled dielectric cavity is suggested for a tunable SERS platform, and its dynamic SERS switching performance is confirmed by demonstrating information encryption through liquid immersion.

Spatiotemporally controlled drug delivery via photothermally driven conformational change of self-integrated plasmonic hybrid nanogels.

BACKGROUND: Spatiotemporal regulation is one of the major considerations for developing a controlled and targeted drug delivery system to treat diseases efficiently. Light-responsive plasmonic nanostructures take advantage due to their tunable optical and photothermal properties by changing size, shape, and spatial arrangement. RESULTS: In this study, self-integrated plasmonic hybrid nanogels (PHNs) are developed for spatiotemporally controllable drug delivery through light-driven conformational change and photothermally-boosted endosomal escape. PHNs are easily synthesized through the simultaneous integration of gold nanoparticles (GNPs), thermo-responsive poly (N-isopropyl acrylamide), and linker molecules during polymerization. Wave-optic simulations reveal that the size of the PHNs and the density of the integrated GNPs are crucial factors in modulating photothermal conversion. Several linkers with varying molecular weights are inserted for the optimal PHNs, and the alginate-linked PHN (A-PHN) achieves more than twofold enhanced heat conversion compared with others. Since light-mediated conformational changes occur transiently, drug delivery is achieved in a spatiotemporally controlled manner. Furthermore, light-induced heat generation from cellular internalized A-PHNs enables pinpoint cytosolic delivery through the endosomal rupture. Finally, the deeper penetration for the enhanced delivery efficiency by A-PHNs is validated using multicellular spheroid. CONCLUSION: This study offers a strategy for synthesizing light-responsive nanocarriers and an in-depth understanding of light-modulated site-specific drug delivery.

Synthesis and discovery of the first potent proteolysis targeting chimaera (PROTAC) degrader of AIMP2-DX2 as a lung cancer drug.

ARS-interacting multifunctional proteins 2 (AIMP2) is known to be a powerful tumour suppressor. However, the target AIMP2-DX2, AIMP2-lacking exon 2, is often detected in many cancer patients and cells. The predominant approach for targeting AIMP-DX2 has been attempted via small molecule mediated inhibition, but due to the lack of satisfactory activity against AIMP2-DX2, new therapeutic strategies are needed to develop a novel drug for AIMP2-DX2. Here, we report the use of the PROTAC strategy that combines small-molecule AIMP2-DX2 inhibitors with selective E3-ligase ligands with optimised linkers. Consequently, candidate compound 45 was found to be a degrader of AIMP2-DX2. Together, these findings demonstrate that our PROTAC technology targeting AIMP2-DX2 would be a potential new strategy for future lung cancer treatment.

Quantum Electrodynamic Behavior of Chlorophyll in a Plasmonic Nanocavity.

Plasmonic nanocavities have been used as a novel platform for studying strong light-matter coupling, opening access to quantum chemistry, material science, and enhanced sensing. However, the biomolecular study of cavity quantum electrodynamics (QED) is lacking. Here, we report the quantum electrodynamic behavior of chlorophyll-a in a plasmonic nanocavity. We construct an extreme plasmonic nanocavity using Au nanocages with various linker molecules and Au mirrors to obtain a strong coupling regime. Plasmon resonance energy transfer (PRET)-based hyperspectral imaging is applied to study the electrodynamic behaviors of chlorophyll-a in the nanocavity. Furthermore, we observe the energy level splitting of chlorophyll-a, similar to the cavity QED effects due to the light-matter interactions in the cavity. Our study will provide insight for further studies in quantum biological electron or energy transfer, electrodynamics, the electron transport chain of mitochondria, and energy harvesting, sensing, and conversion in both biological and biophysical systems.

Optical Detection of Copper Ions via Structural Dissociation of Plasmonic Sugar Nanoprobes.

