Design of Biosensors and Arrays: Toxicity Screening

James F. Rusling

Departments of Chemistry & Pharmacology

University of Connecticut

Storrs, CT, USA

electrode

substrate product

Enzyme, or Label on Ab/AgOr DNA

Apply voltage Measure current prop.to concentration of substrate

Traditional Electrochemical Biosensors

• nanoscale biosensing architecture• patternable nanomaterials for arrays

Adsorbed of chem.bonded

Negativesurface

Polycation soln.,then wash

+ + + + + + + + +

soln. of negative proteinthen wash

+ + + + + + + + +

+ + + + + + + + +

Repeat steps for desired number of layers

Proteinlayer

Polycation layersProteinlayer

Polycation soln.,then wash

Layer-by-layerFilm 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

scale

1

2

3

4

5

6

7

Optical arrays - absorbance or fluorescence

DNA, proteins, pathogenic bacteriaDNA arrays with millions of spots

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

label

antibody

pathogen

Multiple-antibody-nanotube

nanoparticles

Light in

Fluorescence out

Fluorescence from all spots measured simultaneously

array

COOH

COOH

HOOC

HOOC

HOOCHOOC

HOOC

HOOC

HOOCCOOH

COOH

COOH

COOH

COOH

HNO3, 3 H2SO4

Ab2 label

sonicate 6 hr

Carbon nanotube (CNT)

Coupling agents

Functionalizing nanotubes with labels and antibodies

• Single walled (1.4 nm o.d.)and multi-walled

• Highly conductive,flexible, strong,patternable

• Commercially Available

Researchgoal

toxic?

transducer

Biomolecular reporter

test chemical

• ~ 30 % of drug candidates defeated by toxicity• Early screening could save drug development costs

Lipophilic Molecule

styreneCyt P450, O2

Enzyme-activated molecule

O

styrene oxide+DNA

DamagedDNA

Detect by electrochemical sensorValidate by LC-MS/MS

In vitro Toxicity Screening

Funding from NIH, NIEHS

Collaboration with Prof. John Schenkman,Pharmacology, Uconn Health Center

ds-DNA

PDDA orRu-PVP (catalyst)(Ru(bpy)2

2+-PVP)Pyrolytic Graphite

Enzyme

Films for Toxicity Screening

20-40 nm

Negativesurface

Polycation soln.,then wash

+ + + + + + + + +

soln. of negative proteinthen wash

+ + + + + + + + +

+ + + + + + + + +

Repeat steps for desired number of layers

Proteinlayer

Polycation layersProteinlayer

Polycation soln.,then wash

Layer-by-layerFilm 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

Mass: M/A = -F/1.86 x 108

Thickness: d = -(0.016) F

Quality control for enzyme-DNA films

0

1000

2000

3000

4000

5000

6000

0 2 4 6 8 10

- , F Hz

. Layer No and Identity

MPA PDDA DNA Mb DNA Mb DNA Mb DNA / 450P / 450 / 450P P

- / 450ST dsDNA Pcam

- /CT dsDNA Mb

- QCM film growth

E, V

time

E-t waveform

potentiostat

Electrochemical cell

counter

working electrode

N2

inlet

DNA/enzyme film

reference

insulator electrodematerial

Equipment for toxicity biosensors

Square-wavevoltammetry

I measured,then subtracted

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, μA

, E V vs SCE

- Controls no styrene

0 min

5 min

+ 2% styrene

15 min

Bare PG

30 min

15 min

30 min

( / )Mb DNA2 films

1. 37 incubateoC

2. , 50 SWV μ ( )M Ru bpy3

2+

0.2 mM H2O2Enzyme/DNA films

Peak increasemeasures damageof DNA by enzyme-generatedmetabolite

Cyt P450

H2O2R (e.g. styrene)

RO (e.g. styreneoxide)

