sensors
Review
Screening Genotoxicity Chemistry with Microfluidic
Electrochemiluminescent Arrays
Itti Bist 1 , Kiran Bano 1 and James F. Rusling 1,2,3,4, *
1
2
3
4
*
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA; [email protected] (I.B.);
[email protected] (K.B.)
Department of Surgery and Neag Cancer Center, University of Connecticut Health Center, Farmington,
CT 06032, USA
Institute of Material Science, University of Connecticut, Storrs, CT 06269, USA
School of Chemistry, National University of Ireland at Galway, Galway, Ireland
Correspondence: [email protected]
Academic Editor: María Jesús Lobo-Castañón
Received: 7 April 2017; Accepted: 26 April 2017; Published: 3 May 2017
Abstract: 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.
Keywords: electrochemiluminescence; genotoxicity; arrays; cytochrome P450; microfluidics
1. Introduction
Toxic chemicals and their metabolites generated by cytochrome P450 (cyt P450) enzymes and
bioconjugate enzymes can cause DNA damage. Various types of DNA damage can be caused by
toxic compounds, including oxidation of DNA bases, nucleobase adduct formation, depurination of
adducts, and strand breaks [1]. DNA oxidations can occur from ionizing radiation and reactive oxygen
species (ROS) [2–4] with the major product 8-oxo-2-deoxyguanosine (8-oxodG) on DNA [5] being
responsible for mutagenesis. DNA adducts formed by coupling of reactive metabolites and DNA bases
are major biomarkers for cancer risk in humans, so a thorough understanding of reactive metabolites
of chemicals generated by cyt P450s [6] and bioconjugation enzymes is essential for a complete toxicity
pathway assessment of chemicals and drugs.
Electrochemical methods for DNA damage and toxicity detection were pioneered by Paleček [7]
and Fojta [8]. To date, various electrochemical sensors for DNA damage, DNA interactions with drugs,
environmental pollutants, and other potentially genotoxic species have been developed and reviewed
by various authors [9–14]. However, our group is the only one to develop metabolite-generating
microfluidic electrochemiluminescent (ECL) arrays for predicting metabolic genotoxicity chemistry of
xenobiotics. This review focuses on our ECL sensor technology for genotoxicity screening.
Sensors 2017, 17, 1008; doi:10.3390/s17051008
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Our approach as described herein features ECL arrays comprised of microwells containing dense
DNA/enzyme
films that produce metabolites in close proximity to DNA. ECL dyes that
utilize
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damaged DNA as a co-reactant are included in the microwells. Extension of reactive metabolite
damaged
DNA as a co-reactant
are analyses
included can
in the
Extension
of reactive
metabolite
studies
to complementary
LC-MS/MS
be microwells.
done utilizing
DNA/enzyme
films
on magnetic
studies
to complementary
analyses can
be done
utilizing
DNA/enzyme
films
on magnetic
beads
to biocolloid
enzymeLC-MS/MS
reactors [15–18].
This
review
describes
the sensor
arrays,
chemistry,
beads
to
biocolloid
enzyme
reactors
[15–18].
This
review
describes
the
sensor
arrays,
chemistry,
and
and mechanisms of genotoxicity revealed using these ECL arrays.
mechanisms of genotoxicity revealed using these ECL arrays.
2. Metabolic Pathway Involved in Genotoxicity
2. Metabolic Pathway Involved in Genotoxicity
Cyt P450s are important metabolic enzymes that catalyze 75% of all known metabolic reactions
Cyt P450s are important metabolic enzymes that catalyze 75% of all known metabolic reactions
involving drugs and other xenobiotics and can often generate reactive metabolites [19,20]. These reactive
involving drugs and other xenobiotics and can often generate reactive metabolites [19,20]. These
metabolites
can cause DNA
damage,
the and
parent
are then
as genotoxic.
reactive metabolites
can cause
DNA and
damage,
the chemicals
parent chemicals
are described
then described
as
Cyt P450-catalyzed
metabolic pathways
works
through
a complex
catalytic
cycle catalytic
by involving
genotoxic. Cyt P450-catalyzed
metabolic
pathways
works
through
a complex
cycle the
by ferric
III
iron–heme
(P-Fe)
prosthetic
group.
A two-electron
reduction
of the heme
iron breaks
oxygen-Fe
involving
the ferric
iron–heme
(P-Fe)
prosthetic group.
A two-electron
reduction
of thethe
heme
iron
III bond
bondbreaks
usingthe
electrons
shuttled
byusing
cyt P450
reductase
(CPR)
from
NADPH
to(CPR)
cyt P450s.
oxygen-Fe
electrons
shuttled
by cyt
P450
reductase
from Reduction
NADPH to of cyt
III
III
III and protonation
III-hydroperoxo complex
cyt
P450s.
Reduction
of
cyt
P450-Fe
yields
the
cyt
P450-Fe
P450-Fe and protonation yields the cyt P450-Fe -hydroperoxo complex 6 (Scheme 1). This complex
IV=O, 7) that
IV =O,
(Scheme an
1). active
This complex
produces
an active
ferryloxy
(cytoxygen
P450-Fe
then 6produces
ferryloxythen
species
(cyt P450-Fe
7) thatspecies
transfers
to bound
substrate
transfers
oxygen
to
bound
substrate
(RH)
to
oxidize
it,
often
by
an
oxygen
transfer
reaction.
The
(RH) to oxidize it, often by an oxygen transfer reaction. The oxidized group can then serve as a
oxidized group can then serve as a site for attaching a solubilizing conjugate. Our genotoxicity assays
site for attaching a solubilizing conjugate. Our genotoxicity assays can incorporate any human cyt
can incorporate any human cyt P450s and/or metabolic bioconjugation enzymes to mimic these
P450s and/or metabolic bioconjugation enzymes to mimic these events in the human body [15].
events in the human body [15]. Many substrates yield DNA-reactive metabolites, including styrene,
Many
substrateshydrocarbons,
yield DNA-reactive
metabolites,
including
styrene,
polyaromatic
hydrocarbons,
polyaromatic
nitrosamines,
aromatic amines,
tamoxifen,
and
other chemotherapeutic
nitrosamines,
aromatic
amines,
tamoxifen,
and
other
chemotherapeutic
agents
[21–24].
agents [21–24].
Scheme 1. The proposed catalytic pathway for cyt P450-catalyzed metabolic reactions [17,25].
