Vepa Kameswara Rao

Immobilization of acetylcholineesterase-choline oxidase on a gold-platinum bimetallic nanoparticles modified glassy carbon electrode for the sensitive detection of organophosphate pesticides, carbamates and nerve agents.

A novel, highly sensitive amperometric biosensor, based on electrodeposition of gold-platinum bimetallic nanoparticles onto 3-aminopropyltriethoxy silane modified glassy carbon electrode for the detection of paraoxon ethyl, aldicarb, and sarin has been developed. The biosensor consists of acetylcholineesterase (AChE)/choline oxidase (ChOx) immobilized by cross-linking with glutaraldehyde on a modified electrode. The properties of nanoparticles modified electrodes are characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Au and Pt nanoparticles showed excellent electrocatalytic activity with low applied potential for the detection of hydrogen peroxide (H(2)O(2)). The IC(50) and inhibition rate constant (K(i)) values were determined for the inhibitors using immobilized enzymes on modified electrode and the data were compared by spectrophotometric determination of these kinetic parameters using free enzymes in solution. Paraoxon ethyl, sarin, and aldicarb could be detected up to 150-200nM, 40-50nM, and 40-60 microM respectively at 30-40% inhibition level of AChE enzyme and followed linearity in wide range concentration.

Highly Sensitive Amperometric Immunosensor for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Serum of Humans with Malaria: Comparison with a Commercial Kit

ABSTRACT A disposable amperometric immunosensor was developed for the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in the sera of humans with P. falciparum malaria. For this purpose, disposable screen-printed electrodes (SPEs) were modified with multiwall carbon nanotubes (MWCNTs) and Au nanoparticles. The electrodes were characterized by cyclic voltammetry, scanning electron microscopy, and Raman spectroscopy. In order to study the immunosensing performances of modified electrodes, a rabbit anti-PfHRP-2 antibody (as the capturing antibody) was first immobilized on an electrode. Further, the electrode was exposed to a mouse anti-PfHRP-2 antibody from a serum sample (as the revealing antibody), followed by a rabbit anti-mouse immunoglobulin G-alkaline phosphatase conjugate. The immunosensing experiments were performed on bare SPEs, MWCNT-modified SPEs, and Au nanoparticle- and MWCNT-modified SPEs (Nano-Au/MWCNT/SPEs) for the amperometric detection of PfHRP-2 in a solution of 0.1 M diethanolamine buffer, pH 9.8, by applying a potential of 450 mV at the working electrode. Nano-Au/MWCNT/SPEs yielded the highest-level immunosensing performance among the electrodes, with a detection limit of 8 ng/ml. The analytical results of immunosensing experiments with human serum samples were compared with the results of a commercial Paracheck Pf test, as well as the results of microscopy. The specificities, sensitivities, and positive and negative predictive values of the Paracheck Pf and amperometric immunosensors were calculated by taking the microscopy results as the “gold standard.” The Paracheck Pf kit exhibited a sensitivity of 79% (detecting 34 of 43 positive samples; 95% confidence interval [CI], 75 to 86%) and a specificity of 81% (correctly identifying 57 of 70 negative samples; 95% CI, 76 to 92%), whereas the developed amperometric immunosensor showed a sensitivity of 96% (detecting 41 of 43 positive samples; 95% CI, 93 to 98%) and a specificity of 94% (correctly identifying 66 of 70 negative samples; 95% CI, 92 to 99%). The developed method is more sensitive and specific than the Paracheck Pf kit.

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.

Amperometric immunosensor for ricin by using on graphite and carbon nanotube paste electrodes.

Ricin is a scheduled chemical warfare agent and biological warfare agent. Attempts were made for the detection of ricin in water samples by utilizing amperometric immunosensors. These electrodes were made by mixing Paraffin oil with graphite powder and multiwalled carbon nanotubes. The graphite paste electrode (CPE) and multiwalled carbon nanotubes paste electrode (MWCNTPE) were tested for their ability to detect 1-naphthol. A sandwich enzyme linked immunosorbent assay system was used to detect ricin. The detection limit for both electrodes was compared. It was found that the response of amperometric sensor is proportional to the ricin concentration in both the cases and is linear in the range 0.625-25 ng/ml for MWCNTPE and 2.5-25 ng/ml for CPE. The SEM showed that the MWCNTPE has revealed crevices/voids in which the antibodies may get trapped. Spectroscopic experiments proved that MWCNTPE adsorbs antibodies better than CPE. The high sensitivity of MWCNTPE was attributed to its better electrochemical properties rather than to its efficiency to adsorb antibodies.

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW(®) Malaria).

An electrochemical genosensor for Salmonella typhi on gold nanoparticles-mercaptosilane modified screen printed electrode.

In this work, we fabricated a system of integrated self-assembled layer of organosilane 3-mercaptopropyltrimethoxy silane (MPTS) on the screen printed electrode (SPE) and electrochemically deposited gold nanoparticle for Salmonella typhi detection employing Vi gene as a molecular marker. Thiolated DNA probe was immobilized on a gold nanoparticle (AuNP) modified SPE for DNA hybridization assay using methylene blue as redox (electroactive) hybridization indicator, and signal was monitored by differential pulse voltammetry (DPV) method. The modified SPE was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) method. The DNA biosensor showed excellent performances with high sensitivity and good selectivity. The current response was linear with the target sequence concentrations ranging from 1.0 × 10(-11) to 0.5 × 10(-8)M and the detection limit was found to be 50 (± 2.1)pM. The DNA biosensor showed good discrimination ability to the one-base, two-base and three-base mismatched sequences. The fabricated genosensor could also be regenerated easily and reused for three to four times for further hybridization studies.

