Rapid and Multiplexed Nucleic Acid Detection using Programmable Aptamer-Based RNA Switches.

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/µL in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays.

Elimination of Multidrug-Resistant Bacteria by Transition Metal Dichalcogenides Encapsulated by Synthetic Single-Stranded DNA.

Antibiotic-resistant bacteria are a significant and growing threat to human health. Recently, two-dimensional (2D) nanomaterials have shown antimicrobial activity and have the potential to be used as new approaches to treating antibiotic resistant bacteria. In this Research Article, we exfoliate transition metal dichalcogenide (TMDC) nanosheets using synthetic single-stranded DNA (ssDNA) sequences, and demonstrate the broad-spectrum antibacterial activity of MoSe2 encapsulated by the T20 ssDNA sequence in eliminating several multidrug-resistant (MDR) bacteria. The MoSe2/T20 is able to eradicate Gram-positive Escherichia coli and Gram-positive Staphylococcus aureus at much lower concentrations than graphene-based nanomaterials. Eradication of MDR strains of methicillin-resistant S. aureus (MRSA), Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii are shown to occur at at 75 µg mL-1 concentration of MoSe2/T20, and E. coli at 150 µg mL-1. Molecular dynamics simulations show that the thymine bases in the T20 sequence lie flat on the MoSe2 surface and can, thus, form a very good conformal coating and allow the MoSe2 to act as a sharp nanoknife. Electron microscopy shows the MoSe2 nanosheets cutting through the cell membranes, resulting in significant cellular damage and the formation of interior voids. Further assays show the change in membrane potential and reactive oxygen species (ROS) formation as mechanisms of antimicrobial activity of MoSe2/T20. The cellular death pathways are also examined by mRNA expression. This work shows that biocompatible TMDCs, specifically MoSe2/T20, is a potent antimicrobial agent against MDR bacteria and has potential for clinical settings.

Exfoliation of boron carbide into ultrathin nanosheets.

Liquid phase exfoliation (LPE) is a method that can be used to produce bulk quantities of two-dimensional (2D) nanosheets from layered van der Waals (vdW) materials. In recent years, LPE has been applied to several non-vdW materials with anisotropic bonding to produce nanosheets and platelets, but it has not been demonstrated for materials with strong isotropic bonding. In this paper, we demonstrate the exfoliation of boron carbide (B4C), the third hardest known material, into ultrathin nanosheets. B4C has a structure consisting of strongly bonded boron icosahedra and carbon chains, but does not have anisotropic cleavage energies to suggest that it can be readily cleaved into nanosheets. B4C has been widely studied for its very high melting point, high mechanical strength, and chemical stability, as well as its zero- and one-dimensional nanostructured forms. Herein, ultrathin nanosheets are successfully prepared by sonication of B4C powder in organic solvents and are characterized by microscopy and spectroscopy. Density functional theory (DFT) simulations reveal that B4C can be cleaved along several different crystallographic planes with similar energetic favourability, facilititated by an unexpected mechanism of breaking boron icosahedra and forming new boron-rich cage structures at the surface. Atomic force microscopy (AFM) shows that the nanosheets produced by LPE are as thin as 5 nm, with an average thickness of 31.4 nm and average area of 16 000 nm2. Raman spectroscopy shows that many of the nanosheets exhibit additional carbon-rich peaks that change with laser irradiation, which are attributed to atomic rearrangements and amorphization at the nanosheet surfaces, consistent with the diverse cleavage planes. High-resolution transmission electron microscopy (HRTEM) demonstrates that many different cleavage planes exist among the exfoliated nanosheets, in agreement with DFT simulations. This work elucidates the exfoliation mechanism of 2D B4C and suggests that LPE can be applied to generate nanosheets from a variety of non-layered and non-vdW materials.

'Urease immobilized single-kit' for sensing of thiourea-glucose pair employing fluorescence 'Turn off - Turn on' and as an efficient sorbent for selective sample cleanup of thiourea.

The tenfold lowering in binding energy for TU-Tyrosine in immobilized urease (Kb: 4.7 × 103) with respect to the native enzyme (Kb: 6.5 × 104) begets easy desorption of thiourea (TU) by glucose (GL) with an eventual formation of a more strong TU- GL adduct; that rejuvenates the kit-material ready for the subsequent cycle(s). The sorption-desorption heeds fluorescence turn-off and turn-on in DCM for selective sensing of TU- GL pair at their respective linear range of concentration 2.5-26.1 ppm and 2.36-11.57 ppm. The process was found to be static (KSV ≥ 2.25 × 103 L mol-1), exothermic (ΔH: -0.08 kJ mol-1), spontaneous (ΔG: -21.1 kJ mol-1) and marginally entropy gaining (ΔS: 0.07 kJ mol-1 K-1). The 'bulk material' (200 ± 20 µm) brilliantly preconcentrates TU with an enrichment factor of 106.2 after its selective extraction at near-neutral pH from a large volume sample (800 mL) of low concentration (30 ppm). A very dilute solution (0.05 mmol L-1) of GL at minimum volume (6 mL) acts as a stripping agent and provides a longer life (200 cycles with good extraction efficiency) to the material. The method was found to be efficient in the analysis of fruit juice as a real sample.

Exuberant Immobilization of Urease on an Inorganic SiO2 Support Enhances the Enzymatic Activities by 3-fold for Perennial Utilization.

Urease has been covalently immobilized on a 3-D networking silica gel (SG) using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and m-nitroaniline as linker component in a robust methodology and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2] n=4{OSi(CH3)2-NH-C6H4-N═N-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable m-nitroaniline modified SG unit prevents enzyme-enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by 3-fold (for both synthetic and real sample) compared to native enzyme values at neutral pH. Up to 30 days and 30 cycles, this 3-fold activity remains as such but reduces gradually to native enzyme level after 60 days and 60 cycles of reuse.