Recent trends in electrochemical biosensors of superoxide dismutases.

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

A miniaturized electrochemical assay for homocysteine using screen-printed electrodes with cytochrome c anchored gold nanoparticles.

Determination of homocysteine (HcySH) is highly beneficial in human physiology and pathophysiology for diagnosis and prognosis of cardiovascular diseases (CVD). Unfortunately, the practicability of the existing methodologies for the determination of HcySH is limited in terms of sample requirements, preparation time and instrumentation cost. To overcome these limitations, we have developed a new miniaturized electrochemical assay for HcySH in which cytochrome c (cyt c) immobilized on gold nanoparticle (GNP) modified screen printed carbon electrode (SPE) is employed as a biosensing element. The electrochemical characterization of the biosensor (cyt c-GNP-SPE) shows quasi-reversible redox peaks at the potentials 0 and -0.2 V, confirming the cyt c binding. The methodology of quantification is based on the electrochemical oxidation of HcySH by the Fe(3+)/Fe(2+) crevice of cyt c, observed at a potential of +0.56 V. Using the amperometric technique, the detection limit of HcySH is found to be 0.3 ± 0.025 µM in the linear range between 0.4 µM and 700 µM, with a sensitivity of 3.8 ± 0.12 nA µM(-1) cm(-2). The practical application of the present assay is validated through the measurement of HcySH in blood plasma samples and the selectivity is ensured by eliminating the impact of the common interfering biological substrates using a Nafion membrane. This biosensor shows striking analytical properties of good repeatability, reproducibility (2.85% SD) and high stability (83% of its initial current response after 4 weeks). This work paves the way for cheap, efficient and reliable point-of-care biosensors for screening one of the major causes of deaths both in the developed and developing countries.

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes.

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO2(-)) and nitrate (NO3(-)) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP-ZnO-SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 µM with a detection limit of 100 nM and sensitivity of 85.4 nA µM(-1). Furthermore, NO2(-) measurement showed linearity of 100 nM to 1mM with a detection limit of 100 nM for NO2(-) and sensitivity of 96.4 nA µM(-1). Then, the concentration of NO3(-) was measured after its enzymatic conversion into NO2(-). Using this assay, the concentrations of NO, NO2(-), and NO3(-) present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP-ZnO-SPCE and validated with the standard Griess method.

Copper, zinc superoxide dismutase and nitrate reductase coimmobilized bienzymatic biosensor for the simultaneous determination of nitrite and nitrate.

This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2(-)) and nitrate (NO3(-)) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2(-) oxidation observed at +0.8 V was linear from 100 nM to 1mM with a detection limit of 50 nM and sensitivity of 98.5 ± 1.7 nA µM(-1)cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3(-) reduction at -0.76 V exhibiting linear response from 500 nM to 10mM NO3(-) with a detection limit of 200 nM and sensitivity of 84.5 ± 1.56 nA µM(-1)cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2(-) and NO3(-) present in human plasma, whole blood and saliva samples.

Nanomaterial-based electrochemical biosensors for cytochrome c using cytochrome c reductase.

Emerging evidences have pointed out that the release of cytochrome c (cyt c) from mitochondria into cytosol is a critical step in the activation of apoptosis. This article presents a novel approach for the detection of mitochondrial cyt c release for the first time using cytochrome c reductase (CcR) immobilized on nanoparticles decorated electrodes. Two kinds of nanomaterial-based biosensor platforms were used: (a) carbon nanotubes (CNT) incorporated polypyrrole (PPy) matrix on Pt electrode and (b) self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in PPy-Pt. Scanning electron microscope was used to characterize the surface morphologies of the nanomaterial modified electrodes. Cyclic voltammograms of both the biosensors showed reversible redox peaks at -0.45 and -0.34 V vs Ag/AgCl, characteristic of CcR. In comparison, the CcR-CNT biosensor gave a detection limit of 0.5±0.03 µM cyt c, which was 4-fold better than the CcR-GNP biosensor (2±0.03 µM). Moreover, the CcR-CNT biosensor achieved a much larger linear range (1-1000 µM) over the CcR-GNP biosensor (5-600 µM) with 2-fold better sensitivity. The CcR-CNT-PPy-Pt biosensor was further applied to quantify the mitochondrial cyt c released in cytosol of A549 cells upon induction of apoptosis with doxorubicin, the results agreed well with standard western blot analysis.

Virtual electrochemical nitric oxide analyzer using copper, zinc superoxide dismutase immobilized on carbon nanotubes in polypyrrole matrix.

In this work, we have designed and developed a novel and cost effective virtual electrochemical analyzer for the measurement of NO in exhaled breath and from hydrogen peroxide stimulated endothelial cells using home-made potentiostat. Here, data acquisition system (NI MyDAQ) was used to acquire the data from the electrochemical oxidation of NO mediated by copper, zinc superoxide dismutase (Cu,ZnSOD). The electrochemical control programs (graphical user-interface software) were developed using LabVIEW 10.0 to sweep the potential, acquire the current response and process the acquired current signal. The Cu,ZnSOD (SOD1) immobilized on the carbon nanotubes in polypyrrole modified platinum electrode was used as the NO biosensor. The electrochemical behavior of the SOD1 modified electrode exhibited the characteristic quasi-reversible redox peak at the potential, +0.06 V vs. Ag/AgCl. The biological interferences were eliminated by nafion coated SOD1 electrode and then NO was measured selectively. Further, this biosensor showed a wide linear range of response over the concentration of NO from 0.1 µM to 1 mM with a detection limit of 0.1 µM and high sensitivity of 1.1 µA µM(-1). The electroanalytical results obtained here using the developed virtual electrochemical instrument were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.