Design of Biosensors and Arrays: Toxicity Screening
James F. Rusling
Departments of Chemistry & Pharmacology
University of Connecticut
Storrs, CT, USA
electrode
substrate product
Enzyme, or Label on Ab/AgOr DNA
Apply voltage Measure current prop.to concentration of substrate
Traditional Electrochemical Biosensors
• nanoscale biosensing architecture• patternable nanomaterials for arrays
Adsorbed of chem.bonded
Negativesurface
Polycation soln.,then wash
+ + + + + + + + +
soln. of negative proteinthen wash
+ + + + + + + + +
+ + + + + + + + +
Repeat steps for desired number of layers
Proteinlayer
Polycation layersProteinlayer
Polycation soln.,then wash
Layer-by-layerFilm assembly
Lvov, Decher
Lvov, Y. in Nalwa, R.W.; Ed.; Handbook Of Surfaces And Interfaces Of Materials, Vol. 3. Academic, 2001, pp. 170-189.
Stable, easily prepared, versatile
scale
1
2
3
4
5
6
7
Optical arrays - absorbance or fluorescence
DNA, proteins, pathogenic bacteriaDNA arrays with millions of spots
array
label
Primary antibody
Multiple-antibody-Nanotube (CNT)
Antigen = Protein, pathogen or
Secondary antibody
and labels; or multi-label
Bioconjugate on CNT
Fluorescence-based sandwich immunoassay
Antibodies capture
The antigen
spot
label
antibody
pathogen
Multiple-antibody-nanotube
nanoparticles
Light in
Fluorescence out
Fluorescence from all spots measured simultaneously
array
COOH
COOH
HOOC
HOOC
HOOCHOOC
HOOC
HOOC
HOOCCOOH
COOH
COOH
COOH
COOH
HNO3, 3 H2SO4
Ab2 label
sonicate 6 hr
Carbon nanotube (CNT)
Coupling agents
Functionalizing nanotubes with labels and antibodies
• Single walled (1.4 nm o.d.)and multi-walled
• Highly conductive,flexible, strong,patternable
• Commercially Available
Researchgoal
toxic?
transducer
Biomolecular reporter
test chemical
• ~ 30 % of drug candidates defeated by toxicity• Early screening could save drug development costs
Lipophilic Molecule
styreneCyt P450, O2
Enzyme-activated molecule
O
styrene oxide+DNA
DamagedDNA
Detect by electrochemical sensorValidate by LC-MS/MS
In vitro Toxicity Screening
Funding from NIH, NIEHS
Collaboration with Prof. John Schenkman,Pharmacology, Uconn Health Center
ds-DNA
PDDA orRu-PVP (catalyst)(Ru(bpy)2
2+-PVP)Pyrolytic Graphite
Enzyme
Films for Toxicity Screening
20-40 nm
Negativesurface
Polycation soln.,then wash
+ + + + + + + + +
soln. of negative proteinthen wash
+ + + + + + + + +
+ + + + + + + + +
Repeat steps for desired number of layers
Proteinlayer
Polycation layersProteinlayer
Polycation soln.,then wash
Layer-by-layerFilm assembly
Lvov, Decher
Lvov, Y. in Nalwa, R.W.; Ed.; Handbook Of Surfaces And Interfaces Of Materials, Vol. 3. Academic, 2001, pp. 170-189.
Stable, easily prepared, versatile
Mass: M/A = -F/1.86 x 108
Thickness: d = -(0.016) F
Quality control for enzyme-DNA films
0
1000
2000
3000
4000
5000
6000
0 2 4 6 8 10
- , F Hz
. Layer No and Identity
MPA PDDA DNA Mb DNA Mb DNA Mb DNA / 450P / 450 / 450P P
- / 450ST dsDNA Pcam
- /CT dsDNA Mb
- QCM film growth
E, V
time
E-t waveform
potentiostat
Electrochemical cell
counter
working electrode
N2
inlet
DNA/enzyme film
reference
insulator electrodematerial
Equipment for toxicity biosensors
Square-wavevoltammetry
I measured,then subtracted
Enzyme reaction - Incubate:Reactant + H2O2-->metabolite
Analysis by catalytic SWV or electrochemiluminescence
RuL2+ = RuL3+ + e-RuL3+ + DNA-G --> RuL2+ + DNA-G•
Screening Chemical Toxicity
-200
-150
-100
-50
00.4 0.6 0.8 1 1.2
I, μA
, E V vs SCE
- Controls no styrene
0 min
5 min
+ 2% styrene
15 min
Bare PG
30 min
15 min
30 min
( / )Mb DNA2 films
1. 37 incubateoC
2. , 50 SWV μ ( )M Ru bpy3
2+
0.2 mM H2O2Enzyme/DNA films
Peak increasemeasures damageof DNA by enzyme-generatedmetabolite
Cyt P450
H2O2R (e.g. styrene)
RO (e.g. styreneoxide)
-5
0
5
10
15
0 3 6 9
Ip, μA
, t min
450cyt Pcam
I/Δt = rel. rateDNA Damage
styrene
controls
Sensor response vs. reaction time
+ H2O2
DNA-adduct formation increases signals
Martz, E. Trends in Biochemical Sciences. 2002, 27, 107, www.proteinexplorer.org.
