Design of Biosensors and Arrays: Toxicity Screening - PowerPoint PPT Presentation

Loading...

PPT – Design of Biosensors and Arrays: Toxicity Screening PowerPoint presentation | free to download - id: 3d95e8-MzgxN



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Design of Biosensors and Arrays: Toxicity Screening

Description:

Design of Biosensors and Arrays: Toxicity Screening James F. Rusling Departments of Chemistry & Pharmacology University of Connecticut Storrs, CT, USA – PowerPoint PPT presentation

Number of Views:40
Avg rating:3.0/5.0
Slides: 40
Provided by: webUconnE
Learn more at: http://web.uconn.edu
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Design of Biosensors and Arrays: Toxicity Screening


1
Design of Biosensors and Arrays Toxicity
Screening James F. Rusling Departments of
Chemistry Pharmacology University of
Connecticut Storrs, CT, USA
2
Traditional Electrochemical Biosensors
substrate
product
Enzyme, or Label on Ab/Ag Or DNA
Adsorbed of chem. bonded
electrode
Measure current prop. to concentration of
substrate
Apply voltage
nanoscale biosensing architecture patternable
nanomaterials for arrays
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
Optical arrays - absorbance or fluorescence
1 2 3 4 5 6 7
scale
DNA, proteins, pathogenic bacteria DNA arrays
with millions of spots
5
Fluorescence-based sandwich immunoassay
Multiple-antibody- Nanotube (CNT)
array
Antigen Protein, pathogen
spot
or
label
Secondary antibody and labels or
multi-label Bioconjugate on CNT
Primary antibody
Antibodies capture The antigen
6
Fluorescence out
Multiple-antibody- nanotube
Light in
array
pathogen
nanoparticles
label
antibody
Fluorescence from all spots measured
simultaneously
7
Functionalizing nanotubes with labels and
antibodies
COOH
HOOC
COOH
COOH
HOOC
HNO3, 3 H2SO4
Coupling agents
HOOC
COOH
sonicate 6 hr
HOOC
COOH
HOOC
Ab2 label
COOH
HOOC
COOH
Carbon nanotube (CNT)
HOOC
8
toxic?
Research goal
transducer
test chemical
Biomolecular reporter
30 of drug candidates defeated by
toxicity Early screening could save drug
development costs
9
In vitro Toxicity Screening
Lipophilic Molecule
Cyt P450, O2
Enzyme-activated molecule
DNA
Damaged DNA
Detect by electrochemical sensor Validate by
LC-MS/MS
10
Collaboration with Prof. John Schenkman, Pharmacol
ogy, Uconn Health Center
Funding from NIH, NIEHS
11
Films for Toxicity Screening
20-40 nm
PDDA or Ru-PVP (catalyst) (Ru(bpy)22-PVP)
Pyrolytic Graphite
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 -DF/1.86 x 108 Thickness
d -(0.016) DF
Quality control for enzyme-DNA films
14
(No Transcript)
15
Equipment for toxicity biosensors
potentiostat
electrode material
insulator
reference
DNA/enzyme film
N2 inlet
counter
working electrode
E-t waveform
Square-wave voltammetry
E, V
Electrochemical cell
I measured, then subtracted
time
Figure1
16
Screening Chemical Toxicity
Enzyme reaction - Incubate Reactant
H2O2--gtmetabolite
Analysis by catalytic SWV or electrochemiluminesc
ence
RuL2 RuL3 e- RuL3 DNA-G --gt RuL2
DNA-G
17
Enzyme/DNA films
Peak increase measures damage of DNA by
enzyme- generated metabolite
18
Sensor response vs. reaction time
H2O2
19
DNA-adduct formation increases signals
  • Adduct affects base pair binding
  • Exposes guanines by disrupting surrounding base
    pairs with unwinding of helix
  • Guanines more easily oxidized - increased signal

Martz, E. Trends in Biochemical Sciences. 2002,
27, 107, www.proteinexplorer.org.
20
Comparison of toxicity sensors with LC-MS For DNA
damage by methylmethane sulfonate
21
(No Transcript)
22
Equipment for ECL toxicity sensors ECL
electrochemiluminence
Catalytic ECL polymer
23
Incubations with styrene oxide
DNA
24
Incubation of Ru-PVP/DNA films with styrene oxide
25
Direct ECL generation from DNA
RVP-RuL2 PVP-RuL3 e- PVP-RuL3 DNA-G --gt
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 --gt PVP-RuL2
26
Direct ECL generation from DNA
Guanine2
Guanine?
  • ECL can be generated by the formation of
    RuPVP2 through the oxidation of Guanine on
    sensor

RuPVP2
RuPVP3
Guanine
PG Electrode
Dennany, L. Forster, R. J. Rusling, J. F. J.
Am. Chem. Soc., 2003, 125, 5213-5218.
27
BenzoaPyrene-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

BP
7,8 epoxide
anti-BPDE
9,10 epoxide
Cation radical
N2dG adduct-
1 Osborne, M. et al. Benzopyrenes. Cambridge
Univ. Press, 1987. 2 Neilson, A.H. PAHs and
Other Related Compounds, Spriger, 1998.
N7dG adduct-
C8dG adduct-
A7dG adduct-
28
Arrays Which Liver Cytochrome P450s -generate
toxic Benzoapyrene Metabolites?
29
Arrays detect in-vitro DNA damage from
metabolites of different enzymes in DNA/enzyme
films
Rel. turnover rate, 1/min (nmol E)
Drug discovery applications
30
Sensors for oxidative stress via oxidized DNA
Os-PVP
PSS
Pyrolytic Graphite
Ru-PVP
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
CCD camera
Bioactivation/DNA Damage/ECL Detection
DNA Adducts
A. Expose sensor to solution of BP and H2O2.
Enzyme to make Metabolite
B. Detect ECL by applying V
DNA
ECL polymer
ECL generated by production of the photoexcited
Ru(bpy)32 via oxidation of damaged DNA.
33
Equipment for ECL Arrays
34
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 ?M) activated with H2O2 (0.5
    mM) control
  • Apply voltage to generate ECL
  • 10 reproducibility

Eli Hvastkovs
35
Monitoring Multiple Enzymes Simultaneously using
ECL Array
1
Cyt P450s
2E1
cam
1B1
1A2
3
5
7
36
ECL Samples of Different Enzymes Exposed to BP
  • Minutes exposure to 100 ?M BP 0.5 mM H2O2
  • Consistent increase in ECL intensity up to 7-10
    minutes exposure

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
About PowerShow.com