Ramaraja P. Ramasamy

Detection of methyl salicylate using bi-enzyme electrochemical sensor consisting salicylate hydroxylase and tyrosinase

Co-reporter:Yi Fang, Hannah Bullock, Sarah A. Lee, Narendran Sekar, Mark A. Eiteman, William B. Whitman, Ramaraja P. Ramasamy

Biosensors and Bioelectronics 2016 Volume 85() pp:603-610

Publication Date(Web):15 November 2016

DOI:10.1016/j.bios.2016.05.060

•Potentially offer a solution to early detect pest and pathogen infection in crops.•Use bi-enzymatic electrochemical biosensor with carbon nanotubes as transducers.•The basic principles outlined in this work could be extended for other biosensors.Volatile organic compounds have been recognized as important marker chemicals to detect plant diseases caused by pathogens. Methyl salicylate has been identified as one of the most important volatile organic compounds released by plants during a biotic stress event such as fungal pathogen infection. Advanced detection of these marker chemicals could help in early identification of plant diseases and has huge significance for agricultural industry. This work describes the development of a novel bi-enzyme based electrochemical biosensor consisting of salicylate hydroxylase and tyrosinase enzymes immobilized on carbon nanotube modified electrodes. The amperometric detection using the bi-enzyme platform was realized through a series of cascade reactions that terminate in an electrochemical reduction reaction. Electrochemical measurements revealed that the sensitivity of the bi-enzyme sensor was 30.6±2.7 µA cm−2 µM−1 and the limit of detection and limit of quantification were 13 nM (1.80 ppb) and 39 nM (5.39 ppb) respectively. Interference studies showed no significant interference from the other common plant volatile compounds. Synthetic analyte studies revealed that the bi-enzyme based biosensor can be used to reliably detect methyl salicylate released by unhealthy plants.

A novel bi-enzyme electrochemical biosensor for selective and sensitive determination of methyl salicylate

Co-reporter:Yi Fang, Yogeswaran Umasankar, Ramaraja P. Ramasamy

Biosensors and Bioelectronics 2016 81() pp: 39-45

Publication Date(Web):15 July 2016

DOI:10.1016/j.bios.2016.01.095

•A rapid method for crop disease detection is currently unavailable and this work could address the issue.•First attempt on enzyme based amperometric biosensor development for agricultural applications.•Principles of enzyme immobilization and amperometric transduction outlined here could be extended for other biosensors.An amperometric sensor based on a bi-enzyme modified electrode was fabricated to detect methyl salicylate, a volatile organic compound released by pathogen-infected plants via systemic response. The detection is based on cascadic conversion reactions that result in an amperometric electrochemical signal. The bi-enzyme electrode is made of alcohol oxidase and horseradish peroxidase enzymes immobilized on to a carbon nanotube matrix through a molecular tethering method. Methyl salicylate undergoes hydrolysis to form methanol, which is consumed by alcohol oxidase to form formaldehyde while simultaneously reducing oxygen to hydrogen peroxide. The hydrogen peroxide will be further reduced to water by horseradish peroxidase, which results in an amperometric signal via direct electron transfer. The bi-enzyme biosensor was evaluated by cyclic voltammetry and constant potential amperometry using hydrolyzed methyl salicylate as the analyte. The sensitivity of the bi-enzyme biosensor as determined by cyclic voltammetry and constant potential amperometry were 112.37 and 282.82 μA cm−2 mM−1 respectively, and the corresponding limits of detection were 22.95 and 0.98 μM respectively. Constant potential amperometry was also used to evaluate durability, repeatability and interference from other compounds. Wintergreen oil was used for real sample study to establish the application of the bi-enzyme sensor for selective determination of plant pathogen infections.

Recent advances in photosynthetic energy conversion

Co-reporter:Narendran Sekar, Ramaraja P. Ramasamy

Journal of Photochemistry and Photobiology C: Photochemistry Reviews 2015 Volume 22() pp:19-33

