Download presentation
Presentation is loading. Please wait.
Published byErik Randall Modified over 8 years ago
1
Design of Biosensors and Arrays: Toxicity Screening James F. Rusling Departments of Chemistry & Pharmacology University of Connecticut Storrs, CT, USA
2
electrode substrate product Enzyme, or Label on Ab/Ag Or DNA Apply voltage Measure current prop. to concentration of substrate Traditional Electrochemical Biosensors nanoscale biosensing architecture patternable nanomaterials for arrays Adsorbed of chem. bonded
3
Layer-by-layer Film 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
4
scale 12345671234567 Optical arrays - absorbance or fluorescence DNA, proteins, pathogenic bacteria DNA arrays with millions of spots
5
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
6
label antibody pathogen Multiple-antibody- nanotube nanoparticles Light in Fluorescence out Fluorescence from all spots measured simultaneously array
7
COOH HOOC COOH HNO 3, 3 H 2 SO 4 Ab 2 label sonicate 6 hr Carbon nanotube (CNT) Coupling agents Functionalizing nanotubes with labels and antibodies
8
Research goal toxic? transducer Biomolecular reporter test chemical ~ 30 % of drug candidates defeated by toxicity Early screening could save drug development costs
9
Lipophilic Molecule Cyt P450, O 2 Enzyme-activated molecule + DNA Damaged DNA Detect by electrochemical sensor Validate by LC-MS/MS In vitro Toxicity Screening
10
Funding from NIH, NIEHS Collaboration with Prof. John Schenkman, Pharmacology, Uconn Health Center
11
ds-DNA PDDA or Ru-PVP (catalyst) (Ru(bpy) 2 2+ -PVP) Pyrolytic Graphite Enzyme Films for Toxicity Screening 20-40 nm
12
Layer-by-layer Film 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
13
Mass: M/A = - F/1.86 x 10 8 Thickness: d = -(0.016) F Quality control for enzyme-DNA films
15
E, V time E-t waveform potentiostat Electrochemical cell counter working electrode N 2 inlet DNA/enzyme film Figure1 reference insulator electrode material Equipment for toxicity biosensors Square-wave voltammetry I measured, then subtracted
16
Enzyme reaction - Incubate: Reactant + H 2 O 2 -->metabolite Analysis by catalytic SWV or electrochemiluminescence RuL 2+ = RuL 3+ + e- RuL 3+ + DNA-G --> RuL 2+ + DNA-G Screening Chemical Toxicity
17
Enzyme/DNA films Peak increase measures damage of DNA by enzyme- generated metabolite
18
Sensor response vs. reaction time + H 2 O 2
19
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
20
Comparison of toxicity sensors with LC-MS For DNA damage by methylmethane sulfonate
22
Equipment for ECL toxicity sensors ECL = electrochemiluminence Catalytic ECL polymer
23
DNA + Incubations with styrene oxide
24
Incubation of Ru-PVP/DNA films with styrene oxide
25
Direct ECL generation from DNA RVP-RuL 2+ = PVP-RuL 3+ + e- PVP-RuL 3+ + DNA-G --> PVP-RuL 2+ + DNA-G Then? PVP-RuL 3+ oxidizes DNA-G to give Photoexcited PVP-[RuL 2+ ]* Or DNA-G reduces PVP-RuL 2+ to PVP-RuL +, PVP-RuL 3+ + PVP-RuL + --> PVP-[RuL 2+ ]*
26
Direct ECL generation from DNA RuPVP 2+ RuPVP 3+ PG Electrode Guanine Guanine Guanine 2+ RuPVP 2+ * ECL can be generated by the formation of RuPVP 2+* through the oxidation of Guanine on sensor Dennany, L.; Forster, R. J.; Rusling, J. F. J. Am. Chem. Soc., 2003, 125, 5213-5218. h RuPVP 3+
27
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 G T 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 N 2 dG adduct - Cation radical N7dG adduct - C8dG adduct - A7dG adduct -
28
Arrays: Which Liver Cytochrome P450s - generate toxic Benzo[a]pyrene Metabolites?
29
Arrays detect in-vitro DNA damage from metabolites of different enzymes in DNA/enzyme films Mb0.9 P450cam3.0 P450 1A23.5 Rel. turnover rate, 1/min (nmol E) Drug discovery applications
30
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.
31
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
32
Bioactivation/DNA Damage/ECL Detection A. Expose sensor to solution of BP and H 2 O 2. B. Detect ECL by applying V DNA Adducts Enzyme to make Metabolite DNA ECL polymer ECL generated by production of the photoexcited Ru(bpy) 3 2+* via oxidation of damaged DNA. h h h h h CCD camera
33
Equipment for ECL Arrays
34
The ECL Array on graphite w/ Enzyme Exposed to BP + H 2 O 2 Cyt P450 1B1 enzyme in all spots (1 mg/mL) 49 spots allows monitoring damage response of cyt 1B1 to BP (100 M) activated with H 2 O 2 (0.5 mM) + control Apply voltage to generate ECL; ±10% reproducibility Eli Hvastkovs
35
Monitoring Multiple Enzymes Simultaneously using ECL Array 1 357 2E1cam 1B11A2 Cyt P450s
36
ECL Samples of Different Enzymes Exposed to BP Minutes exposure to 100 M BP + 0.5 mM H 2 O 2 Consistent increase in ECL intensity up to 7-10 minutes exposure 1B1 1A2 013571015
37
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?
38
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
39
Thanks to NIH, NSF and ARO for funding! Thanks to all our coworkers and collaborators http://web.uconn.edu/rusling/ Thanks to YOU for listening! Thanks to intangible creative factors +
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.