Heavy metal ions are known to cause environmental pollution and several human diseases because of their inherent toxicity. Among them, Cu2+ is an essential element for the human body, but its continuous exposure and accumulation may cause adverse effects. Thus, copper ion levels in aquatic environments are strictly regulated by international standards. Herein, we demonstrate a simple optical method for detecting Cu2+ using plasmonic sugar nanoprobes (PSNs) composed of gold nanoparticles and polysaccharides. Gold precursors were reduced to nanoparticles and spontaneously embedded in the sugar-based polymeric network with the sulfated residues of carrageenan during the polymerization procedure. Owing to the abundant functional residues of PSNs and their affinity toward Cu2+, we observed the Cu2+-mediated preferential dissociation of the PSNs, resulting in absorbance spectral shifts and scattering shifts of the PSNs. Based on these plasmon band shifts, Cu2+ below the EPA regulation level of 20 µM can be easily detected by the optimized experimental condition. Additionally, the reaction mechanism between the PSNs and Cu2+ was elucidated by indepth spectroscopic analyses, which revealed that the increased binding of Cu2+ to the sulfate groups in the PSNs induces the eventual decomposition of the PSNs.

Colloidal Multiscale Assembly via Photothermally Driven Convective Flow for Sensitive In-Solution Plasmonic Detections.

The assembly of metal nanoparticles and targets to be detected in a small light probe volume is essential for achieving sensitive in-solution surface-enhanced Raman spectroscopy (SERS). Such assemblies generally require either chemical linkers or templates to overcome the random diffusion of the colloids unless the aqueous sample is dried. Here, a facile method is reported to produce 3D multiscale assemblies of various colloids ranging from molecules and nanoparticles to microparticles for sensitive in-solution SERS detection without chemical linkers and templates by exploiting photothermally driven convective flow. The simulations suggest that colloids sub 100 nm in diameter can be assembled by photothermally driven convective flow regardless of density; the assembly of larger colloids up to several micrometers by convective flow is significant only if their density is close to that of water. Consistent with the simulation results, the authors confirm that the photothermally driven convective flow is mainly responsible for the observed coassembly of plasmonic gold nanorods with either smaller molecules or larger microparticles. It is further found that the coassembly with the plasmonic nanoantennae leads to dramatic Raman enhancements of molecules, microplastics, and microbes by up to fivefold of magnitude compared to those measured in solution without the coassembly.

Discovery of benzodioxane analogues as lead candidates of AIMP2-DX2 inhibitors.

Aminoacyl-tRNA synthetase (ARS) interacting multifunctional protein2 (AIMP2) plays a vital role in protein synthesis. However, a splicing variant in which the second of the four exons of AIMP2 is deleted, inhibits the tumor suppression activity of AIMP2. Herein, we describe our discovery of series of potent AIMP2-DX2 inhibitors that are targeting lung cancer. Optimization of series using ligand-based drug design strategy led to discovery of compound 35, a potent AIMP2-DX2 inhibitor that is the most efficacious in H460 and A549 cells. This benzodioxane series may represent good starting points for further lead optimization of the identification potential drug candidates for the AIMP2-DX2 targeted treatment of lung cancer.

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening.

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa.

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103-104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and Km values of 111 ± 8 and 8.0 ± 3.5 µm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 µm and potential antibacterial activity against P. aeruginosa These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

Sensitive and Direct Optical Monitoring of Release and Cellular Uptake of Aqueous CO from CO-Releasing Molecules.

Dynamics of release and cellular uptake of aqueous CO from CO-releasing molecules (CORMs) significantly affect signaling and cell viability. So far, it has been mainly observed by IR, UV-visible, and fluorescence techniques, which suffer from poor sensitivity and slow response time. Here, we show how to directly probe the mass transfer of aqueous CO from CORMs to cells using a fluidic chamber integrated with live cells and Raman reporters of large-area Au@Pd core-shell nanoparticle assembly to emulate a physiologically relevant microenvironment. We sensitively and directly detect CO release from trace CORMs of as low as 100 nM by measuring the Raman transitions of CO via rapid chemisorption onto the surface of the Au@Pd nanoparticles. By using our method, we successfully observe the dynamics of CO release from CORM-2 despite its very short half-life. We also reveal that the initial rate of CO release from CORM-3 is dramatically decreased by tens to hundreds of times when exposed to physiologically relevant pH variations from 7.4 to 2.5, which can be attributed to the acid hydrolysis of the CO ligand. CORM-2 tends to quickly release CO regardless of pH, probably because of its rapid cleavage into two monomeric Ru complexes by the co-solvent. The decrease in the initial rate at lower temperatures is more significant for CORM-3 than for CORM-2. Finally, we observe that the cellular uptake of aqueous CO from CORM-3 by lung cancer cells is approximately 2 times higher than that of normal lung cells.

