■ DISCUSSION
The results above illustrate a novel, low cost, 3-D printed
microfluidic array capable of assessing the genotoxic potential
of environmental samples. The 3-D printed device with
disposable microwell array containing enzyme/DNA/RuPVP
films costs less than $1.00 to fabricate. Advantages of this
device include multianalyte analysis, complete automation, on
chip metabolite generation, and rapid detection of DNA
damage (5 min). These disposable arrays are designed to “plugin”
to a reusable automation platform featuring microcontrollers,
micropumps, and battery that costs $150. The
array here was equipped with 21 detection microwells, but it is
possible to expand to larger size to accommodate multiple
enzymes, multiple analytes, and higher sample throughput.
DNA reactivity related to metabolites from smoke or vapor
extracts measured by the array clearly suggests comparable
genotoxic potential of tobacco and nicotine e-cigarettes when
assayed by the same protocol (Figure 3). Expression of the
signals in terms of levels of known tobacco chemicals with
metabolites having high rates of DNA damage, e.g., NNK
(Figure 3D) provides a reference point to assess the severity of
possible genotoxicity, without having to determine individual
DNA adducts by expensive LC-MS/MS assays. E-cigarette
vapor was reported to have low concentrations of chemicals
with potential to cause DNA damage13 and could be assumed
by some to be a safer alternative to tobacco cigarettes.
However, our results suggest similar DNA damage from ecigarette
vapors and tobacco cigarette smoking.
Results also showed that genotoxic potential for non-nicotine
e-cigarettes is about the same as that for filtered tobacco
cigarettes, and 1.5−2-fold lower than that for e-cigarettes. DNA
reactivity for 20 puffs of an e-cigarette was equivalent to about
83 μM NNK (1.6 μg/mL) (Figure 3C) compared to estimated
levels of NNK in one tobacco cigarette of 46 μM (0.9 μg/mL).
Unfiltered tobacco cigarettes gave DNA reactivity nearly 2
times greater than filtered tobacco cigarettes (Figure 3B and
C). Even non-nicotine e-cigarettes showed significant DNA
reactivity, similar to that of filtered tobacco cigarettes (Figure
3C).
The above results are consistent with recent reports using
conventional assays that found significant DNA strand breaks,
cytotoxic effects, and cell death caused by e-cigarette vapor with
and without nicotine.41,42 Ease of use of e-cigarettes may also
result in elevated use compared to tobacco cigarettes, which can
result in escalated DNA damage. For example, DNA reactivity
as NNK equivalents in vapor extract from two full e-cigarette
cartridges was 1.1 mM, roughly equivalent to 0.9 mM for 20
tobacco cigarettes (SI Table S1).
The arrays also revealed genotoxic potential of water samples
(Figure 4). ECL responses from the untreated wastewater were
about 9 times larger than those for fully recovered water,
suggesting significant presence of genotoxic chemicals.
Successful analysis of samples during midtreatment suggests
that the array can be used to monitor the success of
intermediate stages of water treatment. Expression of these
results in terms of water polluting chemicals that cause
metabolite-related DNA damage again provide a rapid
assessment of relative severity of the contamination (Figure
4D). Calibration range and LOD of 3 μM for standard water
pollutants (Figure 4B) suggests applications in rapid identi-
fication of seriously polluted water. Array results for the
untreated water samples are consistent with reports of
genotoxic chemicals in domestic and industrial wastewater.43
ECL responses from fully reclaimed water did not show
significant genotoxic potential when compared to controls,
suggesting significant removal of genotoxic chemicals.
In summary, we described here a new, portable, low cost,
automated, toxicity screening tool to detect metabolite-related
genotoxicity chemistry from environmental samples. The 3-D
printed array is fast and accurate in sensing effects of possible
genotoxic chemicals. A unique feature is that test chemicals are
converted to their metabolites so that metabolite reactivity
toward genetic material can be measured rapidly and efficiently.
This is a major attribute for assessment of possible genotoxic
consequences of pollutant exposure from relevant samples.
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