Co-reporter:Itti Bist, Snehasis Bhakta, Di Jiang, Tia E. Keyes, Aaron Martin, Robert J. Forster, and James F. Rusling
Analytical Chemistry November 21, 2017 Volume 89(Issue 22) pp:12441-12441
Publication Date(Web):October 30, 2017
DOI:10.1021/acs.analchem.7b03528
Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in SN2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2′-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 106 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD50 and Comet assay results.
Co-reporter:Di Jiang, Spundana Malla, You-jun Fu, Dharamainder Choudhary, and James F. Rusling
Analytical Chemistry December 5, 2017 Volume 89(Issue 23) pp:12872-12872
Publication Date(Web):November 8, 2017
DOI:10.1021/acs.analchem.7b03487
Oxidation of DNA by reactive oxygen species (ROS) yields 8-oxo-7,8-dihydroguanosine (8-oxodG) as primary oxidation product, which can lead to downstream G to T transversion mutations. DNA mutations are nonrandom, and mutations at specific codons are associated with specific cancers, as widely documented for the p53 tumor suppressor gene. Here, we present the first direct LC-MS/MS study (without isotopic labeling or hydrolysis) of primary oxidation sites of p53 exon 7. We oxidized a 32 base pair (bp) double-stranded (ds) oligonucleotide representing exon 7 of the p53 gene. Oxidized oligonucleotides were cut by a restriction endonuclease to provide small strands and enable positions and amounts of 8-oxodG to be determined directly by LC-MS/MS. Oxidation sites on the oligonucleotide generated by two oxidants, catechol/Cu2+/NADPH and Fenton’s reagent, were located and compared. Guanines in codons 243, 244, 245, and 248 were most frequently oxidized by catechol/Cu2+/NADPH with relative oxidation of 5.6, 7.2, 2.6, and 10.7%, respectively. Fenton’s reagent oxidations were more specific for guanines in codons 243 (20.3%) and 248 (10.4%). Modeling of docking of oxidizing species on the ds-oligonucleotide were consistent with the experimental codon oxidation sites. Significantly, codons 244 and 248 are mutational “hotspots” in nonsmall cell and small cell lung cancers, supporting a possible role of oxidation in p53 mutations leading to lung cancer.
Co-reporter:Islam M. Mosa;Ajith Pattammattel;Karteek Kadimisetty;Paritosh Pe;Maher F. El-Kady;Gregory W. Bishop;Marc Novak;Richard B. Kaner;Ashis K. Basu;Challa V. Kumar
Advanced Energy Materials 2017 Volume 7(Issue 17) pp:
Publication Date(Web):2017/09/01
DOI:10.1002/aenm.201700358
Nearly all implantable bioelectronics are powered by bulky batteries which limit device miniaturization and lifespan. Moreover, batteries contain toxic materials and electrolytes that can be dangerous if leakage occurs. Herein, an approach to fabricate implantable protein-based bioelectrochemical capacitors (bECs) employing new nanocomposite heterostructures in which 2D reduced graphene oxide sheets are interlayered with chemically modified mammalian proteins, while utilizing biological fluids as electrolytes is described. This protein-modified reduced graphene oxide nanocomposite material shows no toxicity to mouse embryo fibroblasts and COS-7 cell cultures at a high concentration of 1600 µg mL−1 which is 160 times higher than those used in bECs, unlike the unmodified graphene oxide which caused toxic cell damage even at low doses of 10 µg mL−1. The bEC devices are 1 µm thick, fully flexible, and have high energy density comparable to that of lithium thin film batteries. COS-7 cell culture is not affected by long-term exposure to encapsulated bECs over 4 d of continuous charge/discharge cycles. These bECs are unique, protein-based devices, use serum as electrolyte, and have the potential to power a new generation of long-life, miniaturized implantable devices.
Co-reporter:Snehasis Bhakta;Chandra K. Dixit;Itti Bist;John Macharia;Min Shen;Karteek Kadimisetty;Junkai He;Biswanath Dutta;Steven L. Suib
Chemical Communications 2017 vol. 53(Issue 66) pp:9254-9257
Publication Date(Web):2017/08/15
DOI:10.1039/C7CC03412A
Selective removal of albumin from human serum is an essential step prior to proteomic analyses, especially when using mass spectrometry. Here we report stable synthetic nanopockets on magnetic nanoparticle surfaces that bind to human serum albumin (HSA) with high affinity and specificity. The nanopockets are created by templating HSA on 200 nm silica-coated paramagnetic nanoparticles using polymer layers made using 4 organo-silane monomers. These monomers have amino acid-like side chains providing hydrophobic, hydrophilic and H-bonding interactions that closely mimic features of binding sites on antibodies. The binding capacity of the material was 21 mg HSA g−1, and consistently removed ∼88% albumin from human serum in multiple repeated use.
Co-reporter:C. K. Tang;A. Vaze;J. F. Rusling
Lab on a Chip (2001-Present) 2017 vol. 17(Issue 3) pp:484-489
Publication Date(Web):2017/01/31
DOI:10.1039/C6LC01238H
A low cost three-dimensional (3D) printed clear plastic microfluidic device was fabricated for fast, low cost automated protein detection. The unibody device features three reagent reservoirs, an efficient 3D network for passive mixing, and an optically transparent detection chamber housing a glass capture antibody array for measuring chemiluminescence output with a CCD camera. Sandwich type assays were built onto the glass arrays using a multi-labeled detection antibody-polyHRP (HRP = horseradish peroxidase). Total assay time was ∼30 min in a complete automated assay employing a programmable syringe pump so that the protocol required minimal operator intervention. The device was used for multiplexed detection of prostate cancer biomarker proteins prostate specific antigen (PSA) and platelet factor 4 (PF-4). Detection limits of 0.5 pg mL−1 were achieved for these proteins in diluted serum with log dynamic ranges of four orders of magnitude. Good accuracy vs. ELISA was validated by analyzing human serum samples. This prototype device holds good promise for further development as a point-of-care cancer diagnostics tool.
Co-reporter:Ben Liu; Huiqin Yao; Wenqiao Song; Lei Jin; Islam M. Mosa; James F. Rusling; Steven L. Suib;Jie He
Journal of the American Chemical Society 2016 Volume 138(Issue 14) pp:4718-4721
Publication Date(Web):March 25, 2016
DOI:10.1021/jacs.6b01702
We report a robust, universal “soft” nitriding method to grow in situ ligand-free ultrasmall noble metal nanocatalysts (UNMN; e.g., Au, Pd, and Pt) onto carbon. Using low-temperature urea pretreatment at 300 °C, soft nitriding enriches nitrogen-containing species on the surface of carbon supports and enhances the affinity of noble metal precursors onto these supports. We demonstrated sub-2-nm, ligand-free UNMNs grown in situ on seven different types of nitrided carbons with no organic ligands via chemical reduction or thermolysis. Ligand-free UNMNs supported on carbon showed superior electrocatalytic activity for methanol oxidation compared to counterparts with surface capping agents or larger nanocrystals on the same carbon supports. Our method is expected to provide guidelines for the preparation of ligand-free UNMNs on a variety of supports and, additionally, to broaden their applications in energy conversion and electrochemical catalysis.
Co-reporter:Eli G. Hvastkovs and James F. Rusling
Analytical Chemistry 2016 Volume 88(Issue 9) pp:4584
Publication Date(Web):April 4, 2016
DOI:10.1021/acs.analchem.5b04772
Co-reporter:Brunah A. Otieno, Colleen E. Krause, Abby L. Jones, Richard B. Kremer, and James F. Rusling
Analytical Chemistry 2016 Volume 88(Issue 18) pp:9269
Publication Date(Web):August 24, 2016
DOI:10.1021/acs.analchem.6b02637
Parathyroid hormone-related peptide (PTHrP) is recognized as the major causative agent of humoral hypercalcemia of malignancy (HHM). The paraneoplastic PTHrP has also been implicated in tumor progression and metastasis of many human cancers. Conventional PTHrP detection methods like immunoradiometric assay (IRMA) lack the sensitivity required to measure target peptide levels prior to the development of hypercalcemia. In general, sensitive, multiplexed peptide measurement by immunoassay represents challenges that we address in this paper. We describe here the first ultrasensitive multiplexed peptide assay to measure intact PTHrP 1-173 as well as circulating N-terminal and C-terminal peptide fragments. This versatile approach should apply to almost any collection of peptides that are long enough to present binding sites for two antibodies. To target PTHrP, we employed a microfluidic immunoarray featuring a chamber for online capture of the peptides from serum onto magnetic beads decorated with massive numbers of peptide-specific antibodies and enzyme labels. Magnetic bead-peptide conjugates were then washed and sent to a detection chamber housing an antibody-modified 8-electrode array fabricated by inkjet printing of gold nanoparticles. Limits of detection (LODs) of 150 aM (∼1000-fold lower than IRMA) in 5 μL of serum were achieved for simultaneous detection of PTHrP isoforms and peptide fragments in 30 min. Good correlation for patient samples was found with IRMA (n = 57); r2 = 0.99 assaying PTHrP 1-86 equiv fragments. Analysis by a receiver operating characteristic (ROC) plot gave an area under the curve of 0.96, 80–83% clinical sensitivity, and 96–100% clinical specificity. Results suggest that PTHrP1-173 isoform and its short C-terminal fragments are the predominant circulating forms of PTHrP. This new ultrasensitive, multiplexed assay for PTHrP and fragments is promising for clinical diagnosis, prognosis, and therapeutic monitoring from early to advanced stage cancer patients and to examine underlying mechanisms of PTHrP overproduction.
Co-reporter:Chandra K. Dixit, Karteek Kadimisetty, Brunah A. Otieno, Chi Tang, Spundana Malla, Colleen E. Krause and James F. Rusling
Analyst 2016 vol. 141(Issue 2) pp:536-547
Publication Date(Web):14 Oct 2015
DOI:10.1039/C5AN01829C
Early detection and reliable diagnostics are keys to effectively design cancer therapies with better prognoses. The simultaneous detection of panels of biomarker proteins holds great promise as a general tool for reliable cancer diagnostics. A major challenge in designing such a panel is to decide upon a coherent group of biomarkers which have higher specificity for a given type of cancer. The second big challenge is to develop test devices to measure these biomarkers quantitatively with high sensitivity and specificity, such that there are no interferences from the complex serum or tissue matrices. Lastly, integrating all these tests into a technology that does not require exclusive training to operate, and can be used at point-of-care (POC) is another potential bottleneck in futuristic cancer diagnostics. In this article, we review electrochemistry-based tools and technologies developed and/or used in our laboratories to construct low-cost microfluidic protein arrays for the highly sensitive detection of a panel of cancer-specific biomarkers with high specificity which at the same time has the potential to be translated into POC applications.