Scheme 1. The proposed catalytic pathway for cyt P450-catalyzed metabolic reactions [17,25].
3. Development of ECL-Based Genotoxicity Sensors
3. Development of ECL-Based Genotoxicity Sensors
Our early toxicity sensors combined metabolic enzymes, DNA, and polyions in layer-by-layer
(LBL)
thin films
on graphite
electrodes through
electrostatic
alternately
charged layers (LBL)
Our early
toxicity
sensors combined
metabolic
enzymes,adsorption
DNA, andof
polyions
in layer-by-layer
of
a
redox
ECL
polymer,
DNA,
enzymes,
and
synthetic
polyions
[26].
Metabolites
are
produced
thin films on graphite electrodes through electrostatic adsorption of alternately charged layersin
ofan
a redox
enzyme
reaction
step,
and
DNA
damage
is
subsequently
monitored
using
ECL
and
LC-MS
ECL polymer, DNA, enzymes, and synthetic polyions [26]. Metabolites are produced in an enzyme
bioreactors [27,28]. The ECL dye polycation (bis-2,2′-bipyridyl) ruthenium polyvinylpyridine
reaction step, and DNA
damage is subsequently monitored using ECL and LC-MS bioreactors [27,28].
([Ru(bpy)2(PVP)10]2+ or RuPVP) is oxidized by the electrode at ~1.2 V vs. Ag/AgCl to yield RuIII-PVP, 2+
The ECL dye polycation (bis-2,20 -bipyridyl) ruthenium polyvinylpyridine
([Ru(bpy)2 (PVP)10 ] or
which catalytically oxidizes guanines to guanine radicals (G•). G• in intact DNA
serves as an ECL coIII -PVP, which catalytically
RuPVP)
is
oxidized
by
the
electrode
at
~1.2
V
vs.
Ag/AgCl
to
yield
Ru
reactant to eventually produce excited Ru2+*-PVP, which emits light at 610 nm (Scheme 2). Initially,
oxidizes guanines to guanine radicals (G• ). G• in intact DNA serves as an ECL co-reactant to eventually
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produce
excited Ru2+ *-PVP, which emits light at 610 nm (Scheme 2). Initially, single electrode
ECL
sensors
coated
RuPVP/enzyme/DNA
were
developed
as
shown
in
Scheme
3A.
These
in
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2017,with
17, 1008
3sensors
of 10
single electrode ECL sensors coated with RuPVP/enzyme/DNA were developed as shown in Scheme
some cases
measured
andcases
voltammetry
simultaneously
[29,30].
3A. These
sensors ECL
in some
measured ECL
and voltammetry
simultaneously [29,30].
single electrode ECL sensors coated with RuPVP/enzyme/DNA were developed as shown in Scheme
3A. These sensors in some cases measured ECL and voltammetry simultaneously [29,30].
Scheme 2. (A) Structure of ruthenium polyvinylpyridine (RuPVP); (B) proposed reaction mechanism
Scheme 2. (A) Structure of ruthenium polyvinylpyridine (RuPVP); (B) proposed reaction mechanism
for electrochemiluminescent (ECL) generation using RuPVP and guanines in DNA.
Scheme 2. (A) Structure of(ECL)
ruthenium
polyvinylpyridine
(RuPVP);
(B) proposed
for electrochemiluminescent
generation
using RuPVP
and guanines
in reaction
DNA. mechanism
for electrochemiluminescent (ECL) generation using RuPVP and guanines in DNA.
Scheme 3. Evolution of ECL genotoxicity sensors, starting from a single sensor to a 64-microwell
array. (A) A single-electrode sensor coated with RuPVP/enzyme/DNA films as shown in (D). The
Scheme 3. Evolution of ECL genotoxicity sensors, starting from a single sensor to a 64-microwell
sensor
was placedofabove
optical fiber sensors,
connectedstarting
to a photomultiplier
tube
(PMT)to
fora ECL
detection. array.
Scheme
3. Evolution
ECLan
genotoxicity
from a single
sensor
64-microwell
array. (A) A single-electrode sensor coated with RuPVP/enzyme/DNA films as shown in (D). The
(B)
Array showingsensor
a pyrolytic
graphite
(PG)
chip working electrodefilms
with the
RuPVP/enzyme/DNA
(A) A sensor
single-electrode
coated
with
RuPVP/enzyme/DNA
as
shown
in
(D).
The sensor
was placed above an optical fiber connected to a photomultiplier tube (PMT) for ECL detection.
film spots shown with Ag/AgCl reference and Pt-ring wire counter electrode. After the enzyme
was placed
above
an
optical
fiber
connected
to
a
photomultiplier
tube
(PMT)
for
ECL
detection.
(B) Array showing a pyrolytic graphite (PG) chip working electrode with the RuPVP/enzyme/DNA
reaction driven by NADPH, for ECL readout, the device is placed in a dark box and 1.2 V are applied
(B) Array
a pyrolytic
graphite
(PG) chip
electrode
the RuPVP/enzyme/DNA
film showing
spots shown
with Ag/AgCl
reference
and working
Pt-ring wire
counter with
electrode.
After the enzyme
while a charge-coupled device (CCD) camera measures ECL. (C) Recent ECL microwell PG chip in a
reaction
drivenwith
by NADPH,
for ECL
readout,
the Pt-ring
device is wire
placedcounter
in a darkelectrode.
box and 1.2 After
V are applied
film spots
shown
Ag/AgCl
reference
and
the enzyme
microfluidic reaction chamber with symmetrically placed reference and counter electrodes on the
while
a charge-coupled
device
(CCD)
camerathe
measures
ECL.
(C) Recent
ECL
microwell
PG
chip
inapplied
a
reaction
driven
by
NADPH,
for
ECL
readout,
device
is
placed
in
a
dark
box
and
1.2
V
underside of the top plate. Reactants flow into the chamber, and ECL is then captured by using aare
CCD
microfluidic
reaction
chamber
with
symmetrically
placed
reference
and
counter
electrodes
on
the
while camera
a charge-coupled
in a dark box.device (CCD) camera measures ECL. (C) Recent ECL microwell PG chip in
underside of the top plate. Reactants flow into the chamber, and ECL is then captured by using a CCD
a microfluidic reaction chamber with symmetrically placed reference and counter electrodes on the
camera in a dark box.