Publication Date(Web):March 2015

DOI:10.1016/j.jphotochemrev.2014.09.004

•Photosynthesis can be manipulated for electricity generation in an electrochemical cell.•The process can be used to generate clean power with only water and sunlight as raw materials.•The stability of photosynthetic machineries used must be improved greatly for real applications.•The use of live cells (cyanobacteria) is preferable over isolated photosystems or thylakoids.Photosynthesis is one of the first natural processes evolved by cyanobacteria, algae and green plants to trap light and CO2 in the form of reduced carbon compounds while simultaneously oxidizing water to oxygen. The photosynthetic energy conversion forms the basis for all the existing life today. The photosynthetic energy is being harnessed in many ways using modern technologies for the production of fuels using photosynthetic organisms, generation of direct electricity using photosystems/photosynthetic organisms in photo-bioelectrochemical cells or through photovoltaic systems. While the production of energy rich carbon fuels (ethanol, propanol) from photosynthetic organisms has already been accomplished due to advancement in understanding microbial physiology and metabolism, the photosynthetic hydrogen production as well as direct electricity generation from light is still at its infancy. Recent advances include combining photosystem complexes with hydrogenases for hydrogen production, using isolated thylakoids, photosystems on nanostructured electrodes such as gold nanoparticles, carbon nanotubes, ZnO nanoparticles for electricity generation. Many challenging optimizations on the immobilization methods, catalyst stability and isolation procedures, electron transfer strategies have acquired momentum leading to the production of more stable and higher current densities and power densities in photosynthetic devices. Further, the use of whole cell microorganisms (cyanobacteria, microalgae) rather than their isolated counterparts has produced promising results. The photosynthetic energy conversion has an enormous potential for renewable energy generation in a sustainable and environment friendly manner.

Three Dimensional Carbon Nanosheets as a Novel Catalyst Support for Enzymatic Bioelectrodes

Co-reporter:Yogeswaran Umasankar;D. Bradford Brooks;Billyde Brown;Zhiguo Zhou

Advanced Energy Materials 2014 Volume 4( Issue 6) pp:

Publication Date(Web):

DOI:10.1002/aenm.201301306

A high performance laccase-based biofuel cell cathode is developed using carbon nanosheets (CNS) as the catalyst support and buckypaper (BP) as the substrate electrode. Compared to multiwalled carbon nanotube (MWNT)-based electrodes, CNS-based electrodes exhibit better electrochemical properties for the oxygen reduction reaction (ORR) under biologically relevant conditions. It is shown that CNSs are conformally coated on the nanotubule bundles within the BP and that laccase is intimately attached to the CNS-BP. Electrochemical characterization is carried out to derive the kinetic parameters of the ORR at the laccase-CNS-BP cathode. The laccase-CNS-BP exhibits a steep ORR cathodic wave with a Tafel slope of 19 mV decade^-1. The onset potential obtained for laccase ORR at CNS-BP is 20 mV higher than that of the MWNT-based electrodes, and the laccase-CNS-BP cathode has a higher current density than MWNT electrodes.

Electrochemical detection of p-ethylguaiacol, a fungi infected fruit volatile using metal oxide nanoparticles

Co-reporter:Yi Fang, Yogeswaran Umasankar and Ramaraja P. Ramasamy

Analyst 2014 vol. 139(Issue 15) pp:3804-3810

Publication Date(Web):16 Apr 2014

DOI:10.1039/C4AN00384E

Nanoparticles of TiO2 or SnO2 on screen-printed carbon (SP) electrodes have been developed for evaluating their potential application in the electrochemical sensing of volatiles in fruits and plants. These metal oxide nanoparticle-modified electrodes possess high sensitivity and low detection limit for the detection of p-ethylguaiacol, a fingerprint compound present in the volatile signature of fruits and plants infected with a pathogenic fungus Phytophthora cactorum. The electroanalytical data obtained using cyclic voltammetry and differential pulse voltammetry showed that both SnO2 and TiO2 exhibited high sensitivity (174–188 μA cm−2 mM−1) and low detection limits (35–62 nM) for p-ethylguaiacol detection. The amperometric detection was highly repeatable with RSD values ranging from 2.48 to 4.85%. The interference studies show that other common plant volatiles do not interfere in the amperometric detection signal of p-ethylguaiacol. The results demonstrate that metal oxides are a reasonable alternative to expensive electrode materials such as gold or platinum for amperometric sensor applications.

