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

Collaboration with Prof. John Schenkman,Pharmacology, Uconn Health CenterFunding from NIH, NIEHS

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