Evaluation of indirect sequence-specific magneto-extraction-aided LAMP for fluorescence and electrochemical SARS-CoV-2 nucleic acid detection.

Nucleic acid amplification tests (NAATs) such as quantitative real-time reverse transcriptase PCR (qRT-PCR) or isothermal NAATs (iNAATs) such as loop-mediated isothermal amplification (LAMP) require pure nucleic acid free of any polymerase inhibitors as its substrate. This in turn, warrants the use of spin-column mediated extraction with centralized high-speed centrifuges. Additionally, the utilization of centralized real-time fluorescence readout and TaqMan-like molecular probes in qRT-PCR and real-time LAMP add cost and restrict their deployment. To circumvent these disadvantages, we report a novel sample-to-answer workflow comprising an indirect sequence-specific magneto-extraction (also referred to as magnetocapture, magneto-preconcentration, or magneto-enrichment) for detecting SARS-CoV-2 nucleic acid. It was followed by in situ fluorescence or electrochemical LAMP. After in silico validation of the approach's sequence selectivity against SARS-CoV-2 variants of concern, the comparative performance of indirect and direct magnetocapture in detecting SARS-CoV-2 nucleic acid in the presence of excess host nucleic acid or serum was probed. After proven superior, the sensitivity of the indirect sequence-specific magnetocapture in conjunction with electrochemical LAMP was investigated. In each case, its sensitivity was assessed through the detection of clinically relevant 102 and 103 copies of target nucleic acid. Overall, a highly specific nucleic acid detection method was established that can be accommodated for either centralized real-time SYBR-based fluorescence LAMP or portable electrochemical LAMP.

A batch processed titanium-vanadium oxide nanocomposite based solid-state electrochemical sensor for zeptomolar nucleic acid detection.

Approaching a nucleic acid amplification test (NAAT) based diagnosis of a pathogen from an electrochemistry pathway is a relatively economical, decentralized, and yet highly sensitive route. This work aimed to construct an electrochemical biosensor with a 2-electrode geometry using a transition metal oxide (TMO) based sensing layer. A series of batch-processed TiO2-V2O5 (TVO) nanocomposite-based electrodes were fabricated to probe their electrochemical performance and attain a highly sensitive dual-electrode electrochemical sensor (DEES) compared to pristine V2O5. The XRD analysis of the electrodes confirmed the formation of a nanocomposite, while the XPS analysis correlated the formation of oxygen vacancies with improved electrical conduction measured via EIS and I-V characterization. Furthermore, the work demonstrated the application of the optimized electrode in electrochemical detection of end-point loop-mediated isothermal amplification (LAMP) readout for 101-104 copies (0.1 zeptomoles to 0.1 attomoles) of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) plasmid DNA and in vitro transcribed RNA in an aqueous solution. The device achieved a limit of detection as low as 2.5 and 0.25 copies per µL for plasmid DNA and in vitro transcribed RNA, respectively. The DEES was able to successfully detect in situ LAMP performed on magneto-extracted SARS-CoV-2 plasmid and RNA from (a) an aqueous solution, (b) a sample spiked with excess human genomic DNA, and (c) a serum-spiked sample. The DEES results were then compared with those of real-time fluorescence and commercially available screen-printed electrodes (SPEs).

Analysis of the leaching behavior of elements from coal combustion residues for better management.

In this study, fly ash, pond ash, bottom ash, slurry ash, raw water, pond water, and slurry samples were collected from Bokaro Thermal Power Plant, Bokaro, Jharkhand, India, and studied for the leachability of different elements by acid digestion and shake test at different liquid to solid (L/S) ratios. The raw water, pond water, slurry water, and leachates of acid digestion and shake tests were analyzed for the elements sodium (Na), potassium (K), calcium (Ca), iron (Fe), copper (Cu), cobalt (Co), manganese (Mn), cadmium (Cd), zinc (Zn), lead (Pb), nickel (Ni), and chromium (Cr). Shake test results confirmed that the water got saturated when the L/S ratio was equal to or above 10, indicating no further increase in concentration of elements at the L/S ratio of 10. Leaching behavior of Na from pond ash was not understood in the present study. In the study, the chemical composition showed that all the four types of ashes contain a small fraction of CaO (about 0.37 to 0.90 % by weight) and very high contents of SiO2 (about 55.14 to 58.34 % by weight) and Al2O3 (about 29.44 to 32.81 % by weight) that are the major composition of Portland cement. The study will help to understand the leachability potential of harmful elements present in fly ash, pond ash, bottom ash, and slurry ash under natural conditions and to take regulatory measures to protect the surface water, groundwater, and soil environment.