Co-reporter:Gregory W. Bishop, Jennifer E. Satterwhite-Warden, Itti Bist, Eric Chen, and James F. Rusling
ACS Sensors 2016 Volume 1(Issue 2) pp:197
Publication Date(Web):December 17, 2015
DOI:10.1021/acssensors.5b00156
Clear plastic fluidic devices with ports for incorporating electrodes to enable electrochemiluminescence (ECL) measurements were prepared using a low-cost, desktop three-dimensional (3D) printer based on stereolithography. Electrodes consisted of 0.5 mm pencil graphite rods and 0.5 mm silver wires inserted into commercially available 1/4 in.-28 threaded fittings. A bioimaging system equipped with a CCD camera was used to measure ECL generated at electrodes and small arrays using 0.2 M phosphate buffer solutions containing tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+) with 100 mM tri-n-propylamine (TPA) as the coreactant. ECL signals produced at pencil graphite working electrodes were linear with respect to [Ru(bpy)3]2+ concentration for 9–900 μM [Ru(bpy)3]2+. The detection limit was found to be 7 μM using the CCD camera with exposure time set at 10 s. Electrode-to-electrode ECL signals varied by ±7.5%. Device performance was further evaluated using pencil graphite electrodes coated with multilayer poly(diallyldimethylammonium chloride) (PDDA)/DNA films. In these experiments, ECL resulted from the reaction of [Ru(bpy)3]3+ with guanines of DNA. ECL produced at these thin-film electrodes was linear with respect to [Ru(bpy)3]2+ concentration from 180 to 800 μM. These studies provide the first demonstration of ECL measurements obtained using a 3D-printed closed-channel fluidic device platform. The affordable, high-resolution 3D printer used in these studies enables easy, fast, and adaptable prototyping of fluidic devices capable of incorporating electrodes for measuring ECL.Keywords: 3D-printed fluidics; biosensing; DNA oxidation; electrochemiluminescence; stereolithography
Co-reporter:Chi K. Tang, Abhay Vaze, Min Shen, and James F. Rusling
ACS Sensors 2016 Volume 1(Issue 8) pp:1036
Publication Date(Web):July 21, 2016
DOI:10.1021/acssensors.6b00256
Microchip-based microfluidic electrochemical arrays hold great promise for fast, high-throughput multiplexed detection of cancer biomarker proteins at low cost per assay using relatively simple instrumentation. Here we describe an inexpensive high-throughput electrochemical array featuring 32 individually addressable microelectrodes that is further multiplexed with an 8-port manifold to provide 256 sensors. The gold electrode arrays were fabricated by wet-etching commercial gold compact discs (CD-R) followed by patterned insulation. A print-and-peel method was used to create sub-microliter hydrophobic wells surrounding each sensor to eliminate cross contamination during immobilization of capture antibodies. High-throughput analyses were realized using eight 32-sensor immunoarrays connected to the miniaturized 8-port manifold, allowing 256 measurements in <1 h. This system was used to determine prostate cancer biomarker proteins prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), interleukin-6 (IL-6), and platelet factor-4 (PF-4) in serum. Clinically relevant detection limits (0.05 to 2 pg mL–1) and 5-decade dynamic ranges (sub pg mL–1 to well above ng mL–1) were achieved for these proteins utilizing precapture of analyte proteins on magnetic nanoparticles decorated with enzyme labels and antibodies.Keywords: cancer biomarkers proteins; electrochemical array; high-throughput; immunosensors; sensor fabrication
Co-reporter:Itti Bist, Boya Song, Islam M. Mosa, Tia E. Keyes, Aaron Martin, Robert J. Forster, and James F. Rusling
ACS Sensors 2016 Volume 1(Issue 3) pp:272
Publication Date(Web):January 8, 2016
DOI:10.1021/acssensors.5b00189
Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os(bpy)2(phen-benz-COOH)]2+ (OsNG, {bpy= 2,2′-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl)benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton’s reagent or by ROS formation during redox cycles involving NADPH, CuII, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of CuII, NADPH, and model metabolites.Keywords: DNA oxidation sensor; electrochemiluminescence; microwell array; oxidative stress; [Os(bpy)2(phen-benz-COOH)]2+
Co-reporter:Karteek Kadimisetty, Islam M. Mosa, Spundana Malla, Jennifer E. Satterwhite-Warden, Tyler M. Kuhns, Ronaldo C. Faria, Norman H. Lee, James F. Rusling
Biosensors and Bioelectronics 2016 Volume 77() pp:188-193
Publication Date(Web):15 March 2016
DOI:10.1016/j.bios.2015.09.017
•The first low cost 3D-printed immunoarray for detection of up to 3 proteins is described.•Total materials cost of the array is €1.20, and assay cost is €0.50 when the array is reused.•This is the first report of a protein biosensor powered by a supercapacitor, which is rapidly recharged using an inexpensive solar cell.•Detection of 3 proteins in serum was achieved with detection limits of 300–500 fg mL−1.•Good correlations with single-protein ELISA for 3 proteins in prostate cancer patient serum were obtained.Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing. Prostate cancer biomarker proteins, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet factor-4 (PF-4) in serum were captured on the antibody-coated carbon sensors followed by delivery of detection-antibody-coated Ru(bpy)32+ (RuBPY)-doped silica nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) co-reactant using supercapacitor power and ECL was captured with a CCD camera. The supercapacitor was rapidly photo-recharged between assays using an inexpensive solar cell. Detection limits were 300–500 fg mL−1 for the 3 proteins in undiluted calf serum. Assays of 6 prostate cancer patient serum samples gave good correlation with conventional single protein ELISAs. This technology could provide sensitive onsite cancer diagnostic tests in resource-limited settings with the need for only moderate-level training.
Co-reporter:Spundana Malla, Karteek Kadimisetty, You-Jun Fu, Dharamainder Choudhary, Ingela Jansson, John B. Schenkman and James F. Rusling
Chemical Science 2015 vol. 6(Issue 10) pp:5554-5563
Publication Date(Web):24 Jun 2015
DOI:10.1039/C5SC01403D
Damage to p53 tumor suppressor gene is found in half of all human cancers. Databases integrating studies of large numbers of tumors and cancer cell cultures show that mutation sites of specific p53 codons are correlated with specific types of cancers. If the most frequently damaged p53 codons in vivo correlate with the most frequent chemical damage sites in vitro, predictions of organ-specific cancer risks might result. Herein, we describe LC-MS/MS methodology to reveal codons with metabolite-adducted nucleobases by LC-MS/MS for oligonucleotides longer than 20 base pairs. Specifically, we used a known carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) to determine the most frequently adducted nucleobases within codons. We used a known sequence of 32 base pairs (bp) representing part of p53 exon 7 with 5 possible reactive hot spots. This is the first nucleobase reactivity study of a double stranded DNA p53 fragment featuring more than 20 base pairs with multiple reactive sites. We reacted the 32 bp fragment with benzo[a]pyrene metabolite BPDE that undergoes nucleophilic substitution by DNA bases. Liquid chromatography-mass spectrometry (LC-MS/MS) was used for sequencing of oligonucleotide products from the reacted 32 bp fragment after fragmentation by a restriction endonuclease. Analysis of the adducted p53 fragment compared with unreacted fragment revealed guanines of codons 248 and 244 as most frequently targeted, which are also mutated with high frequency in human tumors. Codon 248 is mutated in non-small cell and small cell lung, head and neck, colorectal and skin cancer, while codon 244 is mutated in small cell lung cancer, all of which involve possible BDPE exposure. Results suggest the utility of this approach for screening of adducted p53 gene by drugs and environmental chemicals to predict risks for organ specific cancers.
Co-reporter:Dhanuka P. Wasalathanthri, Dandan Li, Donghui Song, Zhifang Zheng, Dharamainder Choudhary, Ingela Jansson, Xiuling Lu, John B. Schenkman and James F. Rusling
Chemical Science 2015 vol. 6(Issue 4) pp:2457-2468
Publication Date(Web):12 Feb 2015
DOI:10.1039/C4SC03401E
Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termed genotoxic. Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)2(PVP)10]2+ {(PVP = poly(4-vinylpyridine))} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.
Co-reporter:Dandan Li, You-Jun Fu and James F. Rusling
Chemical Communications 2015 vol. 51(Issue 22) pp:4701-4703
Publication Date(Web):11 Feb 2015
DOI:10.1039/C5CC00420A
We report here label-free metabolite–protein adduct detection and identification employing magnetic beads coated with metabolic enzymes as bioreactors to generate metabolites and possible metabolite–protein adducts for analysis by liquid chromatography-tandem mass spectrometry.
Co-reporter:Snehasis Bhakta, Mohammad Saiful Islam Seraji, Steven L. Suib, and James F. Rusling
ACS Applied Materials & Interfaces 2015 Volume 7(Issue 51) pp:28197
Publication Date(Web):December 4, 2015
DOI:10.1021/acsami.5b11650
Natural antibodies are used widely for important applications such as biomedical analysis, cancer therapy, and directed drug delivery, but they are expensive and may have limited stability. This study describes synthesis of antibody-like binding sites by molecular imprinting on silica nanoparticles (SiNP) using a combination of four organosilane monomers with amino acid-like side chains providing hydrophobic, hydrophilic, and H-bonding interactions with target proteins. This approach provided artificial antibody (AA) nanoparticles with good selectivity and specificity to binding domains on target proteins in a relatively low-cost synthesis. The AAs were made by polymer grafting onto SiNPs for human serum albumin (HSA) and glucose oxidase (GOx). Binding affinity, selectivity, and specificity was compared to several other proteins using adsorption isotherms and surface plasmon resonance (SPR). The Langmuir–Freundlich adsorption model was used to obtain apparent binding constants (KLF) from binding isotherms of HSA (6.7 × 104) and GOx (4.7 × 104) to their respective AAs. These values were 4–300 fold larger compared to a series of nontemplate proteins. SPR binding studies of AAs with proteins attached to a gold surface confirmed good specificity and revealed faster binding for the target proteins compared to nontarget proteins. Target proteins retained their secondary structures upon binding. Binding capacity of AAHSA for HSA was 5.9 mg HSA/g compared to 1.4 mg/g for previously report imprinted silica beads imprinted with poly(aminophenyl)boronic acid. Also, 90% recovery for HSA spiked into 2% calf serum was found for AAHSA.Keywords: human serum albumin (HSA); Langmuir−Freundlich adsorption; molecular imprinting; silica nanoparticles; surface plasmon resonance (SPR)
Co-reporter:Huijing Cai, Yixian Wang, Yun Yu, Michael V. Mirkin, Snehasis Bhakta, Gregory W. Bishop, Amit A. Joshi, and James F. Rusling
Analytical Chemistry 2015 Volume 87(Issue 12) pp:6403
Publication Date(Web):June 4, 2015
DOI:10.1021/acs.analchem.5b01468
Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.
