Electrochemical transformations catalyzed by cytochrome P450s and peroxidases.

Cytochrome P450s (Cyt P450s) and peroxidases are enzymes featuring iron heme cofactors that have wide applicability as biocatalysts in chemical syntheses. Cyt P450s are a family of monooxygenases that oxidize fatty acids, steroids, and xenobiotics, synthesize hormones, and convert drugs and other chemicals to metabolites. Peroxidases are involved in breaking down hydrogen peroxide and can oxidize organic compounds during this process. Both heme-containing enzymes utilize active FeIVO intermediates to oxidize reactants. By incorporating these enzymes in stable thin films on electrodes, Cyt P450s and peroxidases can accept electrons from an electrode, albeit by different mechanisms, and catalyze organic transformations in a feasible and cost-effective way. This is an advantageous approach, often called bioelectrocatalysis, compared to their biological pathways in solution that require expensive biochemical reductants such as NADPH or additional enzymes to recycle NADPH for Cyt P450s. Bioelectrocatalysis also serves as an ex situ platform to investigate metabolism of drugs and bio-relevant chemicals. In this paper we review biocatalytic electrochemical reactions using Cyt P450s including C-H activation, S-oxidation, epoxidation, N-hydroxylation, and oxidative N-, and O-dealkylation; as well as reactions catalyzed by peroxidases including synthetically important oxidations of organic compounds. Design aspects of these bioelectrocatalytic reactions are presented and discussed, including enzyme film formation on electrodes, temperature, pH, solvents, and activation of the enzymes. Finally, we discuss challenges and future perspective of these two important bioelectrocatalytic systems.

Quantitative detection of RAS and KKS peptides in COVID-19 patient serum by stable isotope dimethyl labeling LC-MS.

Angiotensin and kinin metabolic pathways are reported to be altered by many diseases, including COVID-19. Monitoring levels of these peptide metabolites is important for understanding mechanisms of disease processes. In this paper, we report dimethyl labeling of amines in peptides by addition of formaldehyde to samples and deutero-formaldehyde to internal standards to generate nearly identical isotopic standards with 4 m/z units larger per amine group than the corresponding analyte. We apply this approach to rapid, multiplexed, absolute LC-MS/MS quantitation of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) peptides in human blood serum. Limits of detection (LODs) were obtained in the low pg mL-1 range with 3 orders of magnitude dynamic ranges, appropriate for determinations of normal and elevated levels of the target peptides in blood serum and plasma. Accuracy is within ±15% at concentrations above the limit of quantitation, as validated by spike-recovery in serum samples. Applicability was demonstrated by measuring RAS and KKS peptides in serum from COVID-19 patients, but is extendable to any class of peptides or other small molecules bearing reactive -NH2 groups.

Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer.

Parathyroid hormone-related peptide (PTHrP) is related to bone metastasis and hypercalcemia in prostate and breast cancers and should be an excellent biomarker for aggressive forms of these cancers. Current clinical detection protocols for PTHrP are immunoradiometric assay and radioimmunoassay but are not sensitive enough to detect PTHrPs at early stages. We recently evaluated a prostate cancer biomarker panel, including serum monocyte differentiation antigen (CD-14), ETS-related gene protein, pigment epithelial-derived factor, and insulin-like growth factor-1, with promise for identifying aggressive prostate cancers. This panel predicted the need for patient biopsy better than PSA alone. In the present paper, we report an ultrasensitive microfluidic assay for PTHrPs and evaluate their diagnostic value and the value of including them with our prior biomarker panel to diagnose aggressive forms of prostate cancer. The immunoarray features screen-printed carbon sensor electrodes coated with 5 nm glutathione gold nanoparticles with capture antibodies attached. PTHrPs are bound to a secondary antibody attached to a polyhorseradish peroxidase label and delivered to the sensors to provide high sensitivity when activated by H2O2 and a mediator. We obtained an unprecedented 0.3 fg mL-1 limit of detection for PTHrP bioactive moieties PTHrP 1-173 and PTHrP 1-86. We also report the first study of PTHrPs in a large serum pool to identify aggressive malignancies. In assays of 130 human patient serum samples, PTHrP levels distinguished between aggressive and indolent prostate cancers with 83-91% clinical sensitivity and 78-96% specificity. Logistic regression identified the best predictive model as a combination of PTHrP 1-86 and vascular endothelial growth factor-D. PTHrP 1-173 alone also showed a high ability to differentiate aggressive and indolent cancers.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 µL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

Sub-zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors.

We report here a low-cost electrochemical immunoarray with unprecedented sensitivity in the sub-zeptomole range with up to 5 log-decades dynamic range for accurate, multiplexed protein determinations. The microfluidic array features eight carbon sensors coated with a dense layer of 5 nm gold-nanoparticles derivatized with primary antibodies. Analyte proteins are captured by secondary antibody-poly-HPR (horseradish peroxidase) bioconjugates containing 400 HRP enzyme labels, with amplified amperometric peaks developed using H2O2 activator and hydroquinone mediator. Prostate cancer biomarkers prostate specific antigen (PSA), vascular endothelial growth factor-D (VEGF-D), ETS-related gene protein (ERG), and insulin-like growth factor-1 (IGF-1) were measured simultaneously with sub-fg/mL LODs (0.08-0.22 zmol). These proteins were determined in serum of postprostatectomy cancer patients which had much lower levels than prostate cancer patients without surgery. This immunoassay protocol makes thousands of low-abundance proteins accessible to quantitative measurements down to zeptomole levels.

Organ-Specific Screening for Protein Damage Using Magnetic Bead Bioreactors and LC-MS/MS.

A new 96-well plate methodology for fast, enzyme-multiplexed screening for metabolite-protein adducts was developed. Magnetic beads coated with metabolic enzymes were used to make potentially reactive metabolites that can react with test protein in the wells, followed by sample workup in multiple 96-well filter plates for LC-MS/MS analysis. Incorporation of human microsomes from multiple organs and selected supersomes of single cytochrome P450 (cyt P450) enzymes on the magnetic beads provided a broad spectrum of metabolic enzymes. The reacted protein was then isolated, denatured, reduced, alkylated, and digested, and peptides were collected in a sequence of 96-well filter plates for analysis. Method performance was evaluated by trapping acetaminophen reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) with human glutathione S-transferase pi (hGSTP), human serum albumin (HSA), and bovine serum albumin (BSA) as model target proteins. Relative amounts of acetaminophen metabolite and hGSTP adducts were compared with 10 different cyt P450 enzymes. Human liver microsomes and CYP1A2 supersomes showed the highest bioactivation rate for adduct formation, in which all four cysteines of hGSTP reacted with NAPQI. Eight cysteines of HSA and four cysteines of BSA have been detected to react with NAPQI. This method has the potential for fast multienzyme protein adduct screening with high efficiency and accuracy.

Metabolites of Tobacco- and E-Cigarette-Related Nitrosamines Can Drive Cu2+-Mediated DNA Oxidation.

Nitrosamine metabolites resulting from cigarette smoking and E-cigarette (E-cig) vaping cause DNA damage that can lead to genotoxicity. While DNA adducts of metabolites of nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are well-known tobacco-related cancer biomarkers, only a few studies implicate NNN and NNK in DNA oxidation in humans. NNK and NNN were found in the urine of E-cigarette users who never smoked cigarettes. This paper proposes the first chemical pathways of DNA oxidation driven by NNK and NNN metabolites in redox reactions with Cu2+ and NADPH leading to reactive oxygen species (ROS). A microfluidic array with thin films of DNA and metabolic enzymes that make metabolites of NNN and NNK in the presence of Cu2+ and NADPH was used to estimate relative rates of DNA oxidation. Detection by electrochemiluminescence (ECL) employed a new ECL dye [Os(tpy-benz-COOH)2]2+ that is selective for and sensitive to the primary DNA oxidation product 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in DNA. Enzyme-DNA films on magnetic beads were used to produce nitrosamine metabolites that enter ROS-forming redox cycles with Cu2+ and NADPH, and liquid chromatography-mass spectrometry (LC-MS) was used to quantify 8-oxodG and identify metabolites. ROS were detected by optical sensors. Metabolites of NNK and NNN + Cu2+ + NADPH generated relatively high rates of DNA oxidation. Lung is the exposure route in smoking and vaping, human lung tissue contains Cu2+ and NADPH, and lung microsomal enzymes gave the highest rates of DNA oxidation in this study. Also, E-cigarette vapor contains 6-fold more copper than that in cigarette smoke, which could exacerbate DNA oxidation.