We of
also
ECL from
oxidized
DNA in
films
of cationic
polymer
underside
thedemonstrated
top plate. Reactants
flow into
the chamber,
andultrathin
ECL is then
captured
by using
a CCD
[Os(bpy)
2(PVP)10]2+ [PVP = poly(vinyl pyridine)] or OsPVP utilizing 8-oxodG as the co-reactant [31].
camera
in
a
dark
box.
We also demonstrated ECL from oxidized DNA in ultrathin films of cationic polymer
OsPVP combined with RuPVP were assembled into films with DNA or oligonucleotides to detect
[Os(bpy)2(PVP)10]2+ [PVP = poly(vinyl pyridine)] or OsPVP utilizing 8-oxodG as the co-reactant [31].II
both DNA oxidation and nucleobase adducts from chemical damage. Electrochemically oxidized Os
OsPVP
with RuPVP
assembled
intoDNA
films with
DNA or oligonucleotides
to detect
We
alsocombined
demonstrated
ECLwere
from
oxidized
in ultrathin
films of cationic
polymer
sites were generated from films containing 8-oxodG on DNA formed by chemical oxidation using aII
2+ [PVP
both
DNA
oxidation
and
nucleobase
adducts
from
chemical
damage.
Electrochemically
oxidized
Os
[Os(bpy)
(PVP)
]
=
poly(vinyl
pyridine)]
or
OsPVP
utilizing
8-oxodG
as
the
co-reactant
II
2
10
Fenton
reagent
that produced ECL specific for oxidized DNA, and Ru sites gave ECL from [31].
sites
were generated
from films
containing
8-oxodG
DNA formed
by chemical oxidation using a
OsPVP
combined
with
were
assembled
intoonfilms
DNA
chemically damagedRuPVP
DNA, and
possibly
from cleaved
DNA with
strands
[28]. or oligonucleotides to detect
Fenton reagent that produced ECL specific for oxidized DNA, and RuII sites gave ECL from
both DNAMore
oxidation
andwenucleobase
adducts
from chemical
damage.
Electrochemically
oxidized
recently,
have incorporated
microsomal
CPR/cyt
P450 films
on electrodes to mimic
the OsII
chemically damaged DNA, and possibly from cleaved DNA strands [28].
situation
in vivo. from
Cyclicfilms
voltammetry
(CV) 8-oxodG
of this system
closelyformed
mimics the
of the
natural cyt
sites were
generated
containing
on DNA
by details
chemical
oxidation
using a
More recently, we have incorporated microsomal CPR/cyt P450 films on electrodes to mimic the
II
P450
catalytic
pathway.
As
in
Scheme
1,
electrons
flow
from
the
electrode
to
CPR
and
then
to
the
cyt
Fentonsituation
reagentinthat
produced
ECL specific
forofoxidized
DNA,
and
Ru sites
gave of
ECL
chemically
vivo.
Cyclic voltammetry
(CV)
this system
closely
mimics
the details
the from
natural
cyt
P450 as in the in vivo pathway to facilitate efficient catalytic turnover of the cyt P450s [32]. CPR is
damaged
and
possibly
cleaved
DNA strands
[28].
P450DNA,
catalytic
pathway.
Asfrom
in Scheme
1, electrons
flow from
the electrode to CPR and then to the cyt
reduced by the electrode but cyt P450s are not (Scheme 4, Equation (1)). CPRred exists in redox
P450 recently,
as in the in
pathway
to facilitate
efficient catalytic
turnover
of theon
cytelectrodes
P450s [32]. to
CPR
is the
More
wevivo
have
incorporated
microsomal
CPR/cyt
P450 films
mimic
equilibrium with cyt P450FeIII to yield CPRox as a product (Scheme 4, Equation (2)), and
reduced
by
the
electrode
but
cyt
P450s
are
not
(Scheme
4,
Equation
(1)).
CPR
red
exists
in
redox
situation in vivo. Cyclic voltammetry (CV) of this system closely mimics the details of the natural
III to yield CPRox as a product (Scheme 4, Equation (2)), and
equilibrium
cyt P450Fe
cyt P450
catalytic with
pathway.
As in Scheme
1, electrons flow from the electrode to CPR and then to the
cyt P450 as in the in vivo pathway to facilitate efficient catalytic turnover of the cyt P450s [32]. CPR
is reduced by the electrode but cyt P450s are not (Scheme 4, Equation (1)). CPRred exists in redox
Sensors 2017, 17, 1008
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equilibrium with cyt P450FeIII to yield CPRox as a product (Scheme 4, Equation (2)), and electrochemical
oxidation of CPRred (Scheme 4, Equation (3)) competes with this equilibrium. We later found that
Sensors
2017, 17, 1008
4 ofP450s
10
a similar
catalytic
mechanism can be activated in microsomes containing multiple cyt
and
CPRs [26,33,34]. Thus, the microsomal CPR-containing film is a model for the natural cyt P450 catalysis
electrochemical oxidation of CPRred (Scheme 4, Equation (3)) competes with this equilibrium. We later
pathway
[30]that
anda can
be used
in toxicity
sensors
electrochemically
active
cyt P450s.
found
similar
catalytic
mechanism
can beto
activated
in microsomes
containing
multiple cyt
Catalytic
oxidation
of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
the
P450s and
CPRs [26,33,34].
Thus, the microsomal CPR-containing film is a model(NNK)
for the with
natural
cytCPR/cyt
P450 further
catalysis confirmed
pathway [30]the
andelectrode
can be used
in toxicity
sensors
to electrochemically
active
cyt P450s.
P450 films
CPR
cyt P450
pathway
for activation
of cyt
P450s [32,33].
Catalytic
of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
with thelarger
CPR/cytthan that
The formation
rateoxidation
of the product
of this reaction in bulk electrolysis (NNK)
was slightly
P450 films further confirmed the electrode CPR cyt P450 pathway for activation of cyt P450s [32,33].
achieved in the same system using NADPH as electron donor. CV simulations (Scheme 4, Equation (1))
The formation rate of the product of this reaction in bulk electrolysis was slightly larger than that
suggestachieved
an equilibrium
complex between CPR and cyt P450 that facilitates electron transfer [35].
in the same system using NADPH as electron donor. CV simulations (Scheme 4, Equation
The theoretical
(kb ≥ 5kcomplex
= kf /kbCPR
≤ and
0.2, cyt
where
kf and
kb are
forward
and[35].
backward
f and Kbetween
(1)) suggestdata
an equilibrium
P450 that
facilitates
electron
transfer
second-order
chemical
iswhere
the equilibrium
constant)
in favor
of CPRred , which
The theoretical
datarate
(kb ≥constants
5kf and K = kand
f/kb ≤ K
0.2,
kf and kb are forward
andlies
backward
second-order
rate
constantselectrochemically
and K is the equilibrium
constant)
lies in favor
CPRred
, which
is why CPR
is whychemical
CPRox is
reduced
in cyt
P450/CPR
filmsofthat
are
preferential
tooxcyt P450.
