Nanoscale Design of Biosensors for Toxicity Screening and Biomedical

Applications

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

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

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

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

ds-DNA

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

2+-PVP)Pyrolytic Graphite

Enzyme

Films for Toxicity Screening

20-40 nm

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

Thickness: d = -(0.016) F

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 /P450 /P450 /P450

ST-dsDNA/P450cam

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, 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)

0

10

20

30

0 10 20 30 40

Ip, A

t, min

controls

styrene

Cyt P450cam/DNA film + 0.2 M H2O

2

50 M Ru(bpy)3

2+

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+

E, V

time

E-t waveform

potentiostat

ECL cell

counter

working electrode

N2

inlet

DNA/enzyme film

reference

insulator electrodematerial

Equipment for ECL toxicity sensors

Square-wavevoltammetry

I measured,then subtracted

Monochromator/PM tube detector

optical fiber

glass

N N N

Ru

N

N

N

N

8

-30

-20

-10

0

10

20

30

-3

-2

-1

0

1

2

3

0.60.811.2

I, AR

el. E

CL

Inte

nsity

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

b SWV 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+]*

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 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, 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.

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

James F. Rusling and Xin Yu, Depts. of Chemistry and Pharmacology, Univ. Connecticut

Maire O’Connor, Anthony Killard, Malcolm SmythNCSR, Dublin City University

Sang Nyon Kim, Fotis Papadimitrakopoulos Institute of Materials Science, University of Connecticut

Single-walled carbon nanotube forests as a basis for immunosensors

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

• Highly conductive, flexible, strong, patternable

• Commercially Available

Carbon Nanotubes

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)

High conductivity to conduct signal (e’s) from enzyme label to meas. circuit

~1.4 nm diameter, high conductivity

Experimental Procedure for SWNT Forest Assembly

Negatively Negatively

Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.

Acid + U-sound

O

HO

O

HO

O

HO

graphite surface/SWNTwater-soluble carbodiimide (1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide hydrochloride, EDC, or EDC + NHSS

N H

O

g r a p h i t e s u r f a c e /S W N T /

N H

O

N H

O

N H N H N H

Protein Protein

HRP

Covalently Binding Protein to SWNT

Protein

Ends of nanotubes -COOH:

(a) (b)

AFM of SWNT forest with and without antibiotin attached

(a) SWNT forest on smooth silicon and (b) Anti-biotin antibody functionalized SWNT onsmooth silicon

SWNT SWNT + antibody(EDC coupling)

O2

H2O2

PFeIII PFeII PFeII-O2- +

2 2 IIIV

-

-

III 2 O2

H2O2

Possible reduced species in red

Electrochemical Response of Peroxidases

Zhe Zhang, Salem Chouchane, Richard S. Magliozzo,and James F. Rusling, "Direct Voltammetry and Enzyme Catalysis with M. tuberculosis Catalase-Peroxidase, Peroxidases and Catalase in Lipid Films", Anal. Chem., 2002, 74, 163-170.

HRP on electrodes: + H2O2 = current signal

Competitive Immunoassay

Catalytic current should be inversely proportional to the amount of non-labeled Ag, depending on binding constant, Ag was pre-bound on Ab

HRP

H2O2

AbAb

HRP

HRP

HRP

antigen

apply E measure I

H2O + 1/2 O2

SWNT forest

Figure B.1.Competitive enzyme-tagged antigen assay

Anti-biotin/biotin-HRP test system(H2O2 present)

with mediator

0.364

0.366

0.368

0.37

0 20 40 60 80 100 120

I, A

t, s

SWNT/Ab/BSA 30 pmol/mL(nM) Ag-HRP

a With mediatorNo mediator

LOD ~1 pmol/mL

not all the HRP label was communicating with the measuring circuit - soluble mediator shuttles

electrons from HRP label more efficiently

Near LOD

3

25

Sandwich Assay for Human Serum Albumin

H2O2

Ab1

Ag

HRPHRP

HRP HRPHRP

Ab1

Ag

HRP

HPR

Ab2

Apply E measure ISWNT forest Conductive polymer

(SPAN)

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,~50-fold better w/ SPAN

Design approaches to future arrays

1. Layer-by-layer approach general, simple2. Stable films, complex architecture, any surface3. Sensors for toxicity, oxidative stress 4. Ambient T solution processable5. SWNT forests patