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. 2009 Summer:18:34-39.

Electrochemiluminescent Arrays For Toxicity Screening

James F Rusling

Electrochemiluminescent Arrays For Toxicity Screening

James F Rusling. Electrochem Soc Interface. 2009 Summer.
No abstract available

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Figures

Figure 1
Figure 1
Strategies for metabolic toxicity screening (a) The chemistry: reactive metabolite styrene oxide is formed by cyt P450 enzyme and oxygen, then the epoxide reacts with DNA damage as a endpoint that can be measured by ECL arrays and other approaches. (b) Microsome/DNA films used in enzyme/DNA nanoreactors (left) or ECL array chip (right): A layer of cationic Ru-PVP polymer is initially deposited (blue ribbon in circle) followed by layers of negative DNA, polycation, and microsomes as a source of enzymes (membrane is brown, cyt P450 reductase (CPR) = red, and cyt P450 = green; (PEI = polyethyleneimine; RuPVP = [Ru(bpy)2PVP10]2+; bpy = bipyridine). In the first step, NADPH generated by an enzyme reaction in solution reduces CPR, which transfers electrons to cyt P450s. O2 and cyt P450 combine to convert substrate to reactive metabolites that form DNA adducts in the film. In the second step, ECL from the array is detected with a CCD camera upon application of +1.25 V vs. Ag/AgCl and monitors DNA adduct accumulation. Hydrolysis can be used to release labile DNA adducts from the nanoreactor particles for LC-MS analysis to obtain direct structural and formation rate confirmation of the ECL array results.
Figure 2
Figure 2
Two configurations for ECL detection of metabolite-based DNA damage: (A) Sensor for simultaneous ECL detection and voltammetry showing (a) reference, (b) sensor and (c) counter electrodes located in a glass cell with a cylindrical base. Fiber optic (d) is outside cell directly under the sensor leading to a photomultiplier tube. Ru-PVP (top structure), DNA (brown), and enzyme (blue) layers form active sensor film shown on right. (B) ECL arrays featuring (a) array; (b) reference electrode; (c) counter electrode; (d) CCD camera; (e) computer; (f) dark box; (g) potentiostat to apply voltage. Active enzyme/DNA/Ru-PVP films are 20–40 nm thick. In the first operational step, enzyme in the film converts substrates to reactive metabolites in close proximity to DNA. Resulting damage to DNA from these reactive metabolites is detected in the second operational step by ECL at an applied voltage 1.25 V for 20 s.
Figure 3
Figure 3
ECL array results for enzyme reactions with 100 μM benzo[a]pyrene + 0.5 mM H2O2 (a) reconstructed images for different reaction times (0, 1, 3, 5, 7 min.) for cyt P450 enzymes and myoglobin on the same array. Brighter spots indicated more DNA damage. (b) ECL initial/final ratios normalized for the amount of enzyme in each spot estimated by quartz crystal microbalance studies. Adapted with permission from reference , copyright 2007 American Chemical Society.
Figure 4
Figure 4
Reconstructed array data demonstrating ECL from spots of RuPVP/DNA/RLM (labeled RLM) or RuPVP/DNA/h2E1 (labeled h2E1) exposed to 1 mM of denoted substrate using enzymatic NADPH regeneration for denoted time in sec. Data for respective substrates are from same array run. Control is an identical array (RLM; h2E1 was similar (not shown)) not exposed to reaction solutions. Internal controls C1 = 120 s exposure to NADPH solution only; C2 = 120 s exposure to substrate (no NADPH) only. Substrates: 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosopiperidine (NPIP), and N-nitrosopyrrolidine (NPYR). Adapted with permission from reference , copyright 2008 American Chemical Society.
Figure 5
Figure 5
Percent ECL increase vs. reaction time for a) RLM and b) cyt P450 @E1 microsome (h2E1) arrays exposed to NPYR (black circles), NNK (blue squares), NPIP (green diamonds), or styrene (red triangles). Control (open circles, dash) in a) is the ECL increase on a RLM array not exposed to any xenobiotic solution. Adapted with permission from reference , copyright 2008 American Chemical Society.
Scheme 1
Scheme 1
Pathway for ECL generation using TprA as reductant
Scheme 2
Scheme 2
Pathway for ECL using DNA as reductant, G = guanine moiety
Scheme 3
Scheme 3
N-Nitrosopyrrolidine (NPYR) metabolism leading to DNA damage
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