◦
◦
is
reduced
electrochemically
in
cyt
P450/CPR
films
that
are
preferential
to
cyt
P450.
The
difference
in
The difference in formal potential (∆E ) between CPR and cyt P450 is ∆E = [RT/nF] ln(K), where
R is
formal potential (ΔE°) between CPR and cyt P450 is ΔE° = [RT/nF] ln(K), where R is the gas constant, T
the gas constant, T is the absolute temperature, and F is Faraday’s constant. Thus, K < 0.2 predicts
is the absolute temperature, and F is Faraday’s constant. Thus, K < 0.2 predicts that cyt P450 formal
that cytpotential
P450 formal
potential
negative of CPRox oxinisthe
films,
that CPR
ox is more
is ~40 mV
negativeisof~40
CPRmV
ox in the films, so that CPR
more
easilyso
reduced.
O2 present
in easily
II and the equilibrium shifts to the right
reduced.
O2 present
in catalytic
oxidations
reacts
P450-Feshifts
catalytic
oxidations
reacts with
P450-FeII and
thewith
equilibrium
to the right to drive substrate
[32].oxidation [32].
to driveoxidation
substrate
Scheme 4. ErCEo simulation model for cyclic voltammetry (CV) of cyt P450s + cyt P450 reductase
Scheme (CPR).
4. Er CEo simulation model for cyclic voltammetry (CV) of cyt P450s + cyt P450 reductase (CPR).
4. Microfluidic
Arrays
Genotoxicity Screening
Screening
4. Microfluidic
ECL ECL
Arrays
forfor
Genotoxicity
ECL-based
genotoxicity
sensor
arrays
were
first
developedin
inour
our laboratory
laboratory in
ECL-based
genotoxicity
sensor
arrays
were
first
developed
in 2007.
2007.These
These arrays
arrays were fabricated on a single conductive chip instead of multielectrode devices. In these ECL
were fabricated on a2 single 2conductive chip instead of multielectrode devices. In these ECL arrays,
arrays,
a 1-inch
(6.45 cm ) pyrolytic graphite (PG) block was electrically connected to a copper block
2 (6.45
a 1-inch
cm2 ) pyrolytic graphite (PG) block was electrically connected to a copper block via
via silver epoxy and insulated using ethyl acrylate so only the upper conductive PG face was exposed.
silver epoxy
and insulated
using
acrylate
so onlylocations.
the upper
conductive
PG face
was exposed.
RuPVP/enzyme/DNA
films
wereethyl
spotted
on demarcated
Metabolite
generation
was driven
RuPVP/enzyme/DNA
films
were
spottedofon
demarcated
Metabolite
generation
was driven
by manual deposition
and
incubation
reactants
on thelocations.
arrays (Scheme
3B), using
an NADPH
regeneration
systemand
to drive
cyt P450 catalysis.
by manual
deposition
incubation
of reactants on the arrays (Scheme 3B), using an NADPH
These
ECL to
arrays
to our current high-throughput arrays integrated into a
regeneration
system
driveeventually
cyt P450 led
catalysis.
microfluidic reactor. A 64-microwell pattern was computer laser-jet printed and transferred onto a
These ECL arrays eventually led to our current high-throughput arrays integrated into a
cleaned and polished PG chip (2.5 × 2.5 × 0.3 cm) by heat pressing the mask onto the PG chip at 290 °F
microfluidic
A 64-microwell
pattern
was the
computer
laser-jet
printed and transferred
onto
(143 °C)reactor.
for 90 s. The
hydrophobic toner
separating
spots prevents
cross-contamination
of DNA,
a cleaned
and polished
PG solutions
chip (2.5 deposited
× 2.5 × 0.3
heat
mask onto
the PG chip at
enzyme
and polymer
in cm)
each by
well
forpressing
LBL filmthe
formation.
Individual
spots
were constructed
in separating
each microwell
sequential
depositions
of 1 μL
290 ◦ F RuPVP/enzyme/DNA
(143 ◦ C) for 90 s. The
hydrophobic
toner
thebyspots
prevents
cross-contamination
drops
of
the
appropriate
solution.
Then,
the
PG
chip
was
fitted
into
a
microfluidic
reaction
of DNA, enzyme and polymer solutions deposited in each well for LBL film formation. cell
Individual
consisting of top and bottom poly(methylmethacrylate) (PMMA) plates with an optical glass window
RuPVP/enzyme/DNA
spots were constructed in each microwell by sequential depositions of 1 µL
to facilitate ECL light acquisition. Pt counter and Ag/AgCl reference electrode wires were fixed on
drops of
the appropriate solution. Then, the PG chip was fitted into a microfluidic reaction cell
the bottom side of the top PMMA plate. A silicone rubber gasket sandwiched between the PMMA
consisting
ofwas
topused
andtobottom
poly(methylmethacrylate)
(PMMA)
plates
with an the
optical
glass
plates
form a sealed
channel, and a copper plate
was placed
underneath
PG chip
for window
to facilitate
ECL
light acquisition.
Pt counter
Ag/AgCltoreference
electrode
were
electrical
connection.
The microfluidic
device and
was connected
a syringe pump
for a wires
constant
flowfixed on
of reactants
or the
buffers
a fixed concentration
and flow
rate to
better control
reactionbetween
conditionsthe
in PMMA
the bottom
side of
topatPMMA
plate. A silicone
rubber
gasket
sandwiched
the
semi-automated
system
(Scheme
3C).
Metabolites
were
generated
in
situ
by
flowing
test
plates was used to form a sealed channel, and a copper plate was placed underneath the PG chip for
compounds through the chip, and ECL was then generated by applying a potential of 1.25 V vs.
electrical connection. The microfluidic device was connected to a syringe pump for a constant flow of
Ag/AgCl for 180 s and captured by a CCD camera in a dark box.
reactants or buffers at a fixed concentration and flow rate to better control reaction conditions in the
semi-automated system (Scheme 3C). Metabolites were generated in situ by flowing test compounds
through the chip, and ECL was then generated by applying a potential of 1.25 V vs. Ag/AgCl for 180 s
and captured by a CCD camera in a dark box.
