Yun Zhang, Amy M. LaFountain, Nikki Magdaong, Marcel Fuciman, James P. Allen, Harry A. Frank, and James F. Rusling,”Thin Film Voltammetry of Wild Type and Mutant Reaction Center Proteins from Photosynthetic Bacteria“,J. Phys. Chem. B, 2011, 115 (12), pp 3226–3232

Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States ; Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, United States ; Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States

Abstract: Photosynthetic reaction centers (RC) convert light into electrical potential via a series of electron transfers between protein-bound, redox-active cofactors. Direct voltammetry was used to characterize the RC protein from Rhodobacter sphaeroides and mutants with focus on the primary electron donor (P) cofactor. Cyclic voltammetry (CV) and square wave voltammetry (SWV) of lipid and polyion films of RCs revealed similar chemically irreversible processes, and starting, switching, or preconditioning potential of −0.15 V was required to observe a well-defined P/P+ oxidation peak at 0.95 V versus normal hydrogen electrode. An irreversible chemical reaction following voltammetric oxidation led to peak decreases upon multiple scans. Mutant RCs with site-directed amino acid modifications in the vicinity of P displayed shifts of oxidation peak potential correlated with those reported from redox titrations. These studies illustrate the utility of thin film voltammetry in characterizing redox properties of bound cofactors in RC proteins.

 

 

Bhaskara V. Chikkaveeraiah, Hongyun Liu, Vigneshwaran Mani, Fotios Papadimitrakapoulos and James F. Rusling, “A microfluidic electrochemical device for high sensitivity biosensing: Detection of nanomolar hydrogen peroxide”, Electrochem. Commun. 2009, 11, 819-822.

Department of Chemistry, University of Connecticut, Storrs, CT 06269, Department of Chemistry, Beijing Normal University, Beijing 100875, China,  Institute of Materials Science, University of Connecticut, Storrs, CT 06269, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, School of Chemistry, National University of Ireland at Galway, Ireland.

Abstract. We report herein a simple device for rapid biosensing consisting of a single microfluidic channel made from poly(dimethylsiloxane) (PDMS) coupled to an injector, and incorporating a biocatalytic sensing electrode, reference and counter electrodes. The sensing electrode was a gold wire coated with 5 nm glutathione-decorated gold nanoparticles (AuNPs). Sensitive detection of H₂O₂ based on direct bioelectrocatalysis by horseradish peroxidase (HRP) was used for evaluation. HRP was covalently linked the glutathione-AuNPs. This electrode presented quasi-reversible cyclic voltammetry peaks at -0.01 V vs. Ag/AgCl at pH 6.5 for the HRP heme FeIII/FeII couple. Direct electrochemical activity of HRP was used to detect H₂O₂ at high sensitivity with a detection limit of 5 nM in an unmediated system.

 

 

Vigneshwaran Mani, Bhaskara V. Chikkaveeraiah, Vyomesh Patel, J. Silvio Gutkind, and James F. Rusling “Ultrasensitive Immunosensor for Cancer Biomarker Proteins Using Gold Nanoparticle Film Electrodes and Multienzyme-Particle Amplification” ACS Nano, 2009, 3, 585-594 (Supported by NIH)

Department of Chemistry, 55 North Eagleville Road, University of Connecticut, Storrs, Connecticut 06269, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06032, and Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892

Abstract: A densely packed gold nanoparticle  platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab₂) attached to activated carboxyl groups on 1μm diameter magnetic beads. These sensors had sensitivity of 31.5 nA mL ng^-1 and detection limit (DL) of 0.5 pg mL^-1 for prostate specific antigen (PSA) in 10μL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, 8-fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays.

 

 

Bhirde A, Patel V, Gavard J,  Zhang G, Sousa AA,  Masedunskas A, Leapman RD, Weigert R,  Gutkind JS, Rusling JF, “Targeting killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery”. ACSNano 3, 307-316 (2009).

Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, Department of Cell Biology, University of Connecticut Health Center, Farmington,Connecticut 06032, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, and Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20982

Abstract: Carbon nanotube-based drug delivery holds great promise for cancer therapy. Herein we report the first targeted, in vivo killing of cancer cells using a drug-single wall carbon nanotube (SWNT) bioconjugate, and demonstrate efficacy superior to non-targeted   bioconjugates. First line anti-cancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the non-targeted control was SWNT-cisplatin without EGF. Initial in vitro imaging studies with head and neck squamous carcinoma cells (HNSCC) overexpressing EGF receptors (EGFR) using Qdot luminescence and confocal microscopy showed that SWNT-Qdot-EGF bioconjugates internalized rapidly into the cancer cells. Limited uptake occurred for control cells without EGF, and uptake was blocked by siRNA knockdown of EGFR in cancer cells, revealing the importance of EGF-EGFR binding. Three color, two-photon intra-vital Web Enhanced Objects imaging in vivo showed that SWNT-Qdot-EGF injected into live mice was selectively taken up by HNSCC tumors, but SWNT-Qdot controls with no EGF were cleared from the tumor region in <20 min. HNSCC cells treated with SWNT-cisplatin-EGF were also killed selectively, while control systems that did not feature EGF-EGFR binding did not influence cell proliferation. Most significantly, regression of tumor growth was rapid in mice treated with targeted SWNT-cisplatin-EGF relative to non-targeted SWNT-cisplatin.