-5

0

5

10

15

0 3 6 9

Ip, μA

, t min

450cyt Pcam

I/Δt = rel. rateDNA Damage

styrene

controls

Sensor response vs. reaction time

+ H2O2

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

1

1.2

1.4

1.6

1.8

0

20

40

60

0 10 20 30

Incubation in MMS, min

LC-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+

Equipment for ECL toxicity sensorsECL = electrochemiluminence

E, V

time

E-t waveform

potentiostat

ECL cell

counter

working electrode

N2

inlet

DNA/enzyme film

reference

insulator electrodematerial

Square-wavevoltammetry

I measured,then subtracted

Monochromator/PM tube detector

optical fiber

glass

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+

N N NRuNN NN 8

Catalytic ECL polymer

N N NRuNN NN 8

-30

-20

-10

0

10

20

30

-3

-2

-1

0

1

2

3

0.60.811.2

I, μA

, . E V vs SCE

0

0

5

5 10

10

15

15

20

20

25

25 min

ECL

SWV

DNA +

Incubations with styrene oxide

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100

DNA + Styrene OxideDNA + TolueneDNA + Buffer

ECL Final ECLInitial

t, min

aECL output

0

2

4

6

8

0 20 40 60 80 100

DNA + Styrene OxideDNA + TolueneDNA + Buffer

Ip, final

Ip. inital

t, min

bSWV output

Incubation of Ru-PVP/DNA films with styrene oxide

Direct ECL generation from DNA

RVP-RuL2+ = PVP-RuL3+ + e-PVP-RuL3+ + DNA-G --> 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+ --> PVP-[RuL2+]*

Direct ECL generation from DNA

RuPVP2+RuPVP3+

PG Electrode

Guanine

Guanine

Guanine2+

RuPVP2+*• ECL can be generated by the formation of RuPVP2+* through the oxidation of Guanine on sensor

Dennany, L.; Forster, R. J.; Rusling, J. F. J. Am. Chem. Soc., 2003, 125, 5213-5218.

h

RuPVP3+

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

• GT 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

N2dG adduct-

Cation radical

N7dG adduct- C8dG adduct- A7dG adduct-

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-epoxide–DNA (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 μM benzo[a]pyrene and 1 m M H2O2 on the peakcurrent ratios from SWV of PDDA/DNA/(enzyme/DN )A 2 films Control isPDDA/DNA/(Mb/DNA)2 film i 50n μM benzo[a]pyrene alone.

Arrays detect in-vitro DNA damage from metabolites ofdifferent enzymes in DNA/enzyme films

Mb 0.9

P450cam 3.0

P450 1A2 3.5

Rel. turnover rate,1/min (nmol E)

Drug discoveryapplications

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, 8835-8841.0

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, μA

, E V vs SCE

a

b

c oxidized DNA

-ds DNA

no DNA

7pH

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.

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

Bioactivation/DNA Damage/ECL Detection

A. Expose sensor to solution of BP and H2O2.

B. Detect ECL by applying V

DNA Adducts

Enzyme to make Metabolite

DNA

ECL polymer

ECL generated by production of the photoexcited Ru(bpy)32+*

via oxidation of damaged DNA.

h

h

h

h

h

CCD camera

Equipment for ECL Arrays

Eli G. Hvastkovs, Minjeong So, Sadagopan Krishnan, Besnik Bajrami, Maricar Tarun, IngelaJansson, John B. Schenkman, and James F. Rusling "Electrochemiluminescent Arrays forCytochrome P450-activated Genotoxicity Screening. DNA Damage from Benzo[a]pyreneMetabolites", Anal. Chem, 2007, in press.

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 μM) activated with H2O2 (0.5 mM) + control

•Apply voltage to generate ECL;

±10% reproducibility

Eli Hvastkovs

Monitoring Multiple Enzymes Simultaneously using ECL Array

1

3 5 7

2E1 cam

1B11A2

Cyt P450s

ECL Samples of Different Enzymes Exposed to BP

• Minutes exposure to 100 μM BP + 0.5 mM H2O2

• Consistent increase in ECL intensity up to 7-10 minutes exposure

1B1

1A2

0 1 3 5 7 10 15

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?

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

Thanks to NIH, NSF and ARO for funding!

Thanks to all our coworkers and collaboratorshttp://web.uconn.edu/rusling/

Thanks to YOU for listening!

Thanks to intangible creative factors

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