Photocurrent generation by immobilized cyanobacteria via direct electron transport in photo-bioelectrochemical cells

Co-reporter:Narendran Sekar, Yogeswaran Umasankar and Ramaraja P. Ramasamy

Physical Chemistry Chemical Physics 2014 vol. 16(Issue 17) pp:7862-7871

Publication Date(Web):27 Feb 2014

DOI:10.1039/C4CP00494A

Cyanobacteria possess unique and exciting features among photosynthetic microorganisms for energy conversion applications. This study focuses on production of direct electricity using a cyanobacterium called Nostoc sp. (NOS) as a photo-biocatalyst immobilized on carbon nanotubes on the anode of photo-bioelectrochemical cells. By illuminating with light (intensity 76 mW cm−2) the NOS immobilized on a carbon nanotube (CNT) modified electrode generated a photocurrent density of 30 mA m−2 at 0.2 V (vs. Ag/AgCl). The contribution of different photosynthetic pigments in NOS to the light capture was analyzed and chlorophyll-a was found to be the major contributor to light capture followed by phycocyanin. Further investigation using a set of inhibitors revealed that the electrons were redirected predominantly from PSII to the CNT through the plastoquinone pool and quinol oxidase. A rudimentary design photosynthetic electrochemical cell has been constructed using NOS/CNT on the anode and laccase/CNT on the cathode as catalysts. The cell generated a maximum current density of 250 mA m−2 and a peak power density of 35 mW m−2 without any mediator. By the addition of 1,4-benzoquinone as a redox mediator, the electricity generation capability was significantly enhanced with a current density of 2300 mA m−2 and a power density of 100 mW m−2. The power densities achieved in this work are the highest among ‘non-engineered’ cyanobacteria based electrochemical systems reported to date.

Enhanced Electron Transfer in Enzymatic Bioelectrodes by a Poly(vinyl alcohol) N-Methyl-4(4-formylstyryl) Pyridinium Methosulfate Acetal Cationic Polymer

Co-reporter:Dr. Yogeswaran Umasankar ; Ramaraja P. Ramasamy

ChemElectroChem 2014 Volume 1( Issue 11) pp:1834-1839

Publication Date(Web):

DOI:10.1002/celc.201402186

Abstract

A novel method for enzyme immobilization has been developed for use in enzymatic biofuel cell cathodes and anodes. Enzyme immobilization was achieved on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWNTs) using the freestanding cationic polymer poly(vinyl alcohol) N-methyl-4(4′-formylstyryl) pyridiniummethosulfateacetal (PVA-SbQ). The enzymes entrapped within the polymer chains possess the ability to exchange electrons with the electrode through their respective substrates. The enzyme catalysts studied for biofuel cell cathodes were tyrosinase and laccase, and for biofuel cell anodes alcohol dehydrogenase (ADH) and glucose oxidase (GOx). The advantage of using PVA-SbQ for enzyme immobilization was demonstrated by using cyclic voltammetry. The experimental results showed that the freestanding PVA-SbQ polymer chains retained a higher enzyme activity on the MWNT-modified electrodes compared to cross-linked PVA-SbQ chains. Electrochemical impedance spectroscopy was applied to understand the electron-transfer resistance of cross-linked PVA-SbQ. The experimental results also show that enzyme/PVA-SbQ/MWNT electrodes generate a higher current compared to control electrodes without MWNTs or the polymer. We conclude that the PVA-SbQ polymer enhances both enzyme immobilization and electrochemical charge transfer in enzyme bioelectrodes, showing promise for the development of high-performance biofuel cells.

High photo-electrochemical activity of thylakoid–carbon nanotube composites for photosynthetic energy conversion

Co-reporter:Jessica O. Calkins, Yogeswaran Umasankar, Hugh O'Neill and Ramaraja P. Ramasamy

Energy & Environmental Science 2013 vol. 6(Issue 6) pp:1891-1900

Publication Date(Web):09 Apr 2013

DOI:10.1039/C3EE40634B

Spinach thylakoids were immobilized onto multiwalled carbon nanotubes using a molecular tethering chemistry. The resulting thylakoid–carbon nanotube composites showed high photo-electrochemical activity under illumination. Multiple membrane proteins have been observed to participate in direct electron transfer with the electrode, resulting in the generation of photocurrents, the first of its kind reported for natural photosynthetic systems. Upon inclusion of a mediator, the photo-activity was enhanced. The major contributor to the photocurrent was the light-induced water oxidation reaction at the photosystem II complex. The thylakoid–MWNT composite electrode yielded a maximum current density of 68 μA cm−2 and a steady state current density of 38 μA cm−2, which are two orders of magnitude larger than previously reported for similar systems. The high electrochemical activity of the thylakoid–MWNT composites has significant implications for both photosynthetic energy conversion and photofuel production applications. A fuel cell type photosynthetic electrochemical cell developed using a thylakoid–MWNT composite anode and laccase cathode produced a maximum power density of 5.3 μW cm−2, comparable to that of enzymatic fuel cells. The carbon based nanostructured electrode has the potential to serve as an excellent immobilization support for photosynthetic electrochemistry based on the molecular tethering approach as demonstrated in this work.