A low-noise solid-state nanopore platform based on a highly insulating substrate.

A solid-state nanopore platform with a low noise level and sufficient sensitivity to discriminate single-strand DNA (ssDNA) homopolymers of poly-A40 and poly-T40 using ionic current blockade sensing is proposed and demonstrated. The key features of this platform are (a) highly insulating dielectric substrates that are used to mitigate the effect of parasitic capacitance elements, which decrease the ionic current RMS noise level to sub-10 pA and (b) ultra-thin silicon nitride membranes with a physical thickness of 5 nm (an effective thickness of 2.4 nm estimated from the ionic current) are used to maximize the signal-to-noise ratio and the spatial depth resolution. The utilization of an ultra-thin membrane and a nanopore diameter as small as 1.5 nm allow the successful discrimination of 40 nucleotide ssDNA poly-A40 and poly-T40. Overall, we demonstrate that this platform overcomes several critical limitations of solid-state nanopores and opens the door to a wide range of applications in single-molecule-based detection and analysis.

Noise and its reduction in graphene based nanopore devices.

Ionic current fluctuations in graphene nanopore devices are a ubiquitous phenomenon and are responsible for degraded spatial and temporal resolution. Here, we descriptively investigate the impact of different substrate materials (Si and quartz) and membrane thicknesses on noise characteristics of graphene nanopore devices. To mitigate the membrane fluctuations and pin-hole defects, a SiNx membrane is transferred onto the substrate and a pore of approximately 70 nm in diameter is perforated prior to the graphene transfer. Comprehensive noise study reveals that the few layer graphene transferred onto the quartz substrate possesses low noise level and higher signal to noise ratio as compared to single layer graphene, without deteriorating the spatial resolution. The findings here point to improvement of graphene based nanopore devices for exciting opportunities in future single-molecule genomic screening devices.

Fabrication of alpha-Fe2O3 nanopowder modified glassy carbon electrode for applications in electrochemical sensing.

In the present study, Fe2O3 nanopowder has been grown by Ultrasonic mist chemical vapor deposition (UM-CVD), which is a promising method for large area deposition at low temperatures taking in to account of its simplicity, inexpensiveness and safety. Room temperature XRD results revealed prominent hematite phase with intense (104) reflection and was also in agreement with the HR-TEM results. In situ high temperature X-ray diffraction (XRD) studies clearly indicated the change of phase from hematite to magnetite as the temperature increases above 300 degrees C. The surface morphology and particle size distribution of Fe2O3 nanopowder were characterized using field emission scanning electron microscope (FE-SEM) and high resolution transmission electron microscope (HR-TEM), which revealed that the particles were spherical in nature and distributed in range of 50-100 nm. SQUID magnetometry results indicate the ferromagnetic nature of the nanopowder with crystallite size of 6 nm as calculated from M-H curve. Transmittance of approximately 55% and estimated direct band gap of 2.5 eV was observed. Further, the nanopowder was used to modify glassy carbon electrode (GCE) and the modified electrode was found to exhibit electrocatalytic activity for the oxidation of dopamine. It is expected that the nanopowder will exhibit promising applications in the development of sensors.

Fabrication and nanoindentation properties of TiN/NiTi thin films and their applications in electrochemical sensing.

Nanocrystalline TiN/NiTi thin films have been grown on silicon substrate by dc magnetron sputtering to improve the corrosion and mechanical properties of NiTi based shape memory alloys without sacrificing the phase transformation effect. Interestingly, the preferential orientation of the TiN films was observed to change from (1 1 1) to (2 0 0) with change in nature of sputtering gas from 70% Ar+30% N(2) to 100% N(2). In present study the influence of crystallographic orientation of TiN on mechanical and corrosion properties of TiN/NiTi thin films was investigated. TiN (2 0 0)/NiTi films were found to exhibit high hardness, high elastic modulus, and thereby better wear resistance as compared to pure NiTi and TiN (1 1 1)/NiTi films. Electrochemical test revealed that TiN coated NiTi film exhibits better corrosion resistance in 1M NaCl solution as compared to uncoated NiTi film. The application of TiN/NiTi films in the electrochemical sensing of dopamine, which has a critical physiological importance in Parkinson's disease, has been demonstrated. A comparison of voltammetric response of dopamine at silicon based electrodes modified with different nanocrystalline coatings indicated that these films catalyze the oxidation of dopamine.