Co-reporter:Gregory W. Bishop, Jennifer E. Satterwhite, Snehasis Bhakta, Karteek Kadimisetty, Kelsey M. Gillette, Eric Chen, and James F. Rusling
Analytical Chemistry 2015 Volume 87(Issue 10) pp:5437
Publication Date(Web):April 22, 2015
DOI:10.1021/acs.analchem.5b00903
A consumer-grade fused filament fabrication (FFF) 3D printer was used to construct fluidic devices for nanoparticle preparation and electrochemical sensing. Devices were printed using poly(ethylene terephthalate) and featured threaded ports to connect polyetheretherketone (PEEK) tubing via printed fittings prepared from acrylonitrile butadiene styrene (ABS). These devices included channels designed to have 800 μm × 800 μm square cross sections and were semitransparent to allow visualization of the solution-filled channels. A 3D-printed device with a Y-shaped mixing channel was used to prepare Prussian blue nanoparticles (PBNPs) under flow rates of 100 to 2000 μL min–1. PBNPs were then attached to gold electrodes for hydrogen peroxide sensing. 3D-printed devices used for electrochemical measurements featured threaded access ports into which a fitting equipped with reference, counter, and PBNP-modified working electrodes could be inserted. PBNP-modified electrodes enabled amperometric detection of H2O2 in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 μM. These experiments show that a consumer-grade FFF printer can be used to fabricate low-cost fluidic devices for applications similar to those that have been reported with more expensive 3D-printing methods.
Co-reporter:Karteek Kadimisetty, Spundana Malla, Naimish P. Sardesai, Amit A. Joshi, Ronaldo C. Faria, Norman H. Lee, and James F. Rusling
Analytical Chemistry 2015 Volume 87(Issue 8) pp:4472
Publication Date(Web):March 30, 2015
DOI:10.1021/acs.analchem.5b00421
Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica–antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10–100 fg mL–1 were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.
Co-reporter:Jennifer E. Satterwhite-Warden, Dilip K. Kondepudi, James A. Dixon and James F. Rusling
Physical Chemistry Chemical Physics 2015 vol. 17(Issue 44) pp:29891-29898
Publication Date(Web):08 Oct 2015
DOI:10.1039/C5CP04471E
Self-motion of physical-chemical systems is a promising avenue for studying and developing mechanical functions with inanimate systems. In this paper, we investigate spontaneous motion of collections of solid macroscopic benzoquinone (BQ) disks at the air–water interface without intervention of chemical reactions. The BQ particles slowly dissolve and create heterogeneous interfacial tension fields on the water surface that drive the motion. Spontaneous, continuous locomotion was observed between multiple BQ particles, along with coupling, collisions, cycling and collective foraging for interfacial free energy. Analysis of the motion suggests co-operative behavior depends strongly on particle shape.
Co-reporter:Yun Zhang;Nikki M. Magdaong;Min Shen; Harry A. Frank; James F. Rusling
ChemistryOpen 2015 Volume 4( Issue 2) pp:111-114
Publication Date(Web):
DOI:10.1002/open.201402080
Abstract
The need for clean, renewable energy has fostered research into photovoltaic alternatives to silicon solar cells. Pigment–protein complexes in green plants convert light energy into chemical potential using redox processes that produce molecular oxygen. Here, we report the first use of spinach protein photosystem II (PSII) core complex in lipid films in photoelectrochemical devices. Photocurrents were generated from PSII in a ∼2 μm biomimetic dimyristoylphosphatidylcholine (DMPC) film on a pyrolytic graphite (PG) anode with PSII embedded in multiple lipid bilayers. The photocurrent was ∼20 μA cm−2 under light intensity 40 mW cm−2. The PSII–DMPC anode was used in a photobiofuel cell with a platinum black mesh cathode in perchloric acid solution to give an output voltage of 0.6 V and a maximum output power of 14 μW cm−2. Part of this large output is related to a five-unit anode–cathode pH gradient. With catholytes at higher pH or no perchlorate, or using an MnO2 oxygen-reduction cathode, the power output was smaller. The results described raise the possibility of using PSII–DMPC films in small portable power conversion devices.
Co-reporter:Amit A. Joshi, Mark W. Peczuh, Challa V. Kumar and James F. Rusling
Analyst 2014 vol. 139(Issue 24) pp:6589-6589
Publication Date(Web):31 Oct 2014
DOI:10.1039/C4AN90090A
Correction for ‘Ultrasensitive carbohydrate-peptide SPR imaging microarray for diagnosing IgE mediated peanut allergy’ by Amit A. Joshi et al., Analyst, 2014, 139, 5728–5733.
Co-reporter:Amit A. Joshi, Mark W. Peczuh, Challa V. Kumar and James F. Rusling
Analyst 2014 vol. 139(Issue 22) pp:5728-5733
Publication Date(Web):16 Sep 2014
DOI:10.1039/C4AN01544D
Severity of peanut allergies is linked to allergen-specific immunoglobulin E (IgE) antibodies in blood, but diagnostics from assays using glycoprotein allergen mixtures may be inaccurate. Measuring IgEs specific to individual peptide and carbohydrate epitopes of allergenic proteins is promising. We report here the first immunoarray for IgEs utilizing both peptide and carbohydrate epitopes. A surface plasmon resonance imaging (SPRi) microarray was equipped with peptide and β-xylosyl glycoside (BXG) epitopes from major peanut allergen glycoprotein Arachis hypogaea h2 (Ara-h2). A monoclonal anti-IgE antibody was included as positive control. IgEs were precaptured onto magnetic beads loaded with polyclonal anti-IgE antibodies to enhance sensitivity and minimize non-specific binding. As little as 0.1 attomole (0.5 pg mL−1) IgE was detected from dilute serum in 45 min. IgEs binding to Ara-h2 peptide and BXG were quantified in 10 μL of patient serum and correlated with standard ImmunoCAP values.
Co-reporter:C. K. Tang, A. Vaze and J. F. Rusling
Analytical Methods 2014 vol. 6(Issue 22) pp:8878-8881
Publication Date(Web):11 Sep 2014
DOI:10.1039/C4AY01962H
Inexpensive, reusable electrochemical sensor chips were fabricated from gold CDs. All reagents were loaded onto a paper disk sequentially, then placed on the chip to detect cancer biomarker prostate specific antigen (PSA) in serum at pg mL−1 levels in ∼15 min.
Co-reporter:Yun Zhang;Nikki Magdaong;Harry A. Frank
Photosynthesis Research 2014 Volume 120( Issue 1-2) pp:153-167
Publication Date(Web):2014 May
DOI:10.1007/s11120-013-9831-4
Direct protein film voltammetry (PFV) was used to investigate the redox properties of the photosystem II (PSII) core complex from spinach. The complex was isolated using an improved protocol not used previously for PFV. The PSII core complex had high oxygen-evolving capacity and was incorporated into thin lipid and polyion films. Three well-defined reversible pairs of reduction and oxidation voltammetry peaks were observed at 4 °C in the dark. Results were similar in both types of films, indicating that the environment of the PSII-bound cofactors was not influenced by film type. Based on comparison with various control samples including Mn-depleted PSII, peaks were assigned to chlorophyll a (Chl a) (Em = −0.47 V, all vs. NHE, at pH 6), quinones (−0.12 V), and the manganese (Mn) cluster (Em = 0.18 V). PFV of purified iron heme protein cytochrome b-559 (Cyt b-559), a component of PSII, gave a partly reversible peak pair at 0.004 V that did not have a potential similar to any peaks observed from the intact PSII core complex. The closest peak in PSII to 0.004 V is the 0.18 V peak that was found to be associated with a two-electron process, and thus is inconsistent with iron heme protein voltammetry. The −0.47 V peak had a peak potential and peak potential-pH dependence similar to that found for purified Chl a incorporated into DMPC films. The midpoint potentials reported here may differ to various extents from previously reported redox titration data due to the influence of electrode double-layer effects. Heterogeneous electron transfer (hET) rate constants were estimated by theoretical fitting and digital simulations for the −0.47 and 0.18 V peaks. Data for the Chl a peaks were best fit to a one-electron model, while the peak assigned to the Mn cluster was best fit by a two-electron/one-proton model.
Co-reporter:Brunah A. Otieno, Colleen E. Krause, Alina Latus, Bhaskara V. Chikkaveeraiah, Ronaldo C. Faria, James F. Rusling
Biosensors and Bioelectronics 2014 Volume 53() pp:268-274
Publication Date(Web):15 March 2014
DOI:10.1016/j.bios.2013.09.054
•A modular microfluidic system for multiple protein detection is described that incorporates on-line magnetic bead capture of proteins.•Massively labeled, antibody laden magnetic beads capture the proteins and deliver them to a sensor array.•Detection of two proteins in serum was achieved with detection limits of 5 fg mL−1.•Good correlations were found with standard ELISA for two proteins in conditioned media of oral cancer cell lines.•Microfluidic chambers are fabricated with non-lithographic procedures and coupled to inexpensive commercial components.Accurate, sensitive, multiplexed detection of biomarker proteins holds significant promise for personalized cancer diagnostics. Here we describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray. Capture and detection chambers are produced from PDMS on machined molds and do not require lithography. Protein analytes are captured from serum or other biological samples in the stirred capture chamber on the beads held in place magnetically. The beads are subsequently washed free of sample components, and wash solutions sent to waste. Removal of the magnet and valve switching sends the magnetic bead–protein bioconjugates into a detection chamber where they are captured on 8 antibody-decorated gold nanoparticle-film sensors and detected amperometrically. Most steps in the immunoassay including protein capture, washing and measurement are incorporated into the device. In simultaneous assays, the microfluidic system gave ultralow detection limits of 5 fg mL−1 for interleukin-6 (IL-6) and 7 fg mL−1 for IL-8 in serum. Accuracy was demonstrated by measuring IL-6 and IL-8 in conditioned media from oral cancer cell lines and showing good correlations with standard ELISAs. The on-line capture chamber facilitates rapid, sensitive, repetitive protein separation and measurement in 30 min in a semi-automated system adaptable to multiplexed protein detection.
Co-reporter:Yixian Wang, Kaan Kececi, Michael V. Mirkin, Vigneshwaran Mani, Naimish Sardesai and James F. Rusling
Chemical Science 2013 vol. 4(Issue 2) pp:655-663
Publication Date(Web):15 Nov 2012
DOI:10.1039/C2SC21502K
Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP–peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.
Co-reporter:James F. Rusling
Analytical Chemistry 2013 Volume 85(Issue 11) pp:5304
Publication Date(Web):May 1, 2013
DOI:10.1021/ac401058v
Measuring diagnostic panels of multiple proteins promises a new, personalized approach to early detection and therapy of diseases like cancer. Levels of biomarker proteins in patient serum can provide a continually updated record of disease status. Research in electrochemical detection of proteins has produced exquisitely sensitive approaches. Most utilize ELISA-like sandwich immunoassays incorporating various aspects of nanotechnology. Several of these ultrasensitive methodologies have been extended to microfluidic multiplexed protein detection, but engineered solutions are needed to measure more proteins in a single device from a small patient sample such as a drop of blood or tissue lysate. To achieve clinical or point-of-care (POC) use, simplicity and low cost are essential. In multiplexed microfluidic immunoassays, required reagent additions and washing steps pose a significant problem calling for creative engineering. A grand challenge is to develop a general cancer screening device to accurately measure 50–100 proteins in a simple, cost-effective fashion. This will require creative solutions to simplified reagent addition and multiplexing.
