Transcript Document

Group Research Interests - an overview
James F. Rusling
Professor of Chemistry and Pharmacology
University of Connecticut and University
Health Center
Example 1. Toxicity Screening
Lipophilic Molecule
Cyt P450, O2
s tyrene
O
Enzyme-activated molecule
+DNA
styrene oxide
Damaged
DNA
Detect by voltammetry, LC-MS/MS
Films for Toxicity Screening
Enzyme
20-40 nm
ds-DNA
Pyrolytic Graphite
PDDA or
Ru-PVP (catalyst)
(Ru(bpy) 22+-PVP)
Collaboration with Prof. John Schenkman,
Pharmacology, Uconn Health Center
Funding from NIH, NIEHS
Screening Chemical Toxicity
Enzyme reaction - Incubate:
Reactant + H2O2-->metabolite
Analysis by catalytic SWV or
electrochemiluminescence
RuL2+ = RuL3+ + eRuL3+ + DNA-G --> RuL2+ + DNA-G•
-200
0.2 mM H2O2
(Mb/DNA) films
Enzyme/DNA
films
2
-150
I, A
o
1. incubate 37 C
+ 2% styrene
30 min
15 min
2. SWV, 50 M Ru(bpy)
2+
3
-100
5 min
Controls no styrene
30 min
15 min
0 min
-50
Peak increase
measures damage
of DNA by enzymegenerated
metabolite
Bare PG
0
0.4
0.6
0.8
E, V vs SCE
1
1.2
Detection of DNA-styrene oxide adducts
after incubations of films + hydrolysis
Nucleobase adducts
LC-UV
LC-MRM-MS/MS
Comparison of toxicity sensors with LC-MS
For DNA damage by methylmethane sulfonate
1.8
60
LC-MS/MS
1.4
20
1.2
1
Sensor
0
10
20
Incubation in MMS, min
0
30
3
Sensor ratio
40
pmol N7-CH G
1.6
Alternative way to detect DNA damage with light
electrochemiluminescence
- - - - -- - - - - - - - - - - -
Ru-PVP
[Ru(bpy)2-(PVP)10]2+
DNA
DNA
Pyrolytic Graphite
E=1.15 V
Echemdetection
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 .
Arrays: Which Liver Cytochrome P450s -generate toxic
Benzo[a]pyrene Metabolites
Catalytic Current
Benzo[a]pyrene
Benzo[a]pyrene diolepoxideĞDNA (in film)
Cyt P450
cam
Mb/DNA film
ds-DNA
PDDA
Pyrolytic Graphite
Cyt P450cam/DNA film
PDDA/DNA Control
Cyt P4501A2/ DNA film
Electrode array
Arrays detect in-vitro DNA damage from metabolites of
different enzymes with DNA/enzyme films
1.5
1.4
1.3
I p,f / I p,i
Rel. turnover rate,
1/min (nmol Enzyme)
Mb
cyt P450cam
cyt P4501A2
Control
Mb
0.9
1.2
P450cam
3.0
1.1
P450 1A2 3.5
1
Drug development
applications
0.9
0.8
0
5
10
15
20
25
30
35
Incubation time, min
Figure 7. Influence of incubation time with 50 M benzo[a]pyrene and 1 mM H2O2 on the peak
current ratios from SWV of PDDA/DNA/(enzyme/DNA)2 films Control is
PDDA/DNA/(Mb/DNA)2 film in 50 M benzo[a]pyrene alone.
Sensors for oxidative stress via oxidized DNA
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 reage nt
12
Os-PVP
PSS
c oxidized DNA
10
Ru-PVP
pH 7
8
Pyrolytic Graphite
ds-DNA
b
a
I, A
no DNA
6
ECL detection in films:
4
Lynn Dennany, Robert J.
Forster, Blanaid White,
Malcolm Smyth and James F.
Rusling, J. Am. Chem. Soc.,
2004, 126, 8835-8841.
2
0
0
0.2
0.4
0.6
0.8
1
E, V vs SCE
Amos Mugweru, Bingquan Wang , and James F. Rusling “Voltammetric Detection of Oxidized
DNA using Ultrathin Films of Os and Ru Metallopolymers”, Anal. Chem. 2004, 5557-5563.
Single-Walled Carbon Nanotube
Forests: Antigen-Antibody Sensing
SPAN or
Nafion
Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.
End COOH groups allow chemical attachment to proteins (antibodies)
AFM of SWNT forest with and without antibody attached
Collaboration with F. Papadimitrakopoulos (ARO funded)
(a) SWNT
(b)
SWNT + antibody
(EDC coupling)
(a) SWNT forest on smooth silicon and (b) Anti-biotin antibody functionalized SWNT on
smooth silicon
Sandwich Assay for Human
Serum Albumin
H2O2
HRP
HRP
HRP
HRP
HRP
Ag
Ag
Ab2
Ab1
Ab1
HPR
H
R
P
SWNT forest
Apply E
measure I
Detection of Human Serum albumin in
10 L drops on SWNT forest immunosensor
pmol/mL
30
a
3000
I, nA
LOD ~ 10 pmol/mL
1500
20
750
10
150
75
30
15
0
0
100
200
300
400
t, s
500
600
700
Styrene Epoxidation Catalyzed by
Myoglobin-Polyion Films
in Microemulsions (NSF funded)
CROSSLINKED PLL/MbX FILM
Fully Covalently Linked Layers
Stable in microemulsions
Mb
Mb
Mb
NH
NH
NH
O
O
O
NH
Amide Link s
between PLL
and Mb
M yoglobin
NH
NH
--+
- +-+
- +- +-+
-+
+
NH2
NH 2
O
NH2
O
O
NH
H
O
O
Amide Link s
between
Electrode and
PLL
NH
NH
H
O
H
O
O
O
Oxidized graphite surface
-COOH
Groups
+
+
+
+
+
+
+
+
+
-CO-NH-
+ -CO-NH-CO-NH+ -CO-NH+ +
-CO-NH- +
-CO-NH- +
-CO-NH- + -
Catalytic Styrene Epoxidation
+eMbFeIII
Power Supply
Reference electrode
-
-e
II
MbFe
MbFeII-O2
2e-, 2H+
H2O2
•MbFeIV=O
Counter electrode
O2
H2O2 + MbFeII
active oxidant
O2
S tir bar
2
o
Working electrode
MbFeIII +
–Catalytic efficiency controlled by microemulsion composition
Additional Current Projects
• “Green” chiral synthesis using enzyme films in
microemulsions, nanoparticle catalysts (NSF)
• Redox chemistry of complex photosynthetic proteins
in membrane films (with H. Frank, USDA funded)
• detection of Lyme disease vectors (proteins) in tick
saliva (with Uconn Health Center)
• enzyme activity for generating toxic metabolites (with
Uconn Health Center)
• EC-Laser fluorescent detection of DNA damage
• arrays for anticancer biomarkers (NIH)
• molecular dynamics of film construction