Sensors 2017, 17, 1008
5 of 10
Sensors 2017, 17, 1008
5 of 10
Our first task for this microfluidic reactor focused on reactive metabolites of benzo[a]pyrene
(B[a]P). Cyt
includedreactor
rat liver
microsomes
(RLM)
and human
liver microsomes
OurP450
firstenzyme
task for sources
this microfluidic
focused
on reactive
metabolites
of benzo[a]pyrene
(HLM),
supersomes
(genetically
engineered
contain
only one
cytand
P450
+ CPR)
cyt 1B1, 1A1,
(B[a]P).
Cyt P450 enzyme
sources
included rattoliver
microsomes
(RLM)
human
liver of
microsomes
(HLM),
(genetically
engineered
to contain
only
one cyt P450
+ CPR)
of cyt
1B1, 1A1, and
and 1A2
andsupersomes
human liver
S9 fraction.
B[a]P was
initially
oxidized
by cyt
P450s
to B[a]P-7,8-epoxide
1A2 5,
and
human (1)),
liverwhich
S9 fraction.
B[a]Phydrolyzed
was initially
by cyt P450s toinB[a]P-7,8-epoxide
(Scheme
Equation
was then
tooxidized
B[a]P-7,8-dihydrodiol
presence of epoxide
(Scheme
5,
Equation
(1)),
which
was
then
hydrolyzed
to
B[a]P-7,8-dihydrodiol
in
presence
epoxide
hydrolase (EH, Equation (2)). Further oxidation of dihydrodiol by cyt P450 resulted inofthe
formation
hydrolase (EH, Equation (2)). Further oxidation of dihydrodiol by cyt P450 resulted in the formation
of reactive metabolite B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE, Equation (3)), which reacts with
of reactive metabolite B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE, Equation (3)), which reacts with
guanine and adenine bases in DNA to form adducts.
guanine and adenine bases in DNA to form adducts.
cyt P450 / O2
Sensors 2017, 17, 1008
(1)
O
B[a]P
5 of 10
Our first task for this microfluidic reactor
focused on reactive metabolites of benzo[a]pyrene
EH
(2)
(B[a]P). Cyt P450 enzyme sources included rat liver microsomes (RLM) and human liver microsomes
HO
(HLM), supersomes (genetically
engineered to contain only
one cyt P450 + CPR) of cyt 1B1, 1A1, and
O
OH
1A2 and human liver S9 fraction. B[a]P was initiallyO oxidized by cyt P450s to B[a]P-7,8-epoxide
cyt P450 / O2
(3)
(Scheme 5, Equation (1)), which was then hydrolyzed
to B[a]P-7,8-dihydrodiol
in presence of epoxide
HO
hydrolase (EH, Equation (2)).
Further oxidation of dihydrodiol
by
cyt
P450
resulted
in the formation
HO
OH
OH
BPDE
of reactive metabolite B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE, Equation (3)), which reacts with
guanine and
adenine
in DNA
to formofadducts.
Scheme
5. bases
Metabolic
activation
B[a]P by cyt P450 and epoxide hydrolase (EH).
Scheme 5. Metabolic activation of B[a]P by cyt P450 and epoxide hydrolase (EH).
cyt P450 / O2
ECL is normalized for the amount of enzyme in each film (estimated(1)by quartz crystal microbalance)
O
to find the relative rate of DNA damage
by B[a]P metabolites.
The relative rate of DNA damage was
B[a]P
higher when EH was included with cyt P450 in microwells when using HLM, RLM, or supersomes,
suggesting a complete bioactivation of B[a]P toEHproduce DNA-BPDE adducts.
Cyt P450 1B1 was found
(2)
to have the highest rate of DNA damage followed by
HO cyt P450s 1A1 and 1A2 (Figure 1). The formation
O
of DNA adducts in the presence
of B[a]P was confirmedOHby liquid chromatography–mass spectrometry
O
(LC-MS/MS) by determining dG-BPDE andcyt
dA-BPDE
films of DNA/enzyme on
P450 / O2 adducts using similar
(3)
magnetic bead reactors. The HO
ECL arrays can also measure enzyme inhibition, in this case by measuring
HO
OH
a decrease in the %ECL corresponding
to a decrease inOHDNA
damage, in the presence of furafylline
BPDE
Figure
1.
(A)
Recolorized
and
reconstructed
ECL
array
results
for
enzyme
reactions with[36].
oxygenated
and rhapontigenin,
which
are known
inhibitors
of cyt
1A2
andand
1A1,
respectively
Scheme
5. Metabolic
activation
of B[a]P
by cyt
P450
epoxide hydrolase (EH).
25 μM B[a]P + 1% DMSO in pH = 7.4 phosphate buffer with electronic activation of cyt P450s.
Microwells containing RuPVP/enzyme/DNA film assemblies exposed to B[a]P for various reaction
times. Enzyme sources are cyt P450 supersomes, human, and rat liver microsomes (HLM and RLM),
epoxide hydrolase (EH), and human S9 fractions (HS9). (B) Influence of EH on the sensor array,
relative DNA damage rate for 25 μM B[a]P at pH 7.4: (a) supersomes; (b) microsomes. Reproduced
from [36] with permission, copyright Royal Society of Chemistry, 2013.
ECL is normalized for the amount of enzyme in each film (estimated by quartz crystal
microbalance) to find the relative rate of DNA damage by B[a]P metabolites. The relative rate of DNA
damage was higher when EH was included with cyt P450 in microwells when using HLM, RLM, or
supersomes, suggesting a complete bioactivation of B[a]P to produce DNA-BPDE adducts. Cyt P450
1B1 was found to have the highest rate of DNA damage followed by cyt P450s 1A1 and 1A2 (Figure
1). The formation of DNA adducts in the presence of B[a]P was confirmed by liquid chromatography–
1. (A) Recolorized
and reconstructed
ECL array results
fordA-BPDE
enzyme reactions with
oxygenated films
massFigure
spectrometry
(LC-MS/MS)
by determining
and
using
Figure
1. (A) Recolorized
and reconstructed
ECLdG-BPDE
array results
for enzymeadducts
reactions
withsimilar
oxygenated
25 μM B[a]P on
+ 1%
DMSO in
pH reactors.