Co-reporter:Dhanuka P. Wasalathanthri, Spundana Malla, Itti Bist, Chi K. Tang, Ronaldo C. Faria and James F. Rusling
Lab on a Chip 2013 vol. 13(Issue 23) pp:4554-4562
Publication Date(Web):03 Sep 2013
DOI:10.1039/C3LC50698C
A high throughput electrochemiluminescent (ECL) chip was fabricated and integrated into a fluidic system for screening toxicity-related chemistry of drug and pollutant metabolites. The chip base is conductive pyrolytic graphite onto which are printed 64 microwells capable of holding one-μL droplets. Films combining DNA, metabolic enzymes and an ECL-generating ruthenium metallopolymer (RuIIPVP) are fabricated in these microwells. The system runs metabolic enzyme reactions, and subsequently detects DNA damage caused by reactive metabolites. The performance of the chip was tested by measuring DNA damage caused by metabolites of the well-known procarcinogen benzo[a]pyrene (B[a]P). Liver microsomes and cytochrome P450 (cyt P450) enzymes were used with and without epoxide hydrolase (EH), a conjugative enzyme required for multi-enzyme bioactivation of B[a]P. DNA adduct formation was confirmed by determining specific DNA-metabolite adducts using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by capillary liquid chromatography-mass spectrometry (CapLC-MS/MS). The fluidic chip was also used to measure IC50-values of inhibitors of cyt P450s. All results show good correlation with reported enzyme activity and inhibition assays.
Co-reporter:Boya Song, Shenmin Pan, Chi Tang, Dandan Li, and James F. Rusling
Analytical Chemistry 2013 Volume 85(Issue 22) pp:11061
Publication Date(Web):October 9, 2013
DOI:10.1021/ac402736q
Oxidative stress in humans causes damage to biomolecules by generating reactive oxygen species (ROS). DNA can be oxidatively damaged by ROS, which may lead to carcinogenesis. Here we report a microfluidic electrochemical array designed to rapidly detect oxidation in intact DNA in replicate measurements. Sensor arrays were fabricated by wet-chemistry patterning of gold compact discs. The eight-sensor array is incorporated into a 60 μL microfluidic channel connected to a pump and sample valve. The array features 7 nm thick osmium bipyridyl poly(vinylpyridine) chloride [Os(bpy)2(PVP)10Cl]+ films assembled layer-by-layer with polyions onto the gold sensors. 8-Hydroxy-7,8-hydro-2′-deoxyguanosine (8-oxodG) is selectively oxidized by [Os(bpy)2(PVP)10Cl]+ in intact ds-DNA to provide catalytic square wave voltammograms (SWV). The device is easy-to-use, fast, inexpensive, reusable, and can detect one 8-oxodG per 6600 nucleobases. The mass detection limit is 150-fold lower than a previously reported dip-and-read voltammetric sensor for oxidized DNA. Fast assays (<1 min) and moderate sample consumption (15 pmol DNA) suggest potential for research and clinical applications. Practical use is illustrated by detecting DNA oxidation from cigarette smoke and ash extracts in dispersions with NADPH and Cu2+.
Co-reporter:Vigneshwaran Mani, Karteek Kadimisetty, Spundana Malla, Amit A. Joshi, and James F. Rusling
Environmental Science & Technology 2013 Volume 47(Issue 4) pp:1937
Publication Date(Web):January 18, 2013
DOI:10.1021/es304426j
A low cost, microfluidic paper electrochemical device (μPED) was fabricated using screen printing of electrodes and heat transfer of patterned wax paper onto filter paper. The μPED features films of a light-emitting ruthenium metallopolymer, microsomal metabolic enzymes, and DNA to detect potential genotoxic pollutant activity in environmental samples. Unlike conventional analytical methods that detect specific pollutant compounds, the μPED was designed to rapidly measure the presence of genotoxic equivalents in environmental samples with the signal related to benzo[a]pyrene (B[a]P) as a reference standard. The analytical end point is the detection of DNA damage from metabolites produced in the device using an electrochemiluminescence output measured with a charge-coupled device (CCD) camera. Proof-of-concept of this measurement was established for smoke, water, and food samples. The μPED provides a rapid screening tool for on-site environmental monitoring that specifically monitors the genotoxic reactivity of metabolites of toxic compounds present in the samples.
Co-reporter:Dhanuka P. Wasalathanthri, Ronaldo C. Faria, Spundana Malla, Amit A. Joshi, John B. Schenkman and James F. Rusling
Analyst 2013 vol. 138(Issue 1) pp:171-178
Publication Date(Web):11 Oct 2012
DOI:10.1039/C2AN35993F
A multiplexed, microfluidic platform to detect reactive metabolites is described, and its performance is illustrated for compounds metabolized by oxidative and bioconjugation enzymes in multi-enzyme pathways to mimic natural human drug metabolism. The device features four 8-electrode screen printed carbon arrays coated with thin films of DNA, a ruthenium-polyvinylpyridine (RuPVP) catalyst, and multiple enzyme sources including human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9) and N-acetyltransferase (NAT). Arrays are arranged in parallel to facilitate multiple compound screening, enabling up to 32 enzyme reactions and measurements in 20–30 min. In the first step of the assay, metabolic reactions are achieved under constant flow of oxygenated reactant solutions by electrode driven natural catalytic cycles of cyt P450s and cofactor-supported bioconjugation enzymes. Reactive metabolites formed in the enzyme reactions can react with DNA. Relative DNA damage is measured in the second assay step using square wave voltammetry (SWV) with RuPVP as catalyst. Studies were done on chemicals known to require metabolic activation to induce genotoxicity, and results reproduced known features of metabolite DNA-reactivity for the test compounds. Metabolism of benzo[a]pyrene (B[a]P) by cyt P450s and epoxide hydrolase showed an enhanced relative DNA damage rate for DNA compared to cyt P450s alone. DNA damage rates for arylamines by pathways featuring both oxidative and conjugative enzymes at pH 7.4 gave better correlation with rodent genotoxicity metric TD50. Results illustrate the broad utility of the reactive metabolite screening device.
Co-reporter:Shenmin Pan, Dandan Li, Linlin Zhao, John B. Schenkman, and James F. Rusling
Chemical Research in Toxicology 2013 Volume 26(Issue 8) pp:1229
Publication Date(Web):July 23, 2013
DOI:10.1021/tx400147c
There is limited and sometimes contradictory information about the genotoxicity of the polycyclic aromatic hydrocarbon benzo[ghi]perylene (B[ghi]P). Using recently developed metabolic toxicity screening arrays and a biocolloid reactor-LC-MS/MS approach, both featuring films of DNA and human metabolic enzymes, we demonstrated the relatively low reactivity of metabolically activated B[ghi]P toward DNA. Electro-optical toxicity screening arrays showed that B[ghi]P metabolites damage DNA at a 3-fold lower rate than benzo[a]pyrene (B[a]P), whose metabolites have a strong and well-understood propensity for DNA damage. Metabolic studies using magnetic bead biocolloid reactors coated with microsomal enzymes in 96-well plates showed that cyt P450s 1A1 and 1B1 provide high activity for B[ghi]P and B[a]P conversion. Consistent with published results, the major metabolism of B[ghi]P involved oxidations at 3,4 and 11,12 positions, leading to the formation of B[ghi]P 3,4-oxide and B[ghi]P 3,4,11,12-bisoxide. B[ghi]P 3,4-oxide was synthesized and reacted with deoxyadenosine at N6 and N7 positions and with deoxyguanosine at the N2 position. B[ghi]P 3,4-oxide is hydrolytically unstable and transforms into the 3,4-diol or converts to 3- or 4-hydroxy B[ghi]P. LC-MS/MS of reaction products from the magnetic biocolloid reactor particles coated with DNA and human enzymes revealed for the first time that a major DNA adduct results from the reaction between B[ghi]P 3,4,11,12-bisoxide and deoxyguanosine. Results also demonstrated 5-fold lower formation rates of the major DNA adduct for B[ghi]P metabolites compared to B[a]P. Overall, results from both the electro-optical array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P.
Co-reporter:Shenmin Pan, Naimish P. Sardesai, Hongyun Liu, Dandan Li and James F. Rusling
Toxicology Research 2013 vol. 2(Issue 6) pp:375-378
Publication Date(Web):02 Aug 2013
DOI:10.1039/C3TX50022E
Peroxidase enzyme digests of oxidized single-wall carbon nanotubes (SWCNTs) were shown to damage DNA in potentially genotoxic reactions for the first time using an electro-optical array with and without metabolic activation.
Co-reporter:Colleen E. Krause;Brunah A. Otieno;Dr. Alina Latus;Dr. Ronaldo C. Faria;Dr. Vyomesh Patel;Dr. J. Silvio Gutkind;Dr. James F. Rusling
ChemistryOpen 2013 Volume 2( Issue 4) pp:141-145
Publication Date(Web):
DOI:10.1002/open.201300018
Co-reporter:Ruchika Malhotra, Vyomesh Patel, Bhaskara V. Chikkaveeraiah, Bernard S. Munge, Sok Ching Cheong, Rosnah B. Zain, Mannil T. Abraham, Dipak K. Dey, J. Silvio Gutkind, and James F. Rusling
Analytical Chemistry 2012 Volume 84(Issue 14) pp:6249
Publication Date(Web):June 14, 2012
DOI:10.1021/ac301392g
Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5–50 fg·mL–1 range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400 000 enzyme labels and ∼100 000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.
Co-reporter:Vigneshwaran Mani, Dhanuka P. Wasalathanthri, Amit A. Joshi, Challa V. Kumar, and James F. Rusling
Analytical Chemistry 2012 Volume 84(Issue 23) pp:10485
Publication Date(Web):November 3, 2012
DOI:10.1021/ac3028257
We report here the first kinetic characterization of 1 μm diameter superparamagnetic particles (MP) decorated with over 100 000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab2) exhibit irreversible binding with 100-fold increased association rates compared to free antibodies. The estimated upper limit for the dissociation constant of MP-Ab2 from the SPR sensor surface is 5 fM, compared to 3–8 nM for the free antibodies. These results are explained by up to 2000 interactions of MP-Ab2 with protein-decorated surfaces. Findings are consistent with highly efficient capture of protein antigens in solution by the MP-Ab2 and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range. Aggregation of these particles on the SPR chip, probably due to residual magnetic microdomains in the particles, also contributes to ultrasensitive detection and may also help drive the irreversible binding.
Co-reporter:Chi K. Tang, Abhay Vaze and James F. Rusling
Lab on a Chip 2012 vol. 12(Issue 2) pp:281-286
Publication Date(Web):24 Nov 2011
DOI:10.1039/C1LC20833K
A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm2 with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL−1 (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL−1. These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.
Co-reporter:James F. Rusling
The Chemical Record 2012 Volume 12( Issue 1) pp:164-176
Publication Date(Web):
DOI:10.1002/tcr.201100034
Abstract
For this special issue on 90 years of polarography, the following personal account describes how my early research in electrochemistry and polarography in the laboratory of Prof. Petr Zuman led to a major research effort in the determination of proteins for cancer detection and monitoring. It reviews the very recent history of nanoparticle labels and multiplexed detection in protein immunosensors. It then describes our journey of discovery that has led to ultrasensitive protein immunosensors achieved by combining nanostructured electrodes with particles labeled with up to million enzymes that can detect down to as little as 1 fg mL−1 protein in diluted serum. Our most mature multiple protein detection system is a microfluidic device with eight sensors coated with 5-nm gold nanoparticles that uses off-line protein detection with heavily labeled magnetic particles. This approach has led to reliable sub pg mL−1 detection limits for multiple proteins, provides excellent correlation with referee ELISA methods, and is currently being used for validation of panels of biomarkers for oral and prostate cancer. The article ends with a section on future perspectives.