• Adduct affects base pair binding
• Exposes guanines by disrupting surrounding base pairs with “unwinding” of helix
•Guanines more easily oxidized - increased signal
1
1.2
1.4
1.6
1.8
0
20
40
60
0 10 20 30
Incubation in MMS, min
LC-MS/MS
Sensor
Comparison of toxicity sensors with LC-MSFor DNA damage by methylmethane sulfonate
Pyrolytic Graphite
DNA
DNARu-PVP
Echem detectionE=1.15 V
electrochemiluminescence
Lynn Dennany, Robert J. Forster and James F. Rusling,"Simultaneous Direct Electrochemiluminescence and Catalytic Voltammetry Detection of DNA in Ultrathin Films"J. Am. Chem. Soc. 2003, 125, 5213-5218.Collaboration with NCSR, Dublin City Univ.
[Ru(bpy)2-(PVP)10]2+
Equipment for ECL toxicity sensorsECL = electrochemiluminence
E, V
time
E-t waveform
potentiostat
ECL cell
counter
working electrode
N2
inlet
DNA/enzyme film
reference
insulator electrodematerial
Square-wavevoltammetry
I measured,then subtracted
Monochromator/PM tube detector
optical fiber
glass
Pyrolytic Graphite
DNA
DNARu-PVP
Echem detectionE=1.15 V
electrochemiluminescence
Lynn Dennany, Robert J. Forster and James F. Rusling,"Simultaneous Direct Electrochemiluminescence and Catalytic Voltammetry Detection of DNA in Ultrathin Films"J. Am. Chem. Soc. 2003, 125, 5213-5218.Collaboration with NCSR, Dublin City Univ.
[Ru(bpy)2-(PVP)10]2+
N N NRuNN NN 8
Catalytic ECL polymer
N N NRuNN NN 8
-30
-20
-10
0
10
20
30
-3
-2
-1
0
1
2
3
0.60.811.2
I, μA
, . E V vs SCE
0
0
5
5 10
10
15
15
20
20
25
25 min
ECL
SWV
DNA +
Incubations with styrene oxide
0.5
1
1.5
2
2.5
3
0 20 40 60 80 100
DNA + Styrene OxideDNA + TolueneDNA + Buffer
ECL Final ECLInitial
t, min
aECL output
0
2
4
6
8
0 20 40 60 80 100
DNA + Styrene OxideDNA + TolueneDNA + Buffer
Ip, final
Ip. inital
t, min
bSWV output
Incubation of Ru-PVP/DNA films with styrene oxide
Direct ECL generation from DNA
RVP-RuL2+ = PVP-RuL3+ + e-PVP-RuL3+ + DNA-G --> PVP-RuL2+ + DNA-G•
Then? PVP-RuL3+ oxidizes DNA-G• to give
Photoexcited PVP-[RuL2+]*Or
DNA-G• reduces PVP-RuL2+ to PVP-RuL+,PVP-RuL3+ + PVP-RuL+ --> PVP-[RuL2+]*
Direct ECL generation from DNA
RuPVP2+RuPVP3+
PG Electrode
Guanine
Guanine
Guanine2+
RuPVP2+*• ECL can be generated by the formation of RuPVP2+* through the oxidation of Guanine on sensor
Dennany, L.; Forster, R. J.; Rusling, J. F. J. Am. Chem. Soc., 2003, 125, 5213-5218.
h
RuPVP3+
Benzo[a]Pyrene-DNA Adduct Formation• BP Metabolism much more convoluted
• Stable adducts formed from anti-BPDE 1,2
• Depurinating adducts formed from one-electron oxidized BP 2
• GT Transversions of DNA casued by depurinated bases 2
1 Osborne, M. et al. Benzopyrenes. Cambridge Univ. Press, 1987.
2 Neilson, A.H. PAH’s and Other Related Compounds, Spriger, 1998.
BP
7,8 epoxide
9,10 epoxide
anti-BPDE
N2dG adduct-
Cation radical
N7dG adduct- C8dG adduct- A7dG adduct-
Cyt P4501A2/DNA film
Mb/DNA film
PDDA/DNA Control
Cyt P450 cam
Pyrolytic Graphite
Cyt P450cam/DNA film
Catalytic Current
PDDAds-DNA
Benzo[a]pyreneBenzo[a]pyrene diol-epoxide–DNA (in film)
Electrode array
Arrays: Which Liver Cytochrome P450s -generate toxic Benzo[a]pyrene Metabolites?