3D-Printed Immunosensor Arrays for Cancer Diagnostics.

Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers' and clinicians' needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as additive manufacturing, has drastically reduced the time of sensor and sensor array development while offering excellent sensitivity at a fraction of the cost of conventional technologies such as photolithography. In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.

Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 µm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab of antibodies on the beads. Different patterns of digestion rates were found due to proteolysis of the oriented and nonoriented antibodies on MBs. For oriented antibodies, the peptides from outer Fab regions gave a much higher digestion rate than those from Fc regions, while for randomly immobilized antibodies digestion rates for Fab and Fc peptides were similar. This novel approach is a useful and convenient tool to characterize antibody orientation for immunoassays and other applications. The relative degree of orientation can be assessed using a metric Ro denoting amount of Fab marker peptides found divided by Fc + Fab marker peptides × 100%. Oriented antibodies on the MBs also provided more efficient antigen capture compared to randomly immobilized antibodies.

Accessible Telemedicine Diagnostics with ELISA in a 3D Printed Pipette Tip.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 µL pipettes. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.

Particle Flock Motion at Air-Water Interface Driven by Interfacial Free Energy Foraging.

From flocks of birds and sheep to colonies of bacteria, complex patterns and self-motion are found in all hierarchies of nature. Artificial nonliving systems provide useful insight, since living systems are complicated and may involve cognitive issues not found in nonliving matter. Herein, we report naturally flocking irregularly shaped benzoquinone (BQ) particles on the air-water interface that cross a gate. In this open system designed with absence of external control, the particle flock moves by Marangoni "surfing" driven by slow dissolution of weakly surface active BQ postulated to create inhomogeneous interfacial tension fields. The particle flocks move collectively through a gate placed in the air-water interface to the side that has higher interfacial tension. Position-sensitive surface tension measurements used for the first time in a multiparticle Marangoni motion system show unequivocally that flock motion and gate crossing proceed to areas of slightly higher interfacial tension. Flock crossing is accompanied by a low-high differential interfacial tension change from one side of the gate to the other, with the flock moving to the side with higher interfacial tension. Thus, the flocks move because they are foraging for interfacial free energy.

Influence of antibody immobilization strategy on carbon electrode immunoarrays.

We report here the influence of antibody immobilization strategy for protein immunosensors on screen printed carbon electrode arrays in terms of antibody binding activity, analytical sensitivity, limit of detection, and stability. Horseradish peroxidase (HRP) was the model analyte with anti-HRP immobilized on the sensors, and HRP activity was used for detection. Covalently immobilized anti-HRP antibodies on electrodes coated with chitosan, electrochemically reduced graphene oxide (rGO), and dense gold nanoparticle (AuNP) films had only 20-30% of the total immobilized antibodies active for binding. Active antibodies increased to 60% with passively adsorbed antibodies on bare electrodes, to 85% with oriented antibodies using protein A covalently immobilized on AuNP-coated carbon electrode, and to 98% when attached to protein A passively adsorbed onto bare electrodes. Passively adsorbed antibodies on bare electrodes lost activity in 1-2 days, but antibodies immobilized using other strategies remained relatively stable after 5 days. Covalent immobilization gave limits of detection (LOD) of 40 fg mL-1, while passively adsorbed antibodies or protein A on carbon electrodes had LODs 4-8 fg mL-1, but were unstable. Sensitivity was highest for antibodies covalently attached to AuNP electrodes (2.40 nA per log pg per mL) that also had highest antibody coverage, and decreased slightly when protein A on AuNP was used to orient antibodies. Passively adsorbed antibodies and oriented antibodies on protein A gave slightly lower sensitivities. Immobilization strategy or antibody orientation did not have a significant effect on LOD, but dynamic range increased as the number of active antibodies on sensor surfaces increased.

Glucose biosensor based on open-source wireless microfluidic potentiostat.

Wireless potentiostats capable of cyclic voltammetry and amperometry that connect to the Internet are emerging as key attributes of future point-of-care devices. This work presents an "integrated microfluidic electrochemical detector" (iMED) three-electrode multi-potentiostat designed around operational amplifiers connected to a powerful WiFi-based microcontroller as a promising alternative to more expensive and complex strategies reported in the literature. The iMED is integrated with a microfluidic system developed to be controlled by the same microcontroller. The iMED is programmed wirelessly over a standard WiFi network and all electrochemical data is uploaded to an open-source cloud-based server. A wired desktop computer is not necessary for operation or program uploading. This method of integrated microfluidic automation is simple, uses common and inexpensive materials, and is compatible with commercial sample injectors. An integrated biosensor platform contains four screen-printed carbon arrays inside 4 separate microfluidic detection chambers with Pt counter and pseudo Ag/AgCl reference electrodes in situ. The iMED is benchmarked with K3[Fe(CN)6] against a commercial potentiostat and then as a glucose biosensor using glucose-oxidising films of [Os(2,2'-bipyridine)2(polyvinylimidazole)10Cl] prepared on screen-printed electrodes with multi walled carbon nanotubes, poly(ethylene glycol) diglycidyl ether and flavin adenine dinucleotide-dependent glucose dehydrogenase. Potential application of this cost-effective wireless potentiostat approach to modern bioelectronics and point-of-care diagnosis is demonstrated by production of glucose oxidation currents, under pseudo-physiological conditions, using mediating films with lower redox potentials.

Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

Automated 3D-Printed Microfluidic Array for Rapid Nanomaterial-Enhanced Detection of Multiple Proteins.

We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging.

Peanut allergy can be life-threatening and is mediated by allergen-specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope-resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ-chain-specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross-reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 µL each) in a 45 min assay. Excellent correlation of Ara h2-specific IgE values was found between ImmunoCAP assays and the new SPRi method.

A novel and accurate microfluidic assay of CD62L in bladder cancer serum samples.

We report a low-cost, sensitive, bead-based electrochemical immunoarray for soluble L-selectin (or CD62L protein), a potential biomarker for staging bladder cancer. We used a semi-automated modular microfluidic array with online antigen capture on superparamagnetic beads, which were subsequently delivered to a detection chamber housing multiple sensors. The assay was designed to accurately detect CD62L in diluted serum with a limit of detection (LOD) of 0.25 ng mL-1 and a dynamic range of 0.25-100 ng mL-1. The microfluidic array gave significantly better accuracy and higher sensitivity than a standard ELISA kit, which was shown to be subject to significant systematic error at high and low concentration ranges. 31 serum samples from patients with varying grades of bladder cancer and cancer-free controls were analyzed by the immunoarray and ELISA, and the CD62L levels correlated. This work establishes a new accurate assay for determining CD62L levels and highlights the potential of this protein as a biomarker for detecting locoregional progression of bladder cancer.

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

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.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

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.

Screening Genotoxicity Chemistry with Microfluidic Electrochemiluminescent Arrays.

This review describes progress in the development of electrochemiluminescent (ECL) arrays aimed at sensing DNA damage to identify genotoxic chemistry related to reactive metabolites. Genotoxicity refers to chemical or photochemical processes that damage DNA with toxic consequences. Our arrays feature DNA/enzyme films that form reactive metabolites of test chemicals that can subsequently react with DNA, thus enabling prediction of genotoxic chemical reactions. These high-throughput ECL arrays incorporating representative cohorts of human metabolic enzymes provide a platform for determining chemical toxicity profiles of new drug and environmental chemical candidates. The arrays can be designed to identify enzymes and enzyme cascades that produce the reactive metabolites. We also describe ECL arrays that detect oxidative DNA damage caused by metabolite-mediated reactive oxygen species. These approaches provide valuable high-throughput tools to complement modern toxicity bioassays and provide a more complete toxicity prediction for drug and chemical product development.