 

 

Linlin Zhao, Sadagopan Krishnan, Yun Zhang, John B. Schenkman, James F. Rusling, “Differences in Metabolite-mediated Toxicity of Tamoxifen in Rodents vs. Humans Elucidated with DNA/microsomes Electro-optical Arrays and Nanoreactors” Chem. Res. Toxicol., 2009, 22, 341-347. (supported by NIH)

Department of Chemistry, 55 N. Eagleville Rd., University of Connecticut, Storrs, CT 06269, USA.   and Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA

Abstract:  Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsomes films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents vs. humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage endpoint. The arrays feature thin-film spots containing electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)₂PVP₁₀]²⁺; PVP = polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation vs. enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold ECL turnover rate was observed for spots with rat liver microsomes compared to human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing direct measurement of relative formation rate for α-(N²-deoxyguanosinyl)tamoxifen. We observed 2-5 fold more rapid formation rates for three major metabolites, i.e. α-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in causing liver carcinoma in humans, provided the glucuronidation pathway is active.

 

 

Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, John B. Schenkman and James F. Rusling, “Human cyt P450 mediated metabolic toxicity of 4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated using electrochemiluminescent arrays“ Mol. BioSyst., 2009, 5, 163-169. (Supported by NIH)

Department of Chemistry, University of Connecticut, U-60, 55 N. Eagleville Road, Storrs, Connecticut-06269, and Department of Cell Biology and Surgery, University of Connecticut Health Center, Farmington, Connecticut-06032

Abstract: Electrochemiluminescent (ECL) arrays containing polymer ([Ru(bpy)₂(PVP)₁₀]²⁺, PVP = polyvinylpyridine), DNA, and selected enzymes were employed to elucidate cytochrome (cyt) P450 dependent metabolism of the tobacco specific carcinogen, 4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Bioactivated NNK metabolites formed upon H2O2-enzymatic activation were captured as DNA adducts and detected simultaneously from 36 spot arrays by capturing and quantifying emitted ECL with an overhead CCD camera. Increased ECL emission was dependent on NNK exposure time. Of the enzymes tested, the activity toward NNK bioactivation was cyt P450 1A2 > 2E1 > 1B1 ≈ chloroperoxidase (CPO) > myoglobin (Mb) in accordance with reported in vivo studies. Cyt P450/polyion films were also immobilized on 500 nm diameter silica nanospheres for product analysis by LC-MS. Analysis of the nanosphere film reaction media provided ECL array validation and quantitation of the bioactivated NNK hydrolysis product 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) confirming production of reactive metabolites in the films. Chemical screening in this fashion allows rapid clarification of enzymes responsible for genotoxic activation as well as offering insight into cyt P450-related toxicity and mechanisms.

 

 

Sadagopan Krishnan, Besnik Bajrami, Vigneshwaran Mani, Shenmin Pan, James F. Rusling, “Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors“, Electroanal. 2009, 21, 1005-1013. (Supported by NIH)

Department of Chemistry, University of Connecticut, U-60, 55 N. Eagleville Road, Storrs, Connecticut-06269, and Department of Cell Biology and Surgery, University of Connecticut Health Center, Farmington, Connecticut-06032.

Abstract: Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical

development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square wave voltammetry (SWV) using Ru(bpy)₃²⁺ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD₅₀ data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response.

 

 

Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Dharamainder Choudhary, John B. Schenkman, and James F. Rusling, “Synergistic Metabolic Toxicity Screening Using Microsome/DNA Electrochemiluminescent Arrays and Nanoreactors“, Anal. Chem., 2008, 80, 5279-5285. (Supported by NIH)

Department of Chemistry, University of Connecticut, U-60, 55 N. Eagleville Road, Storrs, Connecticut-06269, and Department of Cell Biology and Surgery, University of Connecticut Health Center, Farmington, Connecticut-06032.

Abstract:  Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. Toxic hits uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as “nanoreactors” to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD₅₀ values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.

 

 

Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Ingela Jansson, John B. Schenkman,  and James F. Rusling, “Genotoxicity screening for N-nitroso compounds. Electrochemical and electrochemiluminescent detection of human enzyme-generated DNA damage from N-nitrosopyrrolidine“, Chem.Commun., 2007, 1713-1715. (Supported by NIH).

Department of Chemistry, University of Connecticut, U-60, 55 N. Eagleville Road, Storrs, Connecticut 06269, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032.

Abstract: We report for the first time voltammetric/electrochemiluminescent sensors applied to predict genotoxicity of N-nitroso compounds bioactivated by human cytochrome P450 enzymes.

 

 

Sadagopan Krishnan, and James F. Rusling, “Thin film voltammetry of metabolic enzymes in rat liver microsomes“, Electrochem.Commun. 2007, 9, 2359-2363. (Supported by NIH).

Department of Chemistry, University of Connecticut, U-60, 55 N. Eagleville Road, Storrs, Connecticut-06269, and Department of Cell Biology and Surgery, University of Connecticut Health Center, Farmington, Connecticut-06032.

Abstract. We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of -0.48 V vs SCE at 0.1 Vs^-1 in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation�reduction peak current ratios were ~1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation�reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s^-1. Absence of significant electrocatalytic reduction of O₂ or H₂O₂ and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes.