Automated 3-D Printed Arrays to Evaluate Genotoxic Chemistry: E-Cigarettes and Water Samples

Abstract

A novel, automated, low cost, three-dimensional (3-D) printed microfluidic array was developed to detect DNA damage from metabolites of chemicals in environmental samples. The electrochemiluminescent (ECL) detection platform incorporates layer-by-layer (LbL) assembled films of microsomal enzymes, DNA and an ECL-emitting ruthenium metallopolymer in ∼10 nm deep microwells. Liquid samples are introduced into the array, metabolized by the human enzymes, products react with DNA if possible, and DNA damage is detected by ECL with a camera. Measurements of relative DNA damage by the array assess the genotoxic potential of the samples. The array analyzes three samples simultaneously in 5 min. Measurement of cigarette and e-cigarette smoke extracts and polluted water samples was used to establish proof of concept. Potentially genotoxic reactions from e-cigarette vapor similar to smoke from conventional cigarettes were demonstrated. Untreated wastewater showed a high genotoxic potential compared to negligible values for treated wastewater from a pollution control treatment plant. Reactivity of chemicals known to produce high rates of metabolite-related DNA damage were measured, and array results for environmental samples were expressed in terms of equivalent responses from these standards to assess severity of possible DNA damage. Genotoxic assessment of wastewater samples during processing also highlighted future on-site monitoring applications.

Keywords: 3-D printing; automation; e-cigarettes; electrochemiluminescence; environmental samples; genotoxicity.

Figure 1

Automated genotoxicity screening array: (A) 3-D printed devices without (left) and with (right) microwell chip and counter electrode wires inserted showing the sample chambers containing dye solutions. (B) Microwell-patterned pyrolytic graphite detection array showing the first row holding 1 μL water droplets retained by the hydrophobic microwell boundaries. Each row is fed by a separate sample line. The working array features films of DNA, metabolic enzymes, and RuPVP in each microwell. (C) Assembled array system showing box enclosing electronic microprocessors and micropumps driven by a rechargeable battery and connected to the 3-D printed array below with a wash reservoir (top) containing pH 7.4 buffer.

Figure 2

Array results for tobacco-related standards with DNA-reactive metabolites: (A) recolorized ECL data using arrays featuring RuPVP/enzyme/DNA microwells treated with oxygenated solutions of carcinogens B[a]P, NNK, and NNN and negative control toluene in 1% DMSO + 10 mM phosphate buffer pH 7.4 for 45 s at −0.65 V vs Ag/AgCl, with ECL captured by CCD camera after subsequently applying 1.25 V vs Ag/AgCl for 180 s. (B) Calibration plots of % ECL increase over 1% DMSO control vs concentration of standards. ECL intensity increases proportional to DNA damage that disorders ds-DNA and allows coreactant guanines in the DNA better access to Ru^III sites of RuPVP.

Figure 3

ECL array results comparing extracted vapor from e-cigarettes with extracted smoke from tobacco cigarettes using the conversion that 20 e-cigarette puffs equals smoke from one tobacco cigarette {Abbrev.: tobacco cigarettes (TC), e-cigarettes (EC), nonfiltered (nf) and non-nicotine (nn)}. (A) Recolorized ECL data from arrays. Each row represents microwells containing RuPVP/Enzyme/DNA layers treated with smoke extracted from 1, 3, and 5 TC and nf-TC (equivalent to 20, 60, and 100 puffs of e-cigarette) and 20, 60, and 100 puffs of EC and non-nicotine (nn)-EC in 1% DMSO containing buffer for 45 s under potential of −0.65 V vs Ag/AgCl. ECL captured while applying 1.25 V vs Ag/AgCl for 180 s. (B) % ECL increase over control (1% DMSO in buffer) vs cigarette samples. (C) NNK equivalents from %ECL for different cigarette samples.

Figure 4

Array results for standards with known DNA-reactive metabolites: (A) Recolorized ECL data using arrays featuring RuPVP/enzyme/DNA microwells treated with oxygenated solutions of carcinogens (2-AAF, 2-NA, and aflatoxin B1 and negative control toluene in 1% DMSO + 10 mM phosphate buffer pH 7.4 for 45 s at −0.65 V vs Ag/AgCl, with ECL captured by CCD camera after subsequently applying 1.25 V vs Ag/AgCl for 180 s. (B) Calibration plots of %ECL increase over the blank vs concentration of standards. ECL intensity increases proportional to DNA damage. (C) ECL array results comparing ECL intensities obtained from untreated water (UTW), partially treated water (PTW), and fully treated reclaimed water (RCW) with respect to 1% DMSO controls. Recolorized ECL data from arrays with each row representing microwells containing RuPVP/enzyme/DNA layers treated with UTW, PTW, RCW, and 1% DMSO in buffer for 45 s at −0.65 V vs Ag/AgCL with ECL captured after subsequent application of 1.25 V vs Ag/AgCl for 180 s. (D) Bar graph showing chemical equivalents from %ECL response for different water samples.

Scheme 1. Cytochrome P450 Mediated Bioactivation and DNA Reactivity of Standard Chemicals Used for Cigarette Studies^a

^a(1) Benzo[a]pyrene (B[a]P), metabolized to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide that intercalates and covalently binds predominantly with guanine base in DNA, Adapted from information in ref . (2) 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and (3) N-nitrosonicotine (NNN) form hydroxyl forms before binding to nucleobases within DNA. Adapted from information in ref .

Scheme 2. Cytochrome P450 Mediated Bioactivation and DNA Reactivity of Standard Chemicals Used for Water Samples^a

^a(4) Aflatoxin B1 (AFB1), metabolically activated to its epoxide form that forms covalent adducts with DNA nucleobases. Adapted from information in ref . (5) 2-Acetylaminofluorene (2-AAF). Adapted from information in ref . (6) 2-naphthylamine (2-NA) form acetoxy forms upon bioactivation that form covalent adducts with DNAnucleobases. Adapted from information in ref .