= 7.4 phosphate
buffer
with
electronic
activation
of cyt
P450s.
of
DNA/enzyme
magnetic
bead
The
ECL
arrays
can
also
measure
enzyme
inhibition,
in
25 µM
B[a]P
+
1%
DMSO
in
pH
=
7.4
phosphate
buffer
with
electronic
activation
of
cyt P450s.
Microwells containing RuPVP/enzyme/DNA film assemblies exposed to B[a]P for various reaction
this case by measuring a decrease in the % ECL corresponding to a decrease in DNA damage, in the
Microwells
containing
RuPVP/enzyme/DNA
film
assemblies
to B[a]P(HLM
for various
reaction
times. Enzyme
sources
are cyt P450 supersomes,
human,
and rat exposed
liver microsomes
and RLM),
presence of furafylline and rhapontigenin, which are known inhibitors of cyt 1A2 and 1A1,
times.
Enzyme
sources(EH),
are cyt
supersomes,
andInfluence
rat liver of
microsomes
(HLM array,
and RLM),
epoxide
hydrolase
andP450
human
S9 fractionshuman,
(HS9). (B)
EH on the sensor
respectively [36].
epoxide
hydrolase
(EH), and
S9 fractions
(HS9).
(B)supersomes;
Influence of(b)
EH
on the sensor
array, relative
relative
DNA damage
ratehuman
for 25 μM
B[a]P at pH
7.4: (a)
microsomes.
Reproduced
[36] with
Royal
of Chemistry,
DNAfrom
damage
ratepermission,
for 25 µM copyright
B[a]P at pH
7.4:Society
(a) supersomes;
(b)2013.
microsomes. Reproduced from [36]
with permission, copyright Royal Society of Chemistry, 2013.
ECL is normalized for the amount of enzyme in each film (estimated by quartz crystal
microbalance) to find the relative rate of DNA damage by B[a]P metabolites. The relative rate of DNA
damage was higher when EH was included with cyt P450 in microwells when using HLM, RLM, or
supersomes, suggesting a complete bioactivation of B[a]P to produce DNA-BPDE adducts. Cyt P450
1B1 was found to have the highest rate of DNA damage followed by cyt P450s 1A1 and 1A2 (Figure
1). The formation of DNA adducts in the presence of B[a]P was confirmed by liquid chromatography–
Sensors 2017, 17, 1008
5. ECL Arrays
Organ-Specific Metabolic Toxicity
Sensors for
2017, Assessing
17, 1008
6 of 10
6 of 10
Traditional
toxicity
focuses on the
liver, however,
5. ECL Arrays
forassessment
Assessing Organ-Specific
Metabolic
Toxicity extra-hepatic tissues are also capable
of metabolizing
xenobiotics
into
reactive
metabolites.
The
64-microwell
ECL array
above
Traditional toxicity assessment focuses on the liver, however,
extra-hepatic tissues
are alsodescribed
capable
was further
developedxenobiotics
to evaluate
genotoxic
withdescribed
different
organ
of metabolizing
intopossible
reactive metabolites.
Thechemistry
64-microwelleffect
ECL array
above
was enzymes.
furtherhuman
developed
to evaluate
possible genotoxic
chemistry
with different
Enzymes from
liver,
lung, intestine,
and kidney
wereeffect
compared
in theorgan
arrayenzymes.
(Scheme 6).
Enzymes from human liver, lung, intestine, and kidney were compared in the array (Scheme 6).
Sensors 2017, 17, 1008
6 of 10
5. ECL Arrays for Assessing Organ-Specific Metabolic Toxicity
Traditional toxicity assessment focuses on the liver, however, extra-hepatic tissues are also capable
of metabolizing xenobiotics into reactive metabolites. The 64-microwell ECL array described above was
further developed to evaluate possible genotoxic chemistry effect with different organ enzymes.
Enzymes from human liver, lung, intestine, and kidney were compared in the array (Scheme 6).
Scheme 6. Experimental design of 64-microwell ECL chip with films of RuPVP/enzymes/DNA for
Scheme 6.metabolic
Experimental
design
64-microwell
ECL chip
films
of RuPVP/enzymes/DNA
genotoxicity
assays of
of test
compounds. Symbols:
H = with
human;
L = liver;
C = cytosol; Lu =
lung;genotoxicity
I = intestine; K =assays
kidney; of
numbers
and letters suchSymbols:
as 3A4 indicate
for metabolic
test compounds.
H =supersomes
human; Lcontaining
= liver;the
C = cytosol;
single cytKP450
manifold.
Reproduced
from
[37] with
permission,
copyright supersomes
Royal Society ofcontaining
Lu = lung;denoted
I = intestine;
= kidney;
numbers
and
letters
such
as 3A4 indicate
Chemistry, 2015.
the denoted single cyt P450 manifold. Reproduced from [37] with permission, copyright Royal Society
of Chemistry, 2015.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a known carcinogen, was used as a test
compound to study the organ-specific metabolic-DNA reactions for enzymes from the different organs
Scheme 6. Experimental design of 64-microwell ECL chip with films of RuPVP/enzymes/DNA for
(Figure 2). A decrease
in relative
DNA
damage
rates
in the
cytosolic
enzymes
and their
metabolic genotoxicity
assays
of test
compounds.
Symbols:
H =presence
human; L = of
liver;
C = cytosol;
Lu =
lung;
I = intestine;which
K = kidney;
numbers and letters
3A4 indicate supersomes
the via various
co-factors were
observed,
is consistent
withsuch
theasdetoxification
effectcontaining
of cytosols
single cyt
P450 manifold. Reproduced from [37] with permission, copyright Royal Society of
bioconjugationdenoted
pathways
[37].
Chemistry, 2015.