Thiophen urea derivatives as a new class of hepatitis C virus entry inhibitors.

To develop unique small-molecule inhibitors of hepatitis C virus (HCV), thiophen urea (TU) derivatives were synthesised and screened for HCV entry inhibitory activities. Among them, seven TU compounds exhibited portent anti-viral activities against genotypes 1/2 (EC50 < 30 nM) and subsequently, they were further investigated; based on the pharmacological, metabolic, pharmacokinetic, and safety profiles, J2H-1701 was selected as the optimised lead compound as an HCV entry inhibitor. J2H-1701 possesses effective multi-genotypic antiviral activity. The docking results suggested the potential interaction of J2H-1701 with the HCV E2 glycoprotein. These results suggest that J2H-1701 can be a potential candidate drug for the development of HCV entry inhibitors.

Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs.

Drug repositioning is the only feasible option to immediately address the COVID-19 global challenge. We screened a panel of 48 FDA-approved drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which were preselected by an assay of SARS-CoV. We identified 24 potential antiviral drug candidates against SARS-CoV-2 infection. Some drug candidates showed very low 50% inhibitory concentrations (IC50s), and in particular, two FDA-approved drugs-niclosamide and ciclesonide-were notable in some respects.

Dual Mode Rapid Plasmonic Detections of Chemical Disinfectants (CMIT/MIT) Using Target-Mediated Selective Aggregation of Gold Nanoparticles.

Chemical disinfectants such as 5-chloro-2-methylisothiazol-3(2H)-one/2-methyl-4-isothiazolin-3-one (CMIT/MIT) have been widely used in commercial products and humidifiers to prevent the growth of microorganisms. However, as continuous inhalation of CMIT/MIT is a fatal health risk, the concentration of its commercially available form is strictly regulated. Nonetheless, there are limited reports on effective methods for the quick and easy detection of CMIT/MIT. In this study, we have demonstrated rapid and convenient plasmonic methods for the dual-mode detection of CMIT/MIT using gold nanoplasmonic particles (GNPs) and understood the underlying molecular mechanism via additional analyses with microscopic and spectroscopic tools. In the presence of CMIT/MIT, the GNPs can rapidly aggregate due to molecular specific interactions with their capping agents and resultant reaction products. This target-mediated aggregation of the GNPs is represented by a visible color change of the solution from red to purple within just 3 min. By adjusting the reaction ratio between the CMIT/MIT and the GNPs, we could observe a marked color change at the regulation level (15 ppm) with naked eyes without any instruments. In addition, the concentration-dependent Raman spectral change in the reaction solution allows us to crosscheck the observed colorimetric responses both quantitatively and qualitatively based on molecular fingerprint spectra. Therefore, our detection protocol provides a powerful way to develop a high-throughput screening method to ensure that the level of the CMIT/MIT ingredients remains within the regulatory concentration.

Identification of inhibitors of Bcl-2 family protein-protein interaction by combining the BRET screening platform with virtual screening.

Bcl-2 family proteins play key roles in tumor initiation, progression, and resistance to therapy. Therefore, the protein-protein interactions (PPIs) between the pro-survival proteins, B-cell lymphoma (Bcl)-2 and Bcl-xL, and the pro-apoptotic proteins, Bax and Bak, could be attractive therapeutic targets for anti-cancer drug discovery. Here, we found new small molecules, BIP-A1001 and BIP-A2001 that modulated Bak/Bax and Bcl-xL interactions by combining the Nanoluc/YFP-based bioluminescence resonance energy transfer (BRET) assay with structure based virtual screening. In addition, we chose compounds with similar structures to BIP-A1001 and BIP-A2001 and tested their inhibitory effects using the BRET assay as a dose-response function. The results indicated that identifying compounds that inhibit interactions between Bak/Bax and Bcl-xL could be a promising approach to enhance cancer therapy.

Discovery of thienothiazolocarboxamide analogues as novel anti-tubercular agent.

