High-Precision Viral Detection Using Electrochemical Kinetic Profiling of Aptamer-Antigen Recognition in Clinical Samples and Machine Learning.

High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.

Integrating Water Purification with Electrochemical Aptamer Sensing for Detecting SARS-CoV-2 in Wastewater.

Wastewater analysis of pathogens, particularly SARS-CoV-2, is instrumental in tracking and monitoring infectious diseases in a population. This method can be used to generate early warnings regarding the onset of an infectious disease and predict the associated infection trends. Currently, wastewater analysis of SARS-CoV-2 is almost exclusively performed using polymerase chain reaction for the amplification-based detection of viral RNA at centralized laboratories. Despite the development of several biosensing technologies offering point-of-care solutions for analyzing SARS-CoV-2 in clinical samples, these remain elusive for wastewater analysis due to the low levels of the virus and the interference caused by the wastewater matrix. Herein, we integrate an aptamer-based electrochemical chip with a filtration, purification, and extraction (FPE) system for developing an alternate in-field solution for wastewater analysis. The sensing chip employs a dimeric aptamer, which is universally applicable to the wild-type, alpha, delta, and omicron variants of SARS-CoV-2. We demonstrate that the aptamer is stable in the wastewater matrix (diluted to 50%) and its binding affinity is not significantly impacted. The sensing chip demonstrates a limit of detection of 1000 copies/L (1 copy/mL), enabled by the amplification provided by the FPE system. This allows the integrated system to detect trace amounts of the virus in native wastewater and categorize the amount of contamination into trace (<10 copies/mL), medium (10-1000 copies/mL), or high (>1000 copies/mL) levels, providing a viable wastewater analysis solution for in-field use.

Development of Nucleic-Acid-Based Electrochemical Biosensors for Clinical Applications.

Nucleic acids are remarkable molecules. In addition to Watson-Crick base pairing, the different structural motifs of these molecules can bind non-nucleic acid targets or catalyze chemical reactions. Additionally, nucleic acids are easily modified with different molecules or functional groups. These properties make nucleic acids, particularly DNA, ideally suited for use in electrochemical biosensors, both as biorecognition elements and redox reporter probes. In this Minireview, we will review the historical evolution of nucleic acids as probes in electrochemical biosensors. We will then review the specific examples of nucleic-acid-based biosensors that have been evaluated for clinical use in the areas of infectious disease, cancer, or cardiovascular health.

Efficient bacterial disinfection based on an integrated nanoporous titanium dioxide and ruthenium oxide bifunctional approach.

The increasing lack of drinking water around the globe is of great concern. Although UV irradiation, photocatalysis, and electrocatalysis for bacterial disinfection have been widely explored, the synergistic kinetics involved in these strategies have not been reported to date. Herein, we report on an efficient and cost-effective strategy for the remediation of a model bacterium (E. coli), through the integration of photochemistry and electrochemistry based on a bifunctional electrode, which utilizes titanium (Ti) as the substrate, nanoporous titanium dioxide (TiO2) as a photocatalyst, and ruthenium oxide (RuO2) nanoparticles as an electrocatalyst. The nanoporous TiO2 was grown directly onto a Ti substrate via a three-step anodization process, and its photocatalytic activity was significantly enhanced by a facile electrochemical treatment. A high disinfection rate at 0.62 min-1, with >99.999% bacterial removal within 20 min was achieved using the novel TiO2/Ti/RuO2 bifunctional electrode. Complete bacterial disinfection was attained within 30 min as assessed by a spread plate method. Bacterial survival strategies, including a viable but non-culturable state of the bacteria, were also investigated during the bifunctional treatment process. The novel strategy demonstrated in this study has strong potential to be utilized for water purification and wastewater treatment as an advanced environmentally compatible technology.

Effective immobilization of alcohol dehydrogenase on carbon nanoscaffolds for ethanol biofuel cell.

An efficient approach for immobilizing alcohol dehydrogenase (ADH) while enhancing its electron transfer ability has been developed using poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT) cationic polymer and carbon nanoscaffolds. The carbon nanoscaffolds were comprised of single-walled carbon nanotubes (SWCNTs) wrapped with reduced graphene oxide (rGO). The ADH entrapped within the MADQUAT that was present on the carbon nanoscaffolds exhibited a high electron exchange capability with the electrode through its cofactor ß-nicotinamide adenine dinucleotide hydrate and ß-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NAD+/NADH) redox reaction. The advantages of the carbon nanoscaffolds used as the support matrix and the MADQUAT employed for the entrapment of ADH versus physisorption were demonstrated via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our experimental results showed a higher electron transfer, electrocatalytic activity, and rate constant for MADQUAT entrapped ADH on the carbon nanoscaffolds. The immobilization of ADH using both MADQUAT and carbon nanoscaffolds exhibited strong potential for the development of an efficient bio-anode for ethanol powered biofuel cells.

A high-performance enzyme entrapment platform facilitated by a cationic polymer for the efficient electrochemical sensing of ethanol.

