Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosensors.

Bilirubin oxidases (BODs) [EC 1.3.3.5 - bilirubin: oxygen oxido-reductase] are enzymes that belong to the multicopper oxidase family and can oxidize bilirubin, diphenols, and aryl amines and reduce the oxygen by direct four-electron transfer from the electrode with almost no electrochemical overpotential. Therefore, BOD is a promising bioelectrocatalyst for (self-powered) biosensors and/or enzymatic fuel cells. The advantages of electrochemically active BOD enzymes include selective biosensing, biocatalysis for efficient energy conversion, and electrosynthesis. Owing to the rise in publications and patents, as well as the expanding interest in BODs for a range of physiological conditions, this Review analyzes scientific literature reports on BOD enzymes and current hypotheses on their bioelectrocatalysis. This Review evaluates the specific research outcomes of the BOD in enzyme (protein) engineering, immobilization strategies, and challenges along with their bioelectrochemical properties, limitations, and applications in the fields of (i) biosensors, (ii) self-powered biosensors, and (iii) biofuel cells for powering bioelectronics.

Bimetallic Cu-Zn Zeolitic Imidazolate Frameworks as Peroxidase Mimics for the Detection of Hydrogen Peroxide: Electrochemical and Spectrophotometric Evaluation.

A copper incorporated zeolitic imidazolate framework-8 (ZIF-8) has been synthesized and demonstrated to be a potential material for a peroxidase mimic. The resultant bimetallic Cu-Zn incorporated MOF is used for the dual mode sensing of hydrogen peroxide by following electrochemical as well as spectrophotometric methods. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate, spectrophotometric studies are carried out, and the steady state kinetic parameters are determined for two different concentrations of Cu incorporated ZIF-8 (viz Cu@ZIF-8-1 and Cu@ZIF-8-2). It is found that both Cu@ZIF-8-1 and Cu@ZIF-8-2 exhibit more affinity toward the TMB substrate than the horseradish peroxidase (HRP) enzyme as indicated by the low Km values obtained for the substrate. Also, as the concentration of incorporated Cu increases, Vmax values are also found to be enhanced. Electrochemically, the Cu@ZIF-8 modified glassy carbon electrode (GCE) showed a good response for peroxide detection in the concentration range from 0.5 mM to 5 mM at a working potential of -0.25 V in PBS (pH 7.0) with a limit of detection (LOD) value of 0.46 mM and a sensitivity of 20.25 µA/mM. Further, the chromogenic substrate TMB is successfully immobilized on the electrode surface and subsequently used for the peroxide detection along with Cu@ZIF-8. Here, TMB acts as a mediator and shifted the working potential to 0.1 V in acetate buffer (pH 5.0) in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.499 mM and a sensitivity of 0.097 µA/mM. Interestingly, the same electrode in PBS of pH 7.0 showed a response to peroxide at a working potential of -0.1 V in the concentration range from 0.5 mM to 5 mM with an LOD value of 0.143 mM and a sensitivity of 0.33 µA/mM. Moreover, the applicability of this material for peroxide sensing is evaluated using milk samples, and the proposed material is able to recover the peroxide present in milk. Thus, the bimetallic Cu-Zn MOF can be utilized for the dual mode sensing of peroxide and can be extended for various real time applications.

A machine learning-based approach to determine infection status in recipients of BBV152 (Covaxin) whole-virion inactivated SARS-CoV-2 vaccine for serological surveys.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

Electrochemical biosensor for serogroup specific diagnosis of leptospirosis.

A problem with the current leptospirosis diagnostic methods is the low sensitivity and specificity during the acute phase of illness. Rapid point-of-care (POC) assays with minimal sample utilization and low cost are desired in clinical practice. Here, we report for the first time lipopolysaccharide (LPS) based electrochemical biosensor that offers a rapid, highly sensitive, serogroup specific diagnosis of leptospirosis during the acute stage of infection and also to distinguish from other flu like infections. The proposed sensor is fabricated by the immobilization of LPS onto dodecanethiol (DT) modified gold electrode. Monolayer of DT is attached through covalent bond (Au-S) interaction onto the gold electrode. Thus, leptospiral antibodies from the human serum samples bind to the LPS present on self-assembled monolayer (SAM) of DT and showed a higher RCT value compared to SAM. The detection limit of the developed LPS sensor is estimated to be 100 nM. This biosensor is the first electrochemical sensing platform used for detection of LPS from Leptospira spp. This method is completely a solution-based diagnostic method and therefore it is rapid, simple, and sensitive; thus establishing a key technology towards a useful POC diagnostic strategy in serogroup level and hence an alternative to MAT.

A Nitronaphthalimide Probe for Fluorescence Imaging of Hypoxia in Cancer Cells.

The bioreductive enzymes typically upregulated in hypoxic tumor cells can be targeted for developing diagnostic and drug delivery applications. In this study, a new fluorescent probe 4-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzaldehyde (NIB) based on a nitronaphthalimide skeleton that could respond to nitroreductase (NTR) overexpressed in hypoxic tumors is designed and its application in imaging tumor hypoxia is demonstrated. The docking studies revealed favourable interactions of NIB with the binding pocket of NTR-Escherichia coli. NIB, which is synthesized through a simple and single step imidation of 4-nitro-1,8-naphthalic anhydride displayed excellent reducible capacity under hypoxic conditions as evidenced from cyclic voltammetry investigations. The fluorescence measurements confirmed the formation of identical products (NIB-red) during chemical as well as NTR-aided enzymatic reduction in the presence of NADH. The potential fluorescence imaging of hypoxia based on NTR-mediated reduction of NIB is confirmed using in-vitro cell culture experiments using human breast cancer (MCF-7) cells, which displayed a significant change in the fluorescence colour and intensity at low NIB concentration within a short incubation period in hypoxic conditions.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Magneto-Biosensor for the Detection of Uric Acid Using Citric Acid-Capped Iron Oxide Nanoparticles.