Co-reporter:Sadagopan Krishnan ; Dhanuka Wasalathanthri ; Linlin Zhao ; John B. Schenkman
Journal of the American Chemical Society 2011 Volume 133(Issue 5) pp:1459-1465
Publication Date(Web):January 7, 2011
DOI:10.1021/ja108637s
Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR−cyt P450 complex uniquely poised for substrate conversion.
Co-reporter:Naimish P. Sardesai, John C. Barron, and James F. Rusling
Analytical Chemistry 2011 Volume 83(Issue 17) pp:6698
Publication Date(Web):July 5, 2011
DOI:10.1021/ac201292q
This paper describes fabrication of a novel electrochemiluminescence (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests residing in the bottoms of 10-μL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)3]2+ and secondary antibodies (RuBPY–silica–Ab2) are employed in this system for highly sensitive two-analyte detection. Antibodies to prostate specific antigen (PSA) and interleukin-6 (IL-6) were attached to the same RuBPY–silica–Ab2 particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 in. × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab2 is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which generates excited state [Ru(bpy)3]2+ in the 100-nm particles, and is measured with a charge-coupled device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. The detection limit (DL) for PSA was 1 pg mL–1 and for IL-6 was 0.25 pg mL–1 (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to the detection of two or more proteins.
Co-reporter:Shenmin Pan, Linlin Zhao, John B. Schenkman, and James F. Rusling
Analytical Chemistry 2011 Volume 83(Issue 7) pp:2754
Publication Date(Web):March 11, 2011
DOI:10.1021/ac200050n
Arrays for screening metabolite-generated toxicity utilizing spots containing DNA, enzyme, and electroluminescent (ECL) polymer ([Ru(bpy)2PVP10]2+) were extended to include a fully representative set of metabolic enzymes from human and rat liver microsomes, human and rat liver cytosol, and mouse liver S9 fractions. Array use involves two steps: (1) enzyme activation of the test chemical and metabolite reaction with DNA, and then, (2) capture of ECL resulting from DNA damage using a charge coupled device (CCD) camera. Plots of ECL increase vs enzyme reaction time monitor relative rates of DNA damage and were converted into turnover rates for enzymic production of DNA-reactive metabolites. ECL turnover rates were defined by R, the initial slope of ECL increase versus enzyme reaction time normalized for amounts of enzyme and test chemical. R-values were used to establish correlations for 11 toxic compounds with the standard toxicity metrics rodent liver TD50 and lethal dose (LD50), Ames tests, and Comet assays for in vitro DNA damage. Results support the value of the ECL genotoxicity arrays together with toxicity bioassays for early screening of new chemicals and drug candidates.
Co-reporter:Dhanuka P. Wasalathanthri, Vigneshwaran Mani, Chi K. Tang, and James F. Rusling
Analytical Chemistry 2011 Volume 83(Issue 24) pp:9499
Publication Date(Web):October 31, 2011
DOI:10.1021/ac202269t
A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)2(PVP)10](ClO4) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD50 toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.
Co-reporter:Gary C. Jensen, Colleen E. Krause, Gregory A. Sotzing and James F. Rusling
Physical Chemistry Chemical Physics 2011 vol. 13(Issue 11) pp:4888-4894
Publication Date(Web):07 Jan 2011
DOI:10.1039/C0CP01755H
Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL−1 in calf serum, sensitivity of 11.4 nA pg−1 cm−2, and a linear dynamic range of 20–400 pg mL−1.
Co-reporter:Dr. Sadagopan Krishnan;Vigneshwaran Mani;Dhanuka Wasalathanthri; Challa V. Kumar; James F. Rusling
Angewandte Chemie 2011 Volume 123( Issue 5) pp:1207-1210
Publication Date(Web):
DOI:10.1002/ange.201005607
Co-reporter:Dr. Sadagopan Krishnan;Vigneshwaran Mani;Dhanuka Wasalathanthri; Challa V. Kumar; James F. Rusling
Angewandte Chemie International Edition 2011 Volume 50( Issue 5) pp:1175-1178
Publication Date(Web):
DOI:10.1002/anie.201005607
Co-reporter:Bhaskara V. Chikkaveeraiah, Vigneshwaran Mani, Vyomesh Patel, J. Silvio Gutkind, James F. Rusling
Biosensors and Bioelectronics 2011 Volume 26(Issue 11) pp:4477-4483
Publication Date(Web):15 July 2011
DOI:10.1016/j.bios.2011.05.005
A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL−1 levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL−1 for PSA and 0.30 pg mL−1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.
Co-reporter:Yun Zhang, Amy M. LaFountain, Nikki Magdaong, Marcel Fuciman, James P. Allen, Harry A. Frank, and James F. Rusling
The Journal of Physical Chemistry B 2011 Volume 115(Issue 12) pp:3226-3232
Publication Date(Web):March 8, 2011
DOI:10.1021/jp111680p
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.
Co-reporter:Sadagopan Krishnan, John B. Schenkman, and James F. Rusling
The Journal of Physical Chemistry B 2011 Volume 115(Issue 26) pp:8371-8380
Publication Date(Web):May 17, 2011
DOI:10.1021/jp201235m
Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This Feature Article discusses the direct electrochemistry of cyt P450s in thin films and the utilization of such films for electrochemically driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems.
Co-reporter:Hongyun Liu, Ruchika Malhotra, Mark W. Peczuh and James F. Rusling
Analytical Chemistry 2010 Volume 82(Issue 13) pp:5865
Publication Date(Web):June 11, 2010
DOI:10.1021/ac101110q
Life-threatening allergies to peanuts and tree nuts can be revealed by detecting antibodies (IgEs) to their allergens in patient serum. Herein, we compare several immunosensor-like methodologies for sensitive detection of antibodies to a peptide sequence from the major peanut allergen, Arachis hypogaea 2 (Ara h2). The sensors feature a synthetic peptide layer of the major IgE-binding epitope from Ara h2 attached to a dense gold nanoparticle (AuNP) film on a pyrolytic graphite (PG) electrode. The AuNP−peptide sensor was used to determine model chicken antipeanut antibodies (IgY) in serum. Faradaic and nonfaradaic impedance strategies were compared to amperometric detection. Measurements employed goat antichicken secondary antibodies (Ab2) labeled with horseradish peroxidase (HRP) to bind to IgY on the sensor and provide amplified signals. The best impedimetric sensor configuration featured HPR-catalyzed precipitation of the enzyme product onto the sensor measured by nonfaradaic impedance. This sensor configuration had the best detection limit (DL) of 5 pg mL−1 and the best linear range of over 5 orders of magnitude (from 5 pg mL−1 to 1 μg mL−1) for IgY antibody in undiluted calf serum. This DL was 100-fold lower than label-free impedimetric immunosensors (0.5 ng mL−1) and 60-fold lower than when using HRP−Ab2 in amperometric immunosensors (0.3 ng mL−1).
Co-reporter:Linlin Zhao, John B. Schenkman, and James F. Rusling
Analytical Chemistry 2010 Volume 82(Issue 24) pp:10172
Publication Date(Web):November 19, 2010
DOI:10.1021/ac102317a
An inexpensive, high-throughput genotoxicity screening method was developed by using magnetic particles coated with cytosol/microsome/DNA films as biocolloid reactors in a 96-well plate format coupled with liquid chromatography−mass spectrometry. Incorporation of both microsomal and cytosolic enzymes in the films provides a broad spectrum of metabolic enzymes representing a range of metabolic pathways for bioactivation of chemicals. Reactive metabolites generated via this process are trapped by covalently binding to DNA in the film. The DNA is then hydrolyzed and nucleobase adducts are collected using filters in the bottom for the 96-well plate of analysis by capillary liquid chromatography−tandem mass spectrometry (LC−MS/MS). The magnetic particles facilitate simple and rapid sample preparation and workup. Major DNA adducts from ethylene dibromide, N-acetyl-2-aminofluorene and styrene were identified in proof-of-concept studies. Relative formation rates of DNA adducts correlated well with rodent genotoxicity metric TD50 for the three compounds. This method has the potential for high-throughput genotoxicity screening, providing chemical structure information that is complementary to toxicity bioassays.
Co-reporter:Ruchika Malhotra, Vyomesh Patel, Jose Pedro Vaqué, J. Silvio Gutkind and James F. Rusling
Analytical Chemistry 2010 Volume 82(Issue 8) pp:3118
Publication Date(Web):March 1, 2010
DOI:10.1021/ac902802b
Squamous cell carcinomas of head and neck (HNSCC) are associated with immune, inflammatory, and angiogenic responses involving interleukin-6 (IL-6). This article reports an ultrasensitive electrochemical immunosensor for human IL-6 and proof-of-concept studies of IL-6 detection in HNSCC cells. Single wall carbon nanotube (SWNT) forests with attached capture antibodies (Ab1) for IL-6 were used in an electrochemical sandwich immunoassay protocol using enzyme label horseradish peroxidase (HRP) to measure very low (≤30 pg mL−1) and elevated levels of IL-6. Two levels of multienzyme labeling were used to measure a broad concentration range of IL-6 in a representative panel of HNSCC cells. Secondary antibodies (Ab2) attached to carboxylated multiwall carbon nanotubes with 106 HRP labels per 100 nm gave the highest sensitivity of 19.3 nA mL (pg IL-6)−1 cm−2 and the best detection limit (DL) of 0.5 pg mL−1 (25 fM) for IL-6 in 10 μL of calf serum. For more concentrated samples, biotinylated Ab2 bound to streptavidin-HRP to provide 14−16 labels per antigen was used. These immunosensors accurately measured secreted IL-6 in a wide range of HNSCC cells demonstrated by excellent correlations with standard enzyme-linked immunosorbent assays (ELISA), suggesting that SWNT immunosensors combined with multilabel detection have excellent promise for detecting IL-6 in research and clinical applications.
Co-reporter:James F. Rusling, Challa V. Kumar, J. Silvio Gutkind and Vyomesh Patel
Analyst 2010 vol. 135(Issue 10) pp:2496-2511
Publication Date(Web):08 Jul 2010
DOI:10.1039/C0AN00204F
This critical review evaluates progress toward viable point-of-care protein biomarker measurements for cancer detection and diagnostics. The ability to measure panels of specific, selective cancer biomarker proteins in physicians' surgeries and clinics has the potential to revolutionize cancer detection, monitoring, and therapy. The dream envisions reliable, cheap, automated, technically undemanding devices that can analyze a patient's serum or saliva in a clinical setting, allowing on-the-spot diagnosis. Existing commercial products for protein assays are reliable in laboratory settings, but have limitations for point-of-care applications. A number of ultrasensitive immunosensors and some arrays have been developed, many based on nanotechnology. Multilabel detection coupled with high capture molecule density in immunosensors and arrays seems to be capable of detecting a wide range of protein concentrations with sensitivity ranging into the sub pg mL−1 level. Multilabel arrays can be designed to detect both high and ultralow abundance proteins in the same sample. However, only a few of the newer ultrasensitive methods have been evaluated with real patient samples, which is key to establishing clinical sensitivity and selectivity.