Figure 2. ECL results from genotoxicity study of oxygenated 150 μM NNK in pH 7.4 phosphate buffer
and necessary cofactors exposed to microwells containing RuPVP/enzyme/DNA with electrochemical
activation of cyt P450s at −0.65 V vs. Ag/AgCl (0.14 M KCl) for different reaction times. (a)
Reconstructed, recolorized ECL array images. Control spots contain microsomes exposed to the same
reaction conditions as above without exposure to NNK. Graphs show influence of enzyme reaction
time on % ECL increase for reaction with 150 μM NNK at pH = 7.4, (b) with human organ tissue
enzymes, and (c) with individual cyt P450 supersomes. Reproduced from [37] with permission,
copyright Royal Society of Chemistry, 2015.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a known carcinogen, was used as a test
compound to study the organ-specific metabolic-DNA reactions for enzymes from the different
Figure 2. ECL results from genotoxicity study of oxygenated 150 μM NNK in pH 7.4 phosphate buffer
(Figure
2). Afrom
decrease
in relative DNA
damage
rates in the presence
cytosolic
enzymes
and
Figureorgans
2. ECL
results
genotoxicity
study
of oxygenated
150 µM of
NNK
in pH
7.4 phosphate
and necessary cofactors exposed to microwells containing RuPVP/enzyme/DNA with electrochemical
buffer andactivation
necessary
exposed
microwells
RuPVP/enzyme/DNA
with
of cytcofactors
P450s at −0.65
V vs. to
Ag/AgCl
(0.14 M containing
KCl) for different
reaction times. (a)
Reconstructed,
recolorized
arrayat
images.
Control
contain microsomes
exposed
the same reaction
electrochemical
activation
of cytECL
P450s
−0.65
V vs.spots
Ag/AgCl
(0.14 M KCl)
fortodifferent
reaction conditionsrecolorized
as above without
to NNK. Control
Graphs show
influence
of enzyme
reactionexposed to
times. (a) Reconstructed,
ECLexposure
array images.
spots
contain
microsomes
time on % ECL increase for reaction with 150 μM NNK at pH = 7.4, (b) with human organ tissue
the same reaction conditions as above without exposure to NNK. Graphs show influence of enzyme
enzymes, and (c) with individual cyt P450 supersomes. Reproduced from [37] with permission,
reaction time
on %ECL
increase
for reaction
copyright
Royal Society
of Chemistry,
2015. with 150 µM NNK at pH = 7.4, (b) with human organ
tissue enzymes, and (c) with individual cyt P450 supersomes. Reproduced from [37] with permission,
(NNK), a known carcinogen, was used as a test
copyright 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
Royal Society of Chemistry, 2015.
compound to study the organ-specific metabolic-DNA reactions for enzymes from the different
organs (Figure 2). A decrease in relative DNA damage rates in the presence of cytosolic enzymes and
Sensors 2017, 17, 1008
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Sensors 2017, 17, 1008
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their co-factors were observed, which is consistent with the detoxification effect of cytosols via
various bioconjugation pathways [37].
biocolloid reactors
reactors provided a more
The two-tier approach based on ECL array
array and
and LC-MS/MS
LC-MS/MS biocolloid
detailed profile of possible
genotoxicity
pathways
associated
with
specific
possible genotoxicity pathways associated with specific organs.
organs. Correlations
between DNA damage rates obtained from the cell-free ECL arrays and cell-based Comet assays that
measure DNA damage in cells were found [37].
[37].
6. ECL
ECL Sensor
Sensor Arrays
Arrays for
for DNA
DNA Oxidation
Oxidation
6.
Oxidative stress
stress or
oxidative DNA
DNA damage
damage is
is an
an imbalance
imbalance between
between the
the production
production of
ROS and
and
Oxidative
or oxidative
of ROS
the
ability
of
the
body
to
counteract
their
harmful
effects
[38].
ROS
oxidizes
guanosines
(dG)
in
the ability of the body to counteract their harmful effects [38]. ROS oxidizes guanosines (dG) in the
the
DNA to
to form
form 8-oxodG,
8-oxodG, aa biomarker
biomarker for
for oxidative
oxidative DNA
DNA damage
damage [39].
8-OxodG serves
serves as
as aa co-reactant
co-reactant
DNA
[39]. 8-OxodG
for Osmium
(phen-benz-COOH)]2+2+{bpy
{bpy= =2,2′-bipyridine;
2,20 -bipyridine;phen-benz-COOH
phen-benz-COOH
for
Osmium complex
complex [Os(bpy)
[Os(bpy)22(phen-benz-COOH)]
=
=
(4(1,1phenanthrolin-6-yl)benzoic
acid)}.
ECL
is
generated
due
to
selective
catalytic
oxidation
(4(1,1phenanthrolin-6-yl)benzoic acid)}. ECL is generated due to selective catalytic oxidation
of of
8III sites in the polymer, leading to the formation of a photoexcited OsII * species that
8-oxodG
III sites
oxodG
byby
OsOs
in the polymer, leading to the formation of a photoexcited OsII* species that emits
emits
ECL
[31].
The
64-microwell
array described
the above
wasto
used
to develop
anarray
ECL
ECL [31]. The 64-microwell
array described
in the in
above
sectionsection
was used
develop
an ECL
array
for
oxidative
DNA
damage.
for oxidative DNA damage.
2+
8-oxodG was
in intact
ds-DNA
using using
composite
films of [Os(bpy)
8-oxodG
wasdetected
detected
in intact
ds-DNA
composite
films of 2 (phen-benz-COOH)]
[Os(bpy)2(phen-benz(Scheme
Nafion 7),
polymer,
reduced
graphene
(OsNG)
[40].
A homogeneous
solution of
COOH)]2+7),
(Scheme
Nafionand
polymer,
and
reducedoxide
graphene
oxide
(OsNG)
[40]. A homogeneous
2+
[Os(bpy)of
-Nafion was
drop-casted
into the microwells
of the PG chip
array
2+-Nafion
2 (phen-benz-COOH)]
solution
[Os(bpy)2(phen-benz-COOH)]
was drop-casted
into the microwells
of the
PG
containing
a
layer
of
reduced
graphene
oxide,
followed
by
the
deposition
of
oxidized
DNA
solution
chip array containing a layer of reduced graphene oxide, followed by the deposition of oxidized DNA
on the OsNG-modified
films. Thefilms.
PG chip
placed
microfluidic
device and device
washed,
and
ECL is
solution
on the OsNG-modified
TheisPG
chipin
is the
placed
in the microfluidic
and
washed,
then ECL
captured
using
a CCDusing
camera
whilecamera
applying
0.9 Vapplying
vs. Ag/AgCl.
Fenton's
reagent
was used
as a
and
is then
captured
a CCD
while
0.9 V vs.
Ag/AgCl.
Fenton's
reagent
model
compound
for
DNA
and
polydeoxyguanosine
(polyG)
oxidation,
and
an
increase
in
the
ECL
was used as a model compound for DNA and polydeoxyguanosine (polyG) oxidation, and an
with
the in
increase
in with
the reaction
time was
observed
3). observed (Figure 3).
increase
the ECL
the increase
in the
reaction(Figure
time was
2+.