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
0 5 10 15 20 25 30 35
Mbcyt P450camcyt P4501A2Control
I p,f
/ I p,i
Incubation time, min
Figure 7. Influence of incubation time with 50 μM benzo[a]pyrene and 1 m M H2O2 on the peakcurrent ratios from SWV of PDDA/DNA/(enzyme/DN )A 2 films Control isPDDA/DNA/(Mb/DNA)2 film i 50n μM benzo[a]pyrene alone.
Arrays detect in-vitro DNA damage from metabolites ofdifferent enzymes in DNA/enzyme films
Mb 0.9
P450cam 3.0
P450 1A2 3.5
Rel. turnover rate,1/min (nmol E)
Drug discoveryapplications
Pyrolytic Graphite
Os-PVP
Ru-PVP
PSS
Sensors for oxidative stress via oxidized DNA
ECL detection in films:Lynn Dennany, Robert J. Forster, Blanaid White, Malcolm Smyth and James F. Rusling, Am. Chem. Soc., 2004, 126, 8835-8841.0
2
4
6
8
10
12
0 0.2 0.4 0.6 0.8 1
SWV (10 Hz) of PVP-Ru/PSS/PVP-Os film (a) in buffer; (b) + 0.2 mg/mL CT ds-DNA
(c) + 0.2 mg/mL CT ds-DNA after 80 min. in Fenton reagent
I, μA
, E V vs SCE
a
b
c oxidized DNA
-ds DNA
no DNA
7pH
Amos Mugweru, Bingquan Wang, and James F. Rusling “Vo ltammetric Detection of OxidizedDNA using Ultrathin Films of Os and Ru Metallopolymers”, Anal. Chem. 2004, 5557-5563.
Summary: DNA damage detection/toxicity sensors
• Catalytic voltammetry and ECL toxicity sensors
• sensors produce metabolites, damage DNA
• Can detect 5-10 damaged bases/10,000
• can detect DNA oxidation - 8-oxoguanine (1/6000)
• Future: extensions to many compounds, cyt P450 arrays, ECL arrays, drug toxicity
Bioactivation/DNA Damage/ECL Detection
A. Expose sensor to solution of BP and H2O2.
B. Detect ECL by applying V
DNA Adducts
Enzyme to make Metabolite
DNA
ECL polymer
ECL generated by production of the photoexcited Ru(bpy)32+*
via oxidation of damaged DNA.
h
h
h
h
h
CCD camera
Equipment for ECL Arrays
Eli G. Hvastkovs, Minjeong So, Sadagopan Krishnan, Besnik Bajrami, Maricar Tarun, IngelaJansson, John B. Schenkman, and James F. Rusling "Electrochemiluminescent Arrays forCytochrome P450-activated Genotoxicity Screening. DNA Damage from Benzo[a]pyreneMetabolites", Anal. Chem, 2007, in press.
The ECL Array on graphite w/ Enzyme Exposed to BP + H2O2
• Cyt P450 1B1 enzyme in all spots (1 mg/mL)
• 49 spots allows monitoring damage response of cyt 1B1 to BP (100 μM) activated with H2O2 (0.5 mM) + control
•Apply voltage to generate ECL;
±10% reproducibility
Eli Hvastkovs
Monitoring Multiple Enzymes Simultaneously using ECL Array
1
3 5 7
2E1 cam
1B11A2
Cyt P450s
ECL Samples of Different Enzymes Exposed to BP
• Minutes exposure to 100 μM BP + 0.5 mM H2O2
• Consistent increase in ECL intensity up to 7-10 minutes exposure
1B1
1A2
0 1 3 5 7 10 15
Toxicity screening sensors and arrays
• Electrochemical, ECL, LC-MS toxicity sensors
• Sensors produce metabolites, detect DNA damage
• Detect relative rates of DNA damage beginning at 5-10 damaged bases/10,000 - in several min.
• Multienzyme arrays
• CapLC-MS rates of DNA damage with fmol detection limits of nucleobase adducts
• Future: commercialization? MS kinetics arrays?
Future of bioanalysis with arrays
• high throughput, speed• DNA microarrays for genetic analysis - most developed• proteomics arrays based on fluorescence• cyt P450 genotoxicity arrays• future - arrays for disease diagnostics and treatment• future - electrochemical and ECL arrays for proteins
Thanks to NIH, NSF and ARO for funding!
Thanks to all our coworkers and collaboratorshttp://web.uconn.edu/rusling/
Thanks to YOU for listening!
Thanks to intangible creative factors
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