Co-reporter:Ruchika Malhotra, Fotios Papadimitrakopoulos, and James F. Rusling
Langmuir 2010 Volume 26(Issue 18) pp:15050-15056
Publication Date(Web):August 23, 2010
DOI:10.1021/la102306z
Electrochemical immunosensors using vertically aligned single wall carbon nanotube (SWNT) forests can provide ultrasensitive, accurate cancer biomarker protein assays. Herein we report a systematic investigation of the structure, thickness, and functionality of each layer of these immunosensors using atomic force microscopy (AFM), quartz crystal microbalance (QCM), and scanning white light interferometry (SWLI). This provides a detailed picture of the surface morphology of each layer along with surface concentration and thickness of each protein layer. Results reveal that the major reasons for sensitivity gain can be assigned to the dense packing of carboxylated SWNT forest tips, which translate to a large surface concentration of capture antibodies, together with the high quality of conductive SWNT forests.
Co-reporter:Linlin Zhao, John B. Schenkman and James F. Rusling
Chemical Communications 2009 (Issue 36) pp:5386-5388
Publication Date(Web):17 Aug 2009
DOI:10.1039/B909372A
We demonstrate for the first time the combination of human liver cytosol and microsomal enzyme sources into an electro-optical array to screen for reactive metabolites produced in multi-enzyme metabolic processes.
Co-reporter:Naimish Sardesai, Shenmin Pan and James Rusling
Chemical Communications 2009 (Issue 33) pp:4968-4970
Publication Date(Web):10 Jul 2009
DOI:10.1039/B909220J
We report the first electrochemiluminescent immunosensor combining single-wall carbon nanotube forests with RuBPY–silica–secondary antibody nanoparticles for sensitive detection of cancer biomarker prostate specific antigen.
Co-reporter:Bhaskara V. Chikkaveeraiah, Ashwin Bhirde, Ruchika Malhotra, Vyomesh Patel, J. Silvio Gutkind and James F. Rusling
Analytical Chemistry 2009 Volume 81(Issue 21) pp:9129
Publication Date(Web):September 23, 2009
DOI:10.1021/ac9018022
Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab2) that bind specifically to streptavidin−HRP conjugates provided 14−16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab2−HRP conjugates were sufficient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (ELISA) assays.
Co-reporter:Dominic O. Hull, Besnik Bajrami, Ingela Jansson, John B. Schenkman and James F. Rusling
Analytical Chemistry 2009 Volume 81(Issue 2) pp:716
Publication Date(Web):December 18, 2008
DOI:10.1021/ac802179s
Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)32+-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis−Menten inhibition models were used to determine apparent inhibition constants (KI*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with KI* of 268.2, 142.3, and 204.2 μM, respectively.
Co-reporter:Besnik Bajrami, Linlin Zhao, John B. Schenkman and James F. Rusling
Analytical Chemistry 2009 Volume 81(Issue 24) pp:9921
Publication Date(Web):November 11, 2009
DOI:10.1021/ac9015853
Silica nanoparticle bioreactors featuring thin films of enzymes and polyions were utilized in a novel high-throughput 96-well plate format for drug metabolism profiling. The utility of the approach was illustrated by investigating the metabolism of the drugs diclofenac (DCF), troglitazone (TGZ), and raloxifene, for which we observed known metabolic oxidation and bioconjugation pathways and turnover rates. A broad range of enzymes was included by utilizing human liver (HLM), rat liver (RLM) and bicistronic human-cyt P450 3A4 (bicis.-3A4) microsomes as enzyme sources. This parallel approach significantly shortens sample preparation steps compared to an earlier manual processing with nanoparticle bioreactors, allowing a range of significant enzyme reactions to be processed simultaneously. Enzyme turnover rates using the microsomal bioreactors were 2−3 fold larger compared to using conventional microsomal dispersions, most likely because of better accessibility of the enzymes. Ketoconazole (KET) and quinidine (QIN), substrates specific to cyt P450 3A enzymes, were used to demonstrate applicability to establish potentially toxic drug−drug interactions involving enzyme inhibition and acceleration.
Co-reporter:Bhaskara V. Chikkaveeraiah, Hongyun Liu, Vigneshwaran Mani, Fotios Papadimitrakopoulos, James F. Rusling
Electrochemistry Communications 2009 Volume 11(Issue 5) pp:1092
Publication Date(Web):May 2009
DOI:10.1016/j.elecom.2009.03.021
Co-reporter:Bernard S. Munge, Colleen E. Krause, Ruchika Malhotra, Vyomesh Patel, J. Silvio Gutkind, James F. Rusling
Electrochemistry Communications 2009 Volume 11(Issue 5) pp:1009-1012
Publication Date(Web):May 2009
DOI:10.1016/j.elecom.2009.02.044
Electrochemical immunosensors based on single wall nanotube (SWNT) forests and 5 nm glutathione-protected gold nanoparticles (GSH-AuNP) were developed and compared for the measurement of human cancer biomarker interleukin-6 (IL-6) in serum. Detection was based on sandwich immunoassays using multiple (14–16) horseradish peroxidase labels conjugated to a secondary antibody. Performance was optimized by effective blocking of non-specific binding (NSB) of the labels using bovine serum albumin. The GSH-AuNP immunosensor gave a detection limit (DL) of 10 pg mL−1 IL-6 (500 amol mL−1) in 10 μL calf serum, which was 3-fold better than 30 pg mL−1 found for the SWNT forest immunosensor for the same assay protocol. The GSH-AuNPs platform also gave a much larger linear dynamic range (20–4000 pg mL−1) than the SWNT system (40–150 pg mL−1), but the SWNTs had 2-fold better sensitivity in the low pg mL−1 range.
Co-reporter:Bhaskara V. Chikkaveeraiah, Hongyun Liu, Vigneshwaran Mani, Fotios Papadimitrakopoulos, James F. Rusling
Electrochemistry Communications 2009 Volume 11(Issue 4) pp:819-822
Publication Date(Web):April 2009
DOI:10.1016/j.elecom.2009.02.002
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.
Co-reporter:Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, John B. Schenkman and James F. Rusling
Molecular BioSystems 2009 vol. 5(Issue 2) pp:163-169
Publication Date(Web):12 Dec 2008
DOI:10.1039/B815910F
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 NNKmetabolites formed upon H2O2-enzymatic activation were captured as DNAadducts 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.
Co-reporter:Linlin Zhao, Sadagopan Krishnan, Yun Zhang, John B. Schenkman and James F. Rusling
Chemical Research in Toxicology 2009 Volume 22(Issue 2) pp:341
Publication Date(Web):January 23, 2009
DOI:10.1021/tx8004295
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/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an 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 versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with 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 the direct measurement of the 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.
Co-reporter:Ashwin A. Bhirde, Vyomesh Patel, Julie Gavard, Guofeng Zhang, Alioscka A. Sousa, Andrius Masedunskas, Richard D. Leapman, Roberto Weigert, J. Silvio Gutkind and James F. Rusling
ACS Nano 2009 Volume 3(Issue 2) pp:307
Publication Date(Web):January 13, 2009
DOI:10.1021/nn800551s
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 nontargeted bioconjugates. First line anticancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the nontargeted 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 intravital video 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 nontargeted SWNT-cisplatin.Keywords: carbon nanotubes; cisplatin; EGF; EGFR; intravital two-photon microscopy; nanomedicine; oral cancer; quantum dots
Co-reporter:Vigneshwaran Mani, Bhaskara V. Chikkaveeraiah, Vyomesh Patel, J. Silvio Gutkind and James F. Rusling
ACS Nano 2009 Volume 3(Issue 3) pp:585
Publication Date(Web):February 13, 2009
DOI:10.1021/nn800863w
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 1 μm diameter magnetic beads. These sensors had sensitivity of 31.5 μA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 μL 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.Keywords: cancer biomarkers; gold nanoparticles; immunosensor; multilabel amplification
Co-reporter:James F. Rusling, Gregory Sotzing, Fotios Papadimitrakopoulosa
Bioelectrochemistry 2009 Volume 76(1–2) pp:189-194
Publication Date(Web):September 2009
DOI:10.1016/j.bioelechem.2009.03.011
Detection of multiple cancer biomarker proteins in human serum and tissue at point-of-care is a viable approach for early cancer detection, but presents a major challenge to bioanalytical device development. This article reviews recent approaches developed in our laboratories combining nanoparticle decorated electrodes and multilabeled secondary antibody labeled particles to achieve high sensitivity for the detection of cancer biomarker proteins. Two nanomaterial-based sensor platforms were used: (a) upright single wall carbon nanotube forests and (b) layers of densely packed 5 nm gold nanoparticles. Both platforms feature pendant carboxylate groups for easy attachment of enzymes or antibodies by amidization. In quality performance tests, the biocatalytic responses for determination of hydrogen peroxide of AuNP layers with attached horseradish peroxidase (HRP) on electrodes gave somewhat better detection limit and sensitivity than single wall carbon nanotube (SWNT) forest platforms with HRP attached. Evaluation of these sensors as platforms for sandwich immunoassays for cancer biomarker prostate specific antigen (PSA) in serum showed that both approaches gave accurate results for human serum samples from cancer patients. The best detection limit (0.5 pg mL− 1) and sensitivity were obtained by combining the AuNP immunosensors with binding of 1 μm diameter magnetic particles decorated with secondary antibodies and 7500 HRP labels.
Co-reporter:Minjeong So, John B. Schenkman and James F. Rusling
Chemical Communications 2008 (Issue 36) pp:4354-4356
Publication Date(Web):18 Jul 2008
DOI:10.1039/B805447A
We demonstrate for the first time a biosensor featuring a sequential two-enzyme pathway suitable to screen potentially toxic reactive metabolites generated during metabolism.
Co-reporter:James F. Rusling, Eli G. Hvastkovs, Dominic O. Hull and John B. Schenkman
Chemical Communications 2008 (Issue 2) pp:141-154
Publication Date(Web):30 Aug 2007
DOI:10.1039/B709121B
This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme–poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA–enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite–DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL “spots” of DNA, enzyme and metallopolymer. DNA–enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.
Co-reporter:Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Dharamainder Choudhary, John B. Schenkman and James F. Rusling
Analytical Chemistry 2008 Volume 80(Issue 14) pp:5279
Publication Date(Web):June 19, 2008
DOI:10.1021/ac800763r
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.