On the bio-electrocatalytic activity of tyrosinase for oxygen reduction reaction

Co-reporter:Yogeswaran Umasankar and Ramaraja P. Ramasamy

Catalysis Science & Technology 2013 vol. 3(Issue 10) pp:2546-2549

Publication Date(Web):25 Jun 2013

DOI:10.1039/C3CY00180F

The application of tyrosinase, a type 3 di-nuclear copper enzyme, as a catalyst for electrochemical oxygen reduction reaction (ORR) has been explored. During the reaction, O2 binds to the coupled type 3 copper atoms inside the redox site of tyrosinase and gets reduced to water. Besides the lack of the type 1 copper atom in its structure, unlike the multi-copper oxidases, tyrosinase exhibited direct electro-catalytic activity upon employing the electrode as a pseudo-substrate. The electrocatalytic activity of tyrosinase was further enhanced by using catechol/quinone as the redox shuttle for mediated electron transfer. This enhanced bio-electrocatalytic activity of tyrosinase for ORR has been reported in detail, which could open a new frontier in mediated enzyme reactions for biological fuel cell applications.

Highly sensitive electrochemical detection of methyl salicylate using electroactive gold nanoparticles

Co-reporter:Yogeswaran Umasankar and Ramaraja P. Ramasamy

Analyst 2013 vol. 138(Issue 21) pp:6623-6631

Publication Date(Web):15 Aug 2013

DOI:10.1039/C3AN01295F

Electrochemical sensing of methyl salicylate, a key plant volatile has been achieved using a gold nanoparticle (AuNP) modified screen printed carbon electrode (SPCE). The electrochemical response of planar gold electrodes, SPCE and AuNP–SPCE in alkaline electrolyte in the presence and absence of methyl salicylate were studied to understand the amperometric response of various electrochemical reactions. The reaction mechanism includes hydrolysis of methyl salicylate and the oxidation of negative species. The electrochemical responses were recorded using cyclic voltammetry and differential pulse voltammetry techniques, where the results showed characteristic signals for methyl salicylate oxidation. Among the examined electrodes, AuNP–SPCE possessed three fold better sensitivity than planar gold and 35 times better sensitivity than SPCE (at 0.5 V). The methyl salicylate sensing by AuNP–SPCE possessed <5% variation coefficient for repeatability, one week of stable performance with no more than 15% activity loss even if used multiple times (n = 8). Even in the presence of high concentration of interfering compounds such as cis-3-hexenol, hexyl acetate and cis-hexenyl acetate, AuNP–SPCE retained >95% of its methyl salicylate response. The electroanalytical results of soybean extract showed that AuNP–SPCE can be employed for the determination of methyl salicylate in real samples.

Kinetic and Mechanistic Parameters of Laccase Catalyzed Direct Electrochemical Oxygen Reduction Reaction

Co-reporter:Naga S. Parimi, Yogeswaran Umasankar, Plamen Atanassov, and Ramaraja P. Ramasamy

ACS Catalysis 2012 Volume 2(Issue 1) pp:38

Publication Date(Web):November 16, 2011

DOI:10.1021/cs200527c

This article presents the kinetic studies of oxygen reduction by one of the most important multicopper oxidases (fungal laccase) using the classic tool of electrochemistry: rotating ring-disk electrode (RRDE). Laccase was immobilized on a multiwalled carbon nanotube (MWNT) modified inert disk electrode using 1-pyrenebutanoic acid succinimidyl ester (PBSE), as a tethering agent. The conditions for laccase immobilization on MWNT were optimized to prepare a highly active composite electro-catalyst for O2 reduction. The mechanistic as well as kinetic parameters such as Tafel slopes, number of electrons transferred, electrochemical rate constants (for heterogeneous charge transfer) and electron transfer rate constant were calculated from the RRDE experiment results. The Tafel slope obtained was close to the value of that of ideal four-electron reduction of O2 to water indicating a highly active laccase in the tethered composite. The RRDE results also suggested the presence of intermediate steps in the oxygen reduction reaction. A model pathway for O2 reduction reaction at the laccase composite modified electrode was postulated, and rate constants for individual reactions in the pathway were calculated. The rate constant for four-electron O2 reduction was determined to be 1.46 × 10–3 mol s–1, indicating excellent electro-catalytic activity of the laccase-MWNT composite catalyst.Keywords: bioelectro-catalysis; biological fuel cell; electrochemical kinetics; laccase; oxygen reduction;