Chemiresistive gas sensor for the sensitive detection of nitrogen dioxide based on nitrogen doped graphene nanosheets

In this paper, we report on the development of a chemiresistive sensor for the detection of nitrogen dioxide (NO2) gas at room temperature using nitrogen-doped graphene nanosheets (NGS). The substitution of the nitrogen atoms in the honey-comb structure of graphene enhances the adsorption sites for gas molecules and thereby the sensitivity of the detection of the adsorbed gas molecules increases. Graphene nanosheets (GS) and NGS were prepared by hydrothermal treatment of graphene oxide in the absence and presence of nitrogen precursor respectively. The sensing materials were characterized by FESEM, TEM, XRD, XPS and elemental analysis. The nitrogen content in as-prepared NGS is at around 10%. The thin films of GS and NGS on pre-patterned gold interdigitated electrodes (IDEs) were obtained by the drop-drying method. The NGS coated sensor showed good response for sensing NO2 in comparison to that of GS at room temperature. The recovery of the sensor was greatly accelerated by ultra-violet light illumination. The proposed sensor showed excellent characteristics such as a low detection limit of 120 ppb (at S/N = 3). The effect of humidity on sensor performance was also studied. The proposed sensor also showed excellent selectivity with respect to various common interfering gases.

Application of bimetallic nanoparticles modified screen printed electrode for the detection of organophosphate compounds using an enzyme inhibition approach

A novel, unique gold–platinum bimetallic nanoparticles modified screen printed electrode (SPE) has been developed for the sensitive detection of paraoxon ethyl, carbofuran and simulant of nerve agent diisopropyl fluorophosphate (DFP), based on the enzyme inhibition approach. The gold–platinum nanoparticles were electrochemically deposited on a 3-aminopropyl triethoxy silane modified electrode. Following this acetylcholineesterase (AChE)/choline oxidase (ChOx) bienzymes were immobilized through cross-linking with glutaraldehyde onto a modified electrode. Electrodes were characterized by scanning electron microscopy (SEM), EDX (energy dispersive X-ray analysis) and cyclic voltammetry (CV). The bimetallic alloy nanoparticles have an excellent high surface area and the unique effect of electrocatalytic activity for the oxidation of hydrogen peroxide (H2O2). The IC50 values were determined for the inhibitors using immobilized enzymes on the modified electrode. Paraoxon ethyl, carbofuran, and DFP could be detected up to 0.20 μM, 0.20–0.25 μM, and 0.20–0.25 μM respectively at a 25–30% inhibition level of AChE enzyme (residual enzyme activity) with an incubation time of 10 min.

Relative efficiency of zinc sulfide (ZnS) quantum dots (QDs) based electrochemical and fluorescence immunoassay for the detection of Staphylococcal enterotoxin B (SEB)

In this paper an attempt was made to detect Staphylococcal enterotoxin B (SEB) both by electrochemical and fluorescence immunoassay methods using zinc sulphide (ZnS) QDs. Wet-chemical method was adopted for the preparation of fluorescent ZnS QDs (diameter ∼ 5–10 nm). These QDs were bioconjugated with monoclonal antibodies and then characterized by various method. A detection limit of 0.02 ng mL−1 by fluorescence assay and 1.0 ng mL−1 by electrochemical assay for SEB was achieved. While by sandwich ELISA it is possible to detect 0.24 ng mL−1 only. The sensitivity of all techniques is very good, since the LD50 of SEB is 20 ng kg−1. Electrochemical assay is faster, need low-cost instrument, independent to the size of QDs and found to be one of the best alternative methods as compared to the other existing methods studied herein. The presented method could be expanded to the development of electrochemical and fluorescence biosensors for various agents for field and laboratory use.

Sensitive detection of staphylococcal enterotoxin B (SEB) using quantum dots by various methods with special emphasis on an electrochemical immunoassay approach

Staphylococcal enterotoxin B (SEB) is a potent foodborne pathogen and categorized as a class B type of biological warfare agent. In this research work, SEB is detected by various sensitive analytical methods such as enzyme linked immunosorbent assay (ELISA), quantum dots-based fluorescence linked immunosorbent assay (QDs-FLISA) and square-wave voltammetry (SWV). The obtained results were compared in terms of sensitivity, ease of experimentation and analysis time. For the QD-based detection, fluorescent lead sulfide (PbS) QDs were prepared by a bottom-up approach and characterized by various techniques. Highly specific antibodies against SEB were conjugated with the prepared PbS QDs and were used as revealing antibodies. For the electrochemical detection of SEB, rabbit anti-SEB polyclonal antibodies (primary antibodies) were immobilized on screen-printed electrodes (SPEs) followed by the addition of various concentrations of SEB antigen. These electrodes were further incubated with revealing antibodies. Finally, 1 M HCl solution was added to the SPE to dissolve the PbS QDs which were captured in a sandwiched immunoassay, and resulting Pb2+ ions were determined by the SWV method using a glassy-carbon electrode. The obtained peak current is proportional to the amount of Pb2+ ions which indirectly depends on the SEB concentration. Linearity was observed in the concentration range of 1 ng mL−1 to 1 μg mL−1 of SEB antigen. The limit of detection was found to be 0.01 ng mL−1 for SEB. The results reveal that electrochemical SWV sensing is much easier, faster and provides high sensitivity as compared to the other methods. It is found that the detection limits achieved for sandwich ELISA and QDs-FLISA were 0.24 ng mL−1 and 0.03 ng mL−1 respectively. In addition, the developed SWV method can be implemented for the on-site detection of SEB particularly for civil and defense applications where security is of prime importance.