Group Research Interests - an overview
James F. RuslingProfessor of Chemistry and
PharmacologyUniversity of Connecticut and
University Health Center
Lipophilic Molecule
styreneCyt P450, O2
Enzyme-activated molecule
O
styrene oxide+DNA
DamagedDNA
Detect by voltammetry, LC-MS/MS
Example 1. Toxicity Screening
ds-DNA
PDDA orRu-PVP (catalyst)(Ru(bpy)2
2+-PVP)Pyrolytic Graphite
Enzyme
Films for Toxicity Screening
20-40 nm
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, A
E, V vs SCE
Controls - no styrene
0 min
5 min
+ 2% styrene
15 min
Bare PG
30 min
15 min
30 min
(Mb/DNA)2 films
1. incubate 37 oC
2. SWV, 50 M Ru(bpy)3
2+
0.2 mM H2O2Enzyme/DNA films
Peak increasemeasures damageof DNA by enzyme-generatedmetabolite
Cyt P450
H2O2R (e.g. styrene)
RO (e.g. styreneoxide)
Detection of DNA-styrene oxide adductsafter incubations of films + hydrolysis
LC-MRM-MS/MSLC-UV
Nucleobase adducts
1
1.2
1.4
1.6
1.8
0
20
40
60
0 10 20 30
pm
ol N
7-C
H3 G
Incubation in MMS, min
Se
ns
or
rati
oLC-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+
Alternative way to detect DNA damage with light
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-epoxideĞDNA (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 M benzo[a]pyrene and 1 mM H2O2 on the peakcurrent ratios from SWV of PDDA/DNA/(enzyme/DNA)2 films Control isPDDA/DNA/(Mb/DNA)2 film in 50 M benzo[a]pyrene alone.
Arrays detect in-vitro DNA damage from metabolites ofdifferent enzymes with DNA/enzyme films
Mb 0.9
P450cam 3.0
P450 1A2 3.5
Rel. turnover rate,1/min (nmol Enzyme)
Drug developmentapplications
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, J. 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, A
E, V vs SCE
a
b
c oxidized DNA
ds-DNA
no DNA
pH 7
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.
Single-Walled Carbon Nanotube Forests: Antigen-Antibody Sensing
SPAN orNafion
Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.
End COOH groups allow chemical attachment to proteins (antibodies)
(a) (b)
AFM of SWNT forest with and without antibody attachedCollaboration with F. Papadimitrakopoulos (ARO funded)
(a) SWNT forest on smooth silicon and (b) Anti-biotin antibody functionalized SWNT onsmooth silicon
SWNT SWNT + antibody(EDC coupling)
Sandwich Assay for Human Serum Albumin
H2O2
Ab1
Ag
HRPHRP
HRP HRPHRP
Ab1
Ag
HRP
HPR
Ab2
Apply E measure ISWNT forest
0
10
20
30
0 100 200 300 400 500 600 700
I, nA
t, s
3000
1500
15
15075
30
pmol/mL
750
a
Detection of Human Serum albumin in10 L drops on SWNT forest immunosensor
LOD ~ 10 pmol/mL
Styrene Epoxidation Catalyzed by Myoglobin-Polyion Films
in Microemulsions (NSF funded)
Myoglobin
+
+
+
+
+
+
+
+
--------
+
+
+
+
+
+
+
+
--------
--------
+
+
+
+
+
+
+
+
--------
-CO-NH--CO-NH-
-CO-NH-
-CO-NH-
-CO-NH-
-CO-NH-
-CO-NH-
CROSSLINKED PLL/MbX FILMFully Covalently Linked Layers
O
HO
O
HO
O
HO
Oxidized graphite surface
NH
O
NH
O
NH
O
NH2 NH2 NH2
NH
O
NH
O
NH
O
NH NH NH
Mb Mb Mb
-COOHGroups
Amide LinksbetweenElectrode andPLL
Amide Linksbetween PLLand Mb
Stable in microemulsions
Catalytic Styrene Epoxidation
2
Reference electrode
Working electrodeStir bar
Power Supply
2O2
Counter electrode
O2
2e-, 2H+
H2O2 + MbFeII
H2O2
•MbFeIV=O
active oxidant
+e-
-e-
MbFeIII +
o
MbFeIII MbFeII MbFeII-O2
–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