Entrapment is one of the major approaches for enzyme immobilization; however, it suffers a few critical drawbacks including leakage and high mass transfer resistance to substrates. To address these challenges, herein we report on a new facile and effective enzyme entrapment platform using a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) (MADQUAT) on a single-wall carbon nanotube and reduced graphene oxide (SWCNT-rGO) nanohybrid thin film. To demonstrate this new approach, alcohol dehydrogenase (ADH) is employed as a model enzyme for the entrapment toward the design of an efficient electrochemical biosensor for the detection of ethanol. MADQUAT possesses strong electrostatic affinity with various negatively charged biomolecules; and our FTIR study has shown that there are no structural changes in the enzyme following the entrapment, with an excellent secondary structure association (r = 0.92). Our electrochemical measurements have shown that the entrapped ADH exhibits high ability to exchange electrons in the presence of the NAD+/NADH cofactor and that the SWCNT-rGO nanohybrid significantly enhances the biocatalytic activity of the immobilized ADH and the electrochemical oxidation of NADH in comparison with either SWCNTs or rGO. The ethanol biosensor developed in this study exhibits a fast response, wide linearity range, high sensitivity (26.27 µA mM-1 cm-2), remarkable low limit of detection (0.16 µM), high selectivity and high stability. The optimized biosensor has been further tested with real samples including wine, beer and blood alcohol, showing promising analytical and biomedical applications.

Carbon Nanomaterials Based Electrochemical Sensors/Biosensors for the Sensitive Detection of Pharmaceutical and Biological Compounds.

Electrochemical sensors and biosensors have attracted considerable attention for the sensitive detection of a variety of biological and pharmaceutical compounds. Since the discovery of carbon-based nanomaterials, including carbon nanotubes, C60 and graphene, they have garnered tremendous interest for their potential in the design of high-performance electrochemical sensor platforms due to their exceptional thermal, mechanical, electronic, and catalytic properties. Carbon nanomaterial-based electrochemical sensors have been employed for the detection of various analytes with rapid electron transfer kinetics. This feature article focuses on the recent design and use of carbon nanomaterials, primarily single-walled carbon nanotubes (SWCNTs), reduced graphene oxide (rGO), SWCNTs-rGO, Au nanoparticle-rGO nanocomposites, and buckypaper as sensing materials for the electrochemical detection of some representative biological and pharmaceutical compounds such as methylglyoxal, acetaminophen, valacyclovir, ß-nicotinamide adenine dinucleotide hydrate (NADH), and glucose. Furthermore, the electrochemical performance of SWCNTs, rGO, and SWCNT-rGO for the detection of acetaminophen and valacyclovir was comparatively studied, revealing that SWCNT-rGO nanocomposites possess excellent electrocatalytic activity in comparison to individual SWCNT and rGO platforms. The sensitive, reliable and rapid analysis of critical disease biomarkers and globally emerging pharmaceutical compounds at carbon nanomaterials based electrochemical sensor platforms may enable an extensive range of applications in preemptive medical diagnostics.

Outbreak of pandemic influenza A/H1N1 2009 in Nepal.

BACKGROUND: The 2009 flu pandemic is a global outbreak of a new strain of H1N1 influenza virus. Pandemic influenza A (H1N1) 2009 has posed a serious public health challenge world-wide. Nepal has started Laboratory diagnosis of Pandemic influenza A/H1N1 from mid June 2009 though active screening of febrile travellers with respiratory symptoms was started from April 27, 2009. RESULTS: Out of 609 collected samples, 302 (49.6%) were Universal Influenza A positive. Among the influenza A positive samples, 172(28.3%) were positive for Pandemic influenza A/H1N1 and 130 (21.3%) were Seasonal influenza A. Most of the pandemic cases (53%) were found among young people with ≤ 20 years. Case Fatality Ratio for Pandemic influenza A/H1N1 in Nepal was 1.74%. Upon Molecular characterization, all the isolated pandemic influenza A/H1N1 2009 virus found in Nepal were antigenically and genetically related to the novel influenza A/CALIFORNIA/07/2009-LIKE (H1N1)v type. CONCLUSION: The Pandemic 2009 influenza virus found in Nepal were antigenically and genetically related to the novel A/CALIFORNIA/07/2009-LIKE (H1N1)v type.

Loop-mediated isothermal amplification (LAMP) for the direct detection of human pulmonary infections with environmental (nontuberculosis) mycobacteria.

Most first-line anti-tuberculosis drugs have less in vitro activity against atypical mycobacteria. Loop-mediated isothermal amplification (LAMP) was used for the rapid diagnosis of mycobacterial species. The sensitivity of LAMP was 96.1% (49/51) in smear-positive and culture-positive sputum samples and 85.0% (17/20) in smear-negative and culture-positive samples. Of the 77 total LAMP-positive samples, 75 (97.4%) were identified as Mycobacterium tuberculosis and 2 (2.6%) as M. intracellulare. One of the M. intracellulare-infected cases was identified in a patient with suspected mycobacteriosis and another was found in a follow-up patient.