Scheme 7.
7. Structure
Structure of
of [Os(bpy)
[Os(bpy)2(phen-benz-COOH)]
(phen-benz-COOH)]2+
Scheme
.
2
For array standardization, the amount of 8-oxodG in DNA oxidized by Fenton’s reagent was
For array standardization, the amount of 8-oxodG in DNA oxidized by Fenton’s reagent
measured using UHPLC-MS/MS. The ratio of 8-oxodG to the total amount of dG increased with the
was measured using UHPLC-MS/MS. The ratio of 8-oxodG to the total amount of dG increased
oxidation time in the UHPLC-MS/MS experiments, which was used to obtain relative amounts of 8with the oxidation time in the UHPLC-MS/MS experiments, which was used to obtain relative
oxodG. A calibration curve of % ECL increase vs. relative 8-oxodG concentration from UHPLCamounts of 8-oxodG. A calibration curve of %ECL increase vs. relative 8-oxodG concentration from
MS/MS was used to quantitate 8-oxodG in the array (Figure 3d,e). The array was also used to measure
UHPLC-MS/MS was used to quantitate 8-oxodG in the array (Figure 3d,e). The array was also used to
metabolite-mediated DNA oxidation. This ECL array enabled 64 experiments in one run and only 1
measure metabolite-mediated DNA oxidation. This ECL array enabled 64 experiments in one run and
μg of the DNA is required to perform an experiment. The detection limit was 1500 8-oxodG per 106
only 1 µg of the DNA is required to perform an experiment. The detection limit was 1500 8-oxodG per
nucleobases [40].
106 nucleobases [40].
Sensors 2017, 17, 1008
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Sensors 2017, 17, 1008
8 of 10
Figure 3.3.ECL
ECL
array
for polyG
andoxidation.
DNA oxidation.
(a) Reconstructed
ECL
array data
Figure
array
datadata
for polyG
and DNA
(a) Reconstructed
ECL array data
demonstrating
demonstrating
ECL
from
spots
of
OsNG/polyG
or
DNA
film
treated
with
Fenton’s
reagent
in pH 7.2
ECL from spots of OsNG/polyG or DNA film treated with Fenton’s reagent in pH 7.2 phosphate
phosphate
buffer
saline
for
various
time
intervals.
Controls
are
polynucleotides
treated
only
with
buffer saline for various time intervals. Controls are polynucleotides treated only with buffer, with
4 or with only H2O2. Graphs show relative %ECL increase for (b) polyG and (c)
buffer,
with or
only
FeSO
only
FeSO
with
only
H2 O2 . Graphs show relative %ECL increase for (b) polyG and (c) DNA.
4
DNA.
(d)
UHPLC-MS/MS
ECLcalibration
array calibration
measured
ratio of [8-oxodG]/([dG]+[8(d) UHPLC-MS/MS for ECLfor
array
showingshowing
measured
ratio of [8-oxodG]/([dG]+[8-oxodG])
oxodG])
for
DNA
reacted
with
Fenton’s
reagent
for
different
time
and (e)
then
hydrolyzed.
(e)
for DNA reacted with Fenton’s reagent for different time and then hydrolyzed.
Calibration
plot for
Calibration
plot
for
%
ECL
increase
vs.
relative
amount
of
8-oxodG.
Reprinted
with
permission
from
%ECL increase vs. relative amount of 8-oxodG. Reprinted with permission from [40], copyright (2016),
[40], copyright
(2016),
American Chemical Society.
American
Chemical
Society.
7. Summary
7.
Summary and
and Conclusions
Conclusions
It isisclear
clearthat
that
multiple
in vitro
toxicity
screening
aretoneeded
to reliable
approach
reliable
It
multiple
in vitro
toxicity
screening
tools aretools
needed
approach
predictions
predictions
of
in
vivo
toxicity
risks.
Our
two-tiered
approach
of
ECL
microfluidic
array
and
LCof in vivo toxicity risks. Our two-tiered approach of ECL microfluidic array and LC-MS/MS biocolloid
MS/MS can
biocolloid
provide on
detailed
information
ongenotoxicity
possible pathways
of genotoxicity
reactors
providereactors
detailedcan
information
possible
pathways of
that are not
forthcoming
that
are
not
forthcoming
from
bioassays,
but
are
important
components
of
a
toxicity
assessment.
from bioassays, but are important components of a toxicity assessment. Microfluidic ECL arrays
using
Microfluidic
ECL
arrays
using
RuPVP/DNA/enzyme
films
detect
relative
rates
of
DNA
adduct
RuPVP/DNA/enzyme films detect relative rates of DNA adduct damage from reactive metabolites.
2+ detects DNA oxidation. LCdamage from
reactive metabolites.
[Os(bpy)
2+ detects
[Os(bpy)
DNA2(phen-benz-COOH)]
oxidation. LC-MS/MS
elucidates structures and
2 (phen-benz-COOH)]
MS/MS elucidates
structures
measures the
formationadducts
rates ofand
individual
nucleobase
and
measures
the formation
ratesand
of individual
nucleobase
can also
detect theadducts
amounts
of
can
also
detect
the
amounts
of
8-oxodG
formed.
Molecular
pathway
information
obtained
by
these
8-oxodG formed. Molecular pathway information obtained by these approaches can be integrated with
approaches
can be
integrated
withfuture
other predictions
bioassays toofprovide
better of
future
predictions
of genotoxicity
other
bioassays
to provide
better
genotoxicity
chemical
and drug
candidates.
of chemical and drug candidates.
Acknowledgments: The work described herein and the preparation of this article were supported financially
by
the National Institute
of Environmental
Health
(NIEHS),
NIH,
USA.were
Grant
ES03154.financially
The authors
Acknowledgments:
The work
described herein
and Sciences
the preparation
of this
article
supported
by
thank
the coworkers
collaborators named
joint publications
for their
many
excellent
contributions
this
the National
Instituteand
of Environmental
HealthinSciences
(NIEHS), NIH,
USA.
Grant
ES03154.
The authorsto
thank
research, without which progress would not have been possible.
the coworkers and collaborators named in joint publications for their many excellent contributions to this
Conflicts
of Interest:
Theprogress
authors would
declarenot
no conflict
of interest.
research, without
which
have been
possible.
Conflicts of Interest: The authors declare no conflict of interest.
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