Electroanalytical studies on green leaf volatiles for potential sensor development

Co-reporter:Yogeswaran Umasankar, Glen C. Rains and Ramaraja P. Ramasamy

Analyst 2012 vol. 137(Issue 13) pp:3138-3145

Publication Date(Web):20 Apr 2012

DOI:10.1039/C2AN35350D

An electrochemical study for detecting green leaf plant volatiles from healthy and infected plants has been devised and tested. The electrocatalytic response of plant volatiles at a gold electrode was measured using cyclic voltammetry, amperometric current–time (i–t) analysis, differential pulse voltammetry (DPV) and hydrodynamic experiments. The sensitivity of the gold electrode in i–t analysis was 0.13 mA mM−1 cm−2 for cis-3-hexenol, 0.11 mA mM−1 cm−2 for cis-hexenyl acetate and 0.02 mA mM−1 cm−2 for hexyl acetate. The limits of detection of cis-3-hexenol, cis-hexenyl acetate and hexyl acetate by i–t analysis were 0.5, 0.3 and 0.6 μM, respectively, at a signal to noise ratio of 3. The hydrodynamic studies yielded the electro-kinetic parameters such as diffusivities of plant volatiles in solution and the rate constants for their electrochemical reactions. The DPV and interference studies reveal that the gold electrode possessed high sensitivity for plant volatiles determination in synthetic samples, which imitates both healthy and infected plants.

Photocurrent generation by immobilized cyanobacteria via direct electron transport in photo-bioelectrochemical cells

Co-reporter:Narendran Sekar, Yogeswaran Umasankar and Ramaraja P. Ramasamy

Physical Chemistry Chemical Physics 2014 - vol. 16(Issue 17) pp:NaN7871-7871

Publication Date(Web):2014/02/27

DOI:10.1039/C4CP00494A

Cyanobacteria possess unique and exciting features among photosynthetic microorganisms for energy conversion applications. This study focuses on production of direct electricity using a cyanobacterium called Nostoc sp. (NOS) as a photo-biocatalyst immobilized on carbon nanotubes on the anode of photo-bioelectrochemical cells. By illuminating with light (intensity 76 mW cm−2) the NOS immobilized on a carbon nanotube (CNT) modified electrode generated a photocurrent density of 30 mA m−2 at 0.2 V (vs. Ag/AgCl). The contribution of different photosynthetic pigments in NOS to the light capture was analyzed and chlorophyll-a was found to be the major contributor to light capture followed by phycocyanin. Further investigation using a set of inhibitors revealed that the electrons were redirected predominantly from PSII to the CNT through the plastoquinone pool and quinol oxidase. A rudimentary design photosynthetic electrochemical cell has been constructed using NOS/CNT on the anode and laccase/CNT on the cathode as catalysts. The cell generated a maximum current density of 250 mA m−2 and a peak power density of 35 mW m−2 without any mediator. By the addition of 1,4-benzoquinone as a redox mediator, the electricity generation capability was significantly enhanced with a current density of 2300 mA m−2 and a power density of 100 mW m−2. The power densities achieved in this work are the highest among ‘non-engineered’ cyanobacteria based electrochemical systems reported to date.

On the bio-electrocatalytic activity of tyrosinase for oxygen reduction reaction

Co-reporter:Yogeswaran Umasankar and Ramaraja P. Ramasamy

Catalysis Science & Technology (2011-Present) 2013 - vol. 3(Issue 10) pp:NaN2549-2549

Publication Date(Web):2013/06/25

DOI:10.1039/C3CY00180F

The application of tyrosinase, a type 3 di-nuclear copper enzyme, as a catalyst for electrochemical oxygen reduction reaction (ORR) has been explored. During the reaction, O2 binds to the coupled type 3 copper atoms inside the redox site of tyrosinase and gets reduced to water. Besides the lack of the type 1 copper atom in its structure, unlike the multi-copper oxidases, tyrosinase exhibited direct electro-catalytic activity upon employing the electrode as a pseudo-substrate. The electrocatalytic activity of tyrosinase was further enhanced by using catechol/quinone as the redox shuttle for mediated electron transfer. This enhanced bio-electrocatalytic activity of tyrosinase for ORR has been reported in detail, which could open a new frontier in mediated enzyme reactions for biological fuel cell applications.

Ramaraja P. Ramasamy

TOPICS

Abstract