In order to identify anti-tubercular agents with a novel scaffold, commercial libraries of small organic compounds were screened against a fluorescent strain of Mycobacterium tuberculosis H37Rv, using a dual phenotypic assay. Compounds were assessed against bacteria replicating in broth medium, as well as inside macrophages, and thienothiazolocarboxamide (TTCA) scaffold was identified as hit in both assays, with submicromolar inhibitory concentrations. Derivatives of TTCA were further synthesized and evaluated for their inhibitory effects on M.tuberculosis H37Rv. In the present study we report the structure-activity relationship of these TTCA derivatives. Compounds 28, 32 and 42 displayed good anti-tubercular activities, as well as favorable ADME and PK properties. Compound 42 exhibited excellent oral bioavailability in mice with high distribution to lungs, within 1 h. It was found to be efficacious in a dose dependent manner in a murine model of M. tuberculosis infection. Hence, compound 42 is now under evaluation as a potential lead candidate for treatment of tuberculosis.

Active Surface Hydrophobicity Switching and Dynamic Interfacial Trapping of Microbial Cells by Metal Nanoparticles for Preconcentration and In-Plane Optical Detection.

The surface hydrophobicity of a microbial cell is known to be one of the important factors in its adhesion to an interface. To date, such property has been altered by either genetic modification or external pH, temperature, and nutrient control. Here we report a new strategy to engineer a microbial cell surface and discover the unique dynamic trapping of hydrophilic cells at an air/water interface via hydrophobicity switching. We demonstrate the surface transformation and hydrophobicity switching of Escherichia coli (E. coli) by metal nanoparticles. By employing real-time dark-field imaging, we directly observe that hydrophobic gold nanoparticle-coated E. coli, unlike its naked counterpart, is irreversibly trapped at the air/water interface because of elevated hydrophobicity. We show that our surface transformation method and resulting dynamic interfacial trapping can be generally extended to Gram-positive bateria, Gram-negative bacteria, and fungi. As the dynamic interfacial trapping allows the preconcentration of microbial cells, high intensity of scattering light, in-plane focusing, and near-field enhancement, we are able to directly quantify E. coli as low as 1.0 × 103 cells/ml by using a smartphone with an image analyzer. We also establish the identification of different microbial cells by the characteristic Raman transitions directly measured from the interfacially trapped cells.

Target Engagement and Binding Mode of an Antituberculosis Drug to Its Bacterial Target Deciphered in Whole Living Cells by NMR.

Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc1:aa3, the major respiratory terminal oxidase in mycobacteria. The most advanced IPA called Q203 is currently in clinical trial. Using its derivative IPA317, we identified the atoms of the drug interacting with the cytochrome b in whole cells. NMR data and the self-organizing map algorithm were used to cluster a large set of drug-target complex models. The selected ensemble revealed IPA317 in a transient cavity of the cytochrome b, interacting directly with the residue T313, which is the site of spontaneous mutation conferring resistance to the IPA series. Our approach constitutes a pipeline to obtain atomic information on hit-target interactions in the cellular context.

Optical Detection of Small Metabolites for Biological Gas Conversion by using Metal Nanoparticle Monolayers Produced by Capillary-Assisted Transfer.

Detection of small metabolites is essential for monitoring and optimizing biological gas conversion. Currently, such detection is typically done by liquid chromatography with offline sampling. However, this method often requires large equipment with multiple separation columns and is at risk of serious microbial contamination during sampling. Here we propose real-time optical detection of small metabolites using uniform plasmonic nanoparticles monolayers produced by capillary-assisted transfer. We reproducibly fabricate metal nanoparticles monolayers with a diameter of ∼1 mm for the detection of acetate, butyrate, and glucose by a glass capillary tube. Metal nanoparticles monolayers are not only uniform in terms of average interparticle distance but also structurally stable under dynamic fluidic conditions. The monolayers resistant to fluid shear stress with surface-enhanced Raman scattering are able to reversibly monitor the concentration of acetate and sensitively detect acetate and glucose at levels as low as 10 µM, which is more than 2 orders of magnitude lower than the concentration range of typical biological gas conversion. In addition, structurally similar metabolites such as acetate and butyrate, when mixed, become distinguishable by our method.