In this work, a magneto-biosensor based on iron (II, III) oxide (magnetite, Fe3O4) nanoparticles for the detection of uric acid is developed and demonstrated. These Fe3O4 nanoparticles are successfully synthesized by a co-precipitation method comprising Fe2+ and Fe3+ with ammonium hydroxide, NH4OH, and using citric acid as a surfactant. Comparative studies of Fe3O4 nanoparticles with and without surfactant are also carried out to examine their characteristics. Both types of synthesized iron oxide nanoparticles are characterized by X-ray diffraction, X-ray photoelectron spectroscopy, field emission-scanning electron microscopy, transmission electron microscopy, and vibrating sample magnetometry. The images obtained by field emission-scanning electron microscopy show an average diameter of 30 nm for citric acid-Fe3O4 nanoparticles. The Fourier transform infrared spectra indicate that the carboxylate groups of citric acid are bound onto the surface of magnetite nanoparticles by chemical bonds. For sensing experiments, the synthesized nanoparticles are used to modify the glassy carbon electrode, and the resultant citric acid-Fe3O4 modified glassy carbon electrode is used for the detection of uric acid through cyclic voltammetry. In the case of the citric acid-Fe3O4 nanoparticles-modified glassy carbon electrode, uric acid is oxidized at a less positive potential compared to oxidation using the naked Fe3O4 nanoparticles and a bare glassy carbon electrode. The citric acid-Fe3O4 nanoparticles-modified glassy carbon electrode exhibit a good linear response range for the detection of uric acid of 7.5 µM-0.18 mM, with a lower detection limit of 7.5 µM uric acid. This excellent performance of the fabricated biosensor is attributed to the larger surface area availability of citric acid-Fe3O4 nanoparticles, which promotes constant electron transfer between the modified glassy carbon electrode and the biomolecules (uric acid). The improved electrocatalytic activity of this modified electrode clearly proves that the proposed method is promising for the development of other electrochemical biosensors.

Development of CuAlO2-Encapsulated Reduced Graphene Oxide Nanocomposites: An Efficient and Selective Electrocatalyst for Detection of Neurodegenerative Disorders.

Carbon-based nanomaterials continue to simulate wide interest in diverse disciplines including electrochemical biosensors, which have great ability to function as next-generation clinical diagnostics. Motivated by this point, we for the first time developed a CuAlO2-encapsulated reduced graphene oxide (rGO) nanocomposite by a facile wet-chemical process to modify a glassy carbon electrode for dopamine detection with high selectivity and good sensitivity. The size, shape, phase purity, chemical composition, and surface area were investigated for the samples through transmission electron microscopy, scanning electron microscopy, high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and Brunauer-Emmett-Teller analysis. The electrocatalytic performance was studied using cyclic voltammetry and amperometric technique. The modified rGO/CuAlO2 nanocomposite electrode showed an enhanced electrochemical performance compared to other electrodes and pure CuAlO2 electrodes due to the strong promoting effect between rGO and CuAlO2. Both the oxidation current and concentration were proportional and show a linear range of 9.2 × 10-8 to 1.6 × 10-7 M having a detection limit of 15 nM at S/N = 3. Further, the biosensor successfully neglected the interference of ascorbic and uric acid and exhibited enhanced selectivity, improved sensitivity, and stability toward dopamine formulations. Most obviously, the real-time analysis of the electrochemical biosensor may be proved using the clinical diagnostics in the near future.

Biosynthesis of Copper Oxide (CuO) Nanowires and Their Use for the Electrochemical Sensing of Dopamine.

A facile one-step, eco-friendly, and cost-effective approach for the formation of copper oxide (CuO) nanowires by a green method using saponin-rich Sapindus mukorossi fruit extract (SMFE). The physio-chemical characteristics of the synthesized CuO nanowires have been characterized by X-ray Diffractometry (XRD), X-ray Photoelectron Spectroscopy (XPS), FT-IR (Fourier Transform Infrared Spectroscopy, FE-SEM (Scanning Electron Microscopy), and High-Resolution Transmission Electron Microscopy (HR-TEM). Further, the electrocatalytic activity of the CuO nanowires synthesized with SMFE has been investigated, and they have been used as dopamine (DA) sensors. Because of their unique properties, the CuO nanowires/GCE exhibited remarkable electrochemical response for the detection of DA with enhanced current response. The anodic current demonstrated that the CuO nanowires/GCE linearly detects the concentration of DA over the range of 0.1 µM to 0.105 mM of DA with a regression co-efficient of 0.9960. The obtained results illustrated that the synthesized CuO nanowires can easily stimulate the electron transfer reaction between DA and the nanowires modified electrode with the improvement of the conductivity and stability of the electrode. This remarkable electrocatalytic property of CuO nanowires makes it a unique electrochemical sensor for the detection of DA. Furthermore, the sensor is free from the interference of ascorbic acid, uric acid, and other interfering species. Moreover, the anti-interference performance also showed that the CuO nanowires/GCE could be employed for the determination of DA in real samples with good selectivity and sensitivity.