Co-reporter:Peterson M. Guto, Challa V. Kumar and James F. Rusling
Langmuir 2008 Volume 24(Issue 18) pp:10365-10370
Publication Date(Web):August 9, 2008
DOI:10.1021/la801644e
Microemulsions of oil, water and surfactant were evaluated as media for biocatalysis at high temperatures employing films of polylysine (PLL) and the enzymes horseradish peroxidase (HRP), soybean peroxidase (SBP) and the protein myoglobin (Mb). PLL was covalently linked to oxidized pyrolytic graphite electrodes or carboxylated 500 nm diameter silica nanoparticles, then cross-linked by amidization to HRP, SBP and Mb. The resulting film systems were stable at 90 °C for >12 h in microemulsions. Characterization of the microemulsions by conductivity, viscosity and probe diffusion coefficients suggested that these media have bicontinuous microstructures from 25 to 90 °C. UV circular dichroism and visible spectroscopy confirmed that the enzymes retained near-native conformation in the films at temperatures as high as 90 °C. Oxidation of o-methoxyphenol to 3,3′-dimethoxy-4,4′-biphenoquinone by enzyme-PLL films on silica nanoparticles gave yields 3−5-fold larger in microemulsions at 90 °C compared to the same reaction at 25 °C. The best yields were in CTAB microemulsions and were 3-fold larger than in buffers at 90 °C.
Co-reporter:S. N. Kim;F. Papadimitrakopoulos;J. F. Rusling
Advanced Materials 2007 Volume 19(Issue 20) pp:3214-3228
Publication Date(Web):21 SEP 2007
DOI:10.1002/adma.200700665
The unique electronic and optical properties of carbon nanotubes, in conjunction with their size and mechanically robust nature, make these nanomaterials crucial to the development of next-generation biosensing platforms. In this Review, we present recent innovations in carbon nanotube-assisted biosensing technologies, such as DNA-hybridization, protein-binding, antibody-antigen and aptamers. Following a brief introduction on the diameter- and chirality-derived electronic characteristics of single-walled carbon nanotubes, the discussion is focused on the two major schemes for electronic biodetection, namely biotransistor- and electrochemistry-based sensors. Key fabrication methodologies are contrasted in light of device operation and performance, along with strategies for amplifying the signal while minimizing nonspecific binding. This Review is concluded with a perspective on future optimization based on array integration as well as exercising a better control in nanotube structure and biomolecular integration.
Co-reporter:Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Ingela Jansson, John B. Schenkman and James F. Rusling
Chemical Communications 2007 (Issue 17) pp:1713-1715
Publication Date(Web):03 Apr 2007
DOI:10.1039/B703012F
We report for the first time voltammetric/electrochemiluminescent sensors applied to predict genotoxicity of N-nitroso compounds bioactivated by human cytochrome P450 enzymes.
Co-reporter:Sadagopan Krishnan, James F. Rusling
Electrochemistry Communications 2007 Volume 9(Issue 9) pp:2359-2363
Publication Date(Web):September 2007
DOI:10.1016/j.elecom.2007.07.002
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 V s−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.
Co-reporter:Minjeong So, Eli G. Hvastkovs, John B. Schenkman, James F. Rusling
Biosensors and Bioelectronics 2007 Volume 23(Issue 4) pp:492-498
Publication Date(Web):30 November 2007
DOI:10.1016/j.bios.2007.06.017
Simultaneous optical and voltammetric detection of bioactivated genotoxicity is reported for the first time employing ultrathin films of DNA, model metabolic enzymes, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ on pyrolytic graphite (PG) electrodes. Cytochrome P450cam and myoglobin were used as model monoxygenase enzymes to mimic in vivo processes. Sensor film growth and component amounts were monitored using a quartz crystal microbalance (QCM). Subsequent to the enzyme reaction, DNA damage in the sensor films was measured simultaneously using a simple apparatus combining a standard voltammetry cell coupled with an optical fiber and photomultiplier tube. The model enzyme reaction converted styrene to styrene oxide, which reacts with DNA nucleobases. ECL and SWV signals increased with enzyme reaction time on the scale of several min, and provided relative enzyme turnover rates for DNA damage suitable for toxicity screening applications. Within 1 min, the sensor detects ∼3 damaged bases per 10,000 DNA bases using this simultaneous detection.
Co-reporter:Peterson M. Guto, James F. Rusling
Electrochemistry Communications 2006 Volume 8(Issue 3) pp:455-459
Publication Date(Web):March 2006
DOI:10.1016/j.elecom.2006.01.007
A recent paper [M.T. DeGroot, M. Merkx, M.J.M. Koper, J. Am. Chem. Soc. 127 (2005) 16224] suggests that iron heme is released during casting films of myoglobin (Mb) and didodecyldimethylammonium bromide (DDAB) on pyrolytic graphite (PG) electrodes. Insoluble films of lipids and surfactants have become widely used for protein film electrochemistry, and protein conformation within them is a critical issue. The present communication examines the integrity of myoglobin structure in DDAB films by voltammetry, visible absorption and circular dichroism spectroscopy, and discusses these new data in context with previously reported studies. Voltammetry showed that peak potentials for Mb, horseradish peroxidase and hemin in DDAB films are significantly different, and that Soret band and CD spectra in the films are fully consistent with a near native Mb conformation within the films. The view that pyrolytic graphite surfaces are catalysts for denaturation and heme loss in Mb-DDAB films is discredited by consideration of key results in the literature. On the basis of all results and considerations, this communication confirms that when DDAB films are made from vesicle dispersions at pH 5 to 7 and used in neutral buffers containing NaBr, Mb in the film is in a near-native conformation.
Co-reporter:Khrisna E. Alcantara, James F. Rusling
Electrochemistry Communications 2005 Volume 7(Issue 2) pp:223-226
Publication Date(Web):February 2005
DOI:10.1016/j.elecom.2004.12.016
Rotating disc voltammetry data for the reaction of myoglobin, a model “enzyme” in a thin lipid film, with ferredoxin was shown to fit the Michaelis–Menten kinetic model, but did not fit a simple linear EC’ model. Advantages of this thin-film method are a lipid bilayer environment similar to that of membrane bound enzymes, the tiny amounts of proteins required, and simplicity of instrumentation compared to alternative methodology. The apparent KM for ferredoxin reduction by reduced myoglobin was 112 μM, kcat was 102 s−1, and kcat/KM was 9.1 × 104 M−1 s−1 indicating relatively fast kinetics. Results suggest that this method should be amenable to kinetic studies of enzymes in lipid films with protein reaction partners in solution.
Co-reporter:Jing Yang, Bingquan Wang and James F. Rusling
Molecular BioSystems 2005 vol. 1(Issue 3) pp:251-259
Publication Date(Web):22 Jul 2005
DOI:10.1039/B506111C
Responses from “reagentless” DNA-based electrochemical toxicity sensors to DNA alkylating agents styrene oxide (SO), diepoxybutane (DEB), and methyl methanesulfonate (MMS) were compared to formation rates of total alkylated nucleobases in DNA measured by LC-UV-MS. Sensors utilized a catalytic metallopolymer in DNA films previously exposed to the damage agents. To achieve adequate sensitivity, LC-UV-MS analyses were done on DNA in solution reacted with the damage agents, and subsequently hydrolyzed to nucleosides with enzymes. Sensor response correlated well with nucleobase-adduct formation rates obtained by the molecule-specific analyses. Results confirm that the metallopolymer–DNA film sensors can be used to estimate relative DNA damage rates from nucleobase adduct-forming chemicals. Results from both methods correlated well with animal genotoxicity as estimated by TDLo values, the lowest dose producing carcinogenicity, in mice and rats. These sensors should be useful for rapid, inexpensive screening of moderately and severely genotoxic new chemicals.
Co-reporter:Xin Yu, Sang Nyon Kim, Fotios Papadimitrakopoulos and James F. Rusling
Molecular BioSystems 2005 vol. 1(Issue 1) pp:70-78
Publication Date(Web):24 Mar 2005
DOI:10.1039/B502124C
Vertically aligned arrays of single-wall carbon nanotubes (SWNT forests) on pyrolytic graphite surfaces were developed for amperometric enzyme-linked immunoassays. Improved fabrication of these SWNT forests utilizing aged nanotube dispersions provided higher nanotube density and conductivity. Biosensor performance enhancement was monitored using nanotube-bound peroxidase enzymes showing a 3.5-fold better sensitivity for H2O2 than when using fresh nanotubes to assemble the forests, and improved detection limits. Absence of improvements by electron mediation for detection of H2O2 suggested very efficient electron exchange between nanotubes and enzymes attached to their ends. Protein immunosensors were made by attaching antibodies to the carboxylated ends of nanotube forests. Utilizing casein/detergent blocking to minimize non-specific binding, a detection limit of 75 pmol mL−1
(75 nM) was achieved for human serum albumin (HSA) in unmediated sandwich immunosensors using horseradish peroxidase (HRP) labels. Mediation of the immunosensors dramatically lowered the detection limit to 1 pmol mL−1
(1 nM), providing significantly better performance than alternative methods. In the immunosensor case, the average distance between HRP labels and nanotube ends is presumably too large for efficient direct electron exchange, but this situation can be overcome by electron mediation.
Co-reporter:James F. Rusling
Biosensors and Bioelectronics 2004 Volume 20(Issue 5) pp:1022-1028
Publication Date(Web):15 November 2004
DOI:10.1016/j.bios.2004.06.033
Films of DNA, enzymes, polyions, and catalytic redox polyions of nanometer thickness on electrodes can provide active elements for sensors for screening the toxicity of chemicals and their metabolites, and for oxidative stress. The unifying feature of this approach involves layer-by-layer electrostatic assembly of films designed to detect DNA damage. Films containing DNA and enzymes enable detection of structural damage to DNA as a basis for toxicity screening. These films bioactivate chemicals to their metabolites, which can then react with DNA, mimicking toxicity pathways in the human liver. Metallopolyions that catalyze DNA oxidation can be incorporated into DNA/enzyme films leading to “reagentless” sensors. These sensors are suitable for detecting relative DNA damage rates in <5 min of the enzyme reactions.Films of the osmium polymer [Os(bpy)2(PVP)10Cl]+ [poly(vinylpyridine), PVP] can be used to monitor DNA oxidation selectively. Such films may be applicable to determination of oxidized DNA as a clinical biomarker for oxidative stress. Inclusion of the analogous ruthenium metallopolymer in the sensor provides a monitor for oxidation of other nucleobases.
Co-reporter:Xin Yu, Debjit Chattopadhyay, Izabela Galeska, Fotios Papadimitrakopoulos, James F. Rusling
Electrochemistry Communications 2003 Volume 5(Issue 5) pp:408-411
Publication Date(Web):May 2003
DOI:10.1016/S1388-2481(03)00076-6
This communication reports the first example, to our knowledge, of enzymes covalently attached onto the ends of vertically oriented single-wall carbon nanotube (SWNT) forest arrays used as electrodes. Quasi-reversible FeIII/FeII voltammetry was observed for the iron heme enzymes myoglobin and horseradish peroxidase coupled to carboxylated ends of the nanotube forests by amide linkages. Results suggest that the “trees” in the nanotube forest behaved electrically similar to a metal, conducting electrons from the external circuit to the redox sites of the enzymes. Electrochemically manifested peroxidase activity of myoglobin and horseradish peroxidase attached to the SWNT forests was demonstrated, with detection limits for hydrogen peroxide in buffer solutions of ∼100 nM. These prototype SWNT-forest biosensors are easy to prepare, and enzyme layers were stable for weeks.
Co-reporter:James F. Rusling, Robert J. Forster
Journal of Colloid and Interface Science 2003 Volume 262(Issue 1) pp:1-15
Publication Date(Web):1 June 2003
DOI:10.1016/S0021-9797(03)00148-6
Supramolecular redox-active assemblies on electrodes are of fundamental interest and can be used to create functioning devices such as sensors, biosensors, and bioreactors. The ability of redox-active films to mediate electron transfer reactions in 3-D dramatically increases the sensitivity with which target molecules can be determined. Metallopolyion hydrogel films immobilized on electrode surfaces exhibit many properties that are reminiscent of those shown by redox-active proteins. This review discusses the electrochemical properties and applications of such films, including mediating electron transfer between electrodes and oxidase enzymes. In addition, polyion–protein films grown layer by layer have certain advantages in device fabrication, including facilitating direct electron transfer for many proteins, mechanical stability, use of tiny amounts of protein, and control of film architecture. This review presents examples of iron heme proteins in films grown layer by layer by alternate electrostatic adsorption for catalytic reduction of hydrogen peroxide and trichloroacetic acid and for oxidation of styrene.
Co-reporter:Carmelita Estavillo, Zhongqing Lu, Ingela Jansson, John B. Schenkman, James F. Rusling
Biophysical Chemistry 2003 Volume 104(Issue 1) pp:291-296
Publication Date(Web):1 May 2003
DOI:10.1016/S0301-4622(02)00383-6
Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H2O2-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At −0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H2O2 which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H2O2 (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450cam gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450cam, and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.
Co-reporter:Bernard Munge;Carmelita Estavillo;John B. Schenkman
ChemBioChem 2003 Volume 4(Issue 1) pp:
Publication Date(Web):3 JAN 2003
DOI:10.1002/cbic.200390018
The catalytic and electrochemical properties of myoglobin and cytochrome P450camin films constructed with alternate polyion layers were optimized with respect to film thickness, polyion type, and pH. Electrochemical and hydrogen peroxide driven epoxidation of styrene catalyzed by the proteins was used as the test reaction. Ionic synthetic organic polymers such as poly(styrene sulfonate), as opposed to SiO2nanoparticles or DNA, supported the best catalytic and electrochemical performance. Charge transport involving the iron heme proteins was achieved over 40–320 nm depending on the polyion material and is likely to involve electron hopping facilitated by extensive interlayer mixing. However, very thin films (ca. 12–25 nm) gave the largest turnover rates for the catalytic epoxidation of styrene, and thicker films were subject to reactant transport limitations. Classical bell-shaped activity/pH profiles and turnover rates similar to those obtained in solution suggest that films grown layer-by-layer are applicable to turnover rate studies of enzymes for organic oxidations. Major advantages include enhanced enzyme stability and the tiny amount of protein required.
Co-reporter:Mohamed Sharafeldin, Gregory W. Bishop, Snehasis Bhakta, Abdelhamid El-Sawy, Steven L. Suib, James F. Rusling
Biosensors and Bioelectronics (15 May 2017) Volume 91() pp:
Publication Date(Web):15 May 2017
DOI:10.1016/j.bios.2016.12.052
•Fe3O4@graphene oxide for multiplexed electrochemical detection of protein analytes.•Antibodies decorated Fe3O4@GO for magnetic separation of protein analytes.•Detection limits (LOD) of low fg/mL for prostate cancer biomarker proteins.•Dynamic range and LOD tailored to each protein sample requirements.Ultrasensitive mediator-free electrochemical detection for biomarker proteins was achieved at low cost using a novel composite of Fe3O4 nanoparticles loaded onto graphene oxide (GO) nano-sheets (Fe3O4@GO). This paramagnetic Fe3O4@GO composite (1 µm size range) was decorated with antibodies against prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA), and then used to first capture these biomarkers and then deliver them to an 8-sensor detection chamber of a microfluidic immunoarray. Screen-printed carbon sensors coated with electrochemically reduced graphene oxide (ERGO) and a second set of antibodies selectively capture the biomarker-laden Fe3O4@GO particles, which subsequently catalyze hydrogen peroxide reduction to detect PSA and PSMA. Accuracy was confirmed by good correlation between patient serum assays and enzyme-linked immuno-sorbent assays (ELISA). Excellent detection limits (LOD) of 15 fg/mL for PSA and 4.8 fg/mL for PSMA were achieved in serum. The LOD for PSA was 1000-fold better than the only previous report of PSA detection using Fe3O4. Dynamic ranges were easily tunable for concentration ranges encountered in serum samples by adjusting the Fe3O4@GO Concentration. Reagent cost was only $0.85 for a single 2-protein assay.
Co-reporter:Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Ingela Jansson, John B. Schenkman and James F. Rusling
Chemical Communications 2007(Issue 17) pp:NaN1715-1715
Publication Date(Web):2007/04/03
DOI:10.1039/B703012F
We report for the first time voltammetric/electrochemiluminescent sensors applied to predict genotoxicity of N-nitroso compounds bioactivated by human cytochrome P450 enzymes.
Co-reporter:Naimish Sardesai, Shenmin Pan and James Rusling
Chemical Communications 2009(Issue 33) pp:
Publication Date(Web):
DOI:10.1039/B909220J
Co-reporter:James F. Rusling, Eli G. Hvastkovs, Dominic O. Hull and John B. Schenkman
Chemical Communications 2008(Issue 2) pp:NaN154-154
Publication Date(Web):2007/08/30
DOI:10.1039/B709121B
This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme–poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA–enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite–DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL “spots” of DNA, enzyme and metallopolymer. DNA–enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.
Co-reporter:Dandan Li, You-Jun Fu and James F. Rusling
Chemical Communications 2015 - vol. 51(Issue 22) pp:NaN4703-4703
Publication Date(Web):2015/02/11
DOI:10.1039/C5CC00420A
We report here label-free metabolite–protein adduct detection and identification employing magnetic beads coated with metabolic enzymes as bioreactors to generate metabolites and possible metabolite–protein adducts for analysis by liquid chromatography-tandem mass spectrometry.
Co-reporter:Spundana Malla, Karteek Kadimisetty, You-Jun Fu, Dharamainder Choudhary, Ingela Jansson, John B. Schenkman and James F. Rusling
Chemical Science (2010-Present) 2015 - vol. 6(Issue 10) pp:NaN5563-5563
Publication Date(Web):2015/06/24
DOI:10.1039/C5SC01403D
Damage to p53 tumor suppressor gene is found in half of all human cancers. Databases integrating studies of large numbers of tumors and cancer cell cultures show that mutation sites of specific p53 codons are correlated with specific types of cancers. If the most frequently damaged p53 codons in vivo correlate with the most frequent chemical damage sites in vitro, predictions of organ-specific cancer risks might result. Herein, we describe LC-MS/MS methodology to reveal codons with metabolite-adducted nucleobases by LC-MS/MS for oligonucleotides longer than 20 base pairs. Specifically, we used a known carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) to determine the most frequently adducted nucleobases within codons. We used a known sequence of 32 base pairs (bp) representing part of p53 exon 7 with 5 possible reactive hot spots. This is the first nucleobase reactivity study of a double stranded DNA p53 fragment featuring more than 20 base pairs with multiple reactive sites. We reacted the 32 bp fragment with benzo[a]pyrene metabolite BPDE that undergoes nucleophilic substitution by DNA bases. Liquid chromatography-mass spectrometry (LC-MS/MS) was used for sequencing of oligonucleotide products from the reacted 32 bp fragment after fragmentation by a restriction endonuclease. Analysis of the adducted p53 fragment compared with unreacted fragment revealed guanines of codons 248 and 244 as most frequently targeted, which are also mutated with high frequency in human tumors. Codon 248 is mutated in non-small cell and small cell lung, head and neck, colorectal and skin cancer, while codon 244 is mutated in small cell lung cancer, all of which involve possible BDPE exposure. Results suggest the utility of this approach for screening of adducted p53 gene by drugs and environmental chemicals to predict risks for organ specific cancers.
Co-reporter:Gary C. Jensen, Colleen E. Krause, Gregory A. Sotzing and James F. Rusling
Physical Chemistry Chemical Physics 2011 - vol. 13(Issue 11) pp:NaN4894-4894
Publication Date(Web):2011/01/07
DOI:10.1039/C0CP01755H
Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL−1 in calf serum, sensitivity of 11.4 nA pg−1 cm−2, and a linear dynamic range of 20–400 pg mL−1.
Co-reporter:Minjeong So, John B. Schenkman and James F. Rusling
Chemical Communications 2008(Issue 36) pp:
Publication Date(Web):
DOI:10.1039/B805447A
Co-reporter:Linlin Zhao;John B. Schenkman
Chemical Communications 2009(Issue 36) pp:
Publication Date(Web):2009/09/02
DOI:10.1039/B909372A
We demonstrate for the first time the combination of human liver cytosol and microsomal enzyme sources into an electro-optical array to screen for reactive metabolites produced in multi-enzyme metabolic processes.
Co-reporter:Dhanuka P. Wasalathanthri, Dandan Li, Donghui Song, Zhifang Zheng, Dharamainder Choudhary, Ingela Jansson, Xiuling Lu, John B. Schenkman and James F. Rusling
Chemical Science (2010-Present) 2015 - vol. 6(Issue 4) pp:NaN2468-2468
Publication Date(Web):2015/02/12
DOI:10.1039/C4SC03401E
Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termed genotoxic. Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)2(PVP)10]2+ {(PVP = poly(4-vinylpyridine))} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.
Co-reporter:Yixian Wang, Kaan Kececi, Michael V. Mirkin, Vigneshwaran Mani, Naimish Sardesai and James F. Rusling
Chemical Science (2010-Present) 2013 - vol. 4(Issue 2) pp:NaN663-663
Publication Date(Web):2012/11/15
DOI:10.1039/C2SC21502K
Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP–peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.
Co-reporter:Jennifer E. Satterwhite-Warden, Dilip K. Kondepudi, James A. Dixon and James F. Rusling
Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 44) pp:NaN29898-29898
Publication Date(Web):2015/10/08
DOI:10.1039/C5CP04471E
Self-motion of physical-chemical systems is a promising avenue for studying and developing mechanical functions with inanimate systems. In this paper, we investigate spontaneous motion of collections of solid macroscopic benzoquinone (BQ) disks at the air–water interface without intervention of chemical reactions. The BQ particles slowly dissolve and create heterogeneous interfacial tension fields on the water surface that drive the motion. Spontaneous, continuous locomotion was observed between multiple BQ particles, along with coupling, collisions, cycling and collective foraging for interfacial free energy. Analysis of the motion suggests co-operative behavior depends strongly on particle shape.
Co-reporter:
Analytical Methods (2009-Present) 2014 - vol. 6(Issue 22) pp:
Publication Date(Web):
DOI:10.1039/C4AY01962H
Inexpensive, reusable electrochemical sensor chips were fabricated from gold CDs. All reagents were loaded onto a paper disk sequentially, then placed on the chip to detect cancer biomarker prostate specific antigen (PSA) in serum at pg mL−1 levels in ∼15 min.
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