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.The measurement of protein biomarkers holds significant promise for early cancer detection. 1 , 2 Detection of multiple protein biomarkers provides more reliable diagnostics than single biomarker measurements.1 -3 Proteins in serum can be measured with enzyme-linked immunosorbent assays (ELISA), 4 radioimmunoassay (RIA),5 electrophoretic immunoassay6 and mass spectrometry-based proteomics.7 However, there is a clear need for simple, rapid, sensitive, accurate and low cost protein detection suitable for point of care use.Electrochemiluminescence (ECL) is initiated when tris (2,2′-bipyridyl)ruthenium(II), [Ru-(bpy) 3 ] 2+ , or RuBPY, in its oxidized form reacts with a suitable sacrificial reductant, and has been used for detection of DNA and proteins. [8][9][10] Magnetic bead ECL methods have been commercialized. 11,12 Dye doped silica nanoparticles show promise as labels in ultrasensitive ECL bioassays. 13 RuBPY can be trapped inside mesoporous silica nanoparticles synthesized in water-in-oil (W/ O) microemulsions. 14 A single nanoparticle can encapsulate thousands of RuBPY to provide large signal amplification. Surfaces of the particles can be decorated with attached antibodies or proteins and arrays utilizing ECL can be fabricated using analytical spots on a simple conductive chip with a single connection to a power source and readout with a CCD camera, as we demonstrated with DNA-based toxicity screening arrays.9 This approach is simpler than the commercial bead-based ECL assays, which require sophisticated and expensive sample manipulation and measurement systems.15In this communication, we combine ECL nanoparticle labels with a single-wall carbon nanotube (SWCNT) forest platform in a sandwich immunoassay procedure for protein detection. The SWCNT forests feature self-assembled 20-30 nm long terminally carboxylated SWCNT standing in upright bundles on a thin Nafion-iron oxide layer on a pyrolytic graphite † Electronic supplementary information (ESI) available: Experimental details, Fig. S1 16 and provide a large conductive, functionalized surface area for attachment of capture antibodies in immunoassays.16b We have used SWCNT forests to build ultrasensitive amperometric immunosensors and detected PSA in serum using multiple enzyme labels.17 However, arrays using this strategy involve microfabricated multi-electrode chips as well as multi-electrode potentiostats, which are not necessary in ECL arrays.9In related work, [Ru-(bpy) 3 ] 2+ immobilized on composite films of disordered CNTs in combination with Nafion, 18a partially sulfonated polystyrene 18b and Eastman-AQ polymers 18c were used for ECL determination of tripropylamine 18 with a detection limit of 3 pM. 18c SWCNT forests with large carboxylated surface areas combined with immunoassays using RuBPY-silica-secondary ant...

Arrays for screening metabolite-generated toxicity utilizing spots containing DNA, enzyme and electroluminescent (ECL) polymer ([Ru(bpy) 2 PVP 10 ] 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, then (2) capture of ECL resulting from DNA damage using a 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 DNAreactive 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 TD 50 and LD 50 , 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.

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 8-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. 7,8-dihydro-8-oxoguanine (8-oxodG) in ds-DNA 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+.

Peroxidase enzyme digests of oxidized single-wall carbon nanotubes (SWCNT) were shown to damage DNA in potentially genotoxic reactions for the first time using an electro-optical array with and without metabolic activation.

There is limited and sometimes contradictory information about the genotoxicity of 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 relatively low reactivity of metabolically activated B[ghi]P towards 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 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 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 ECL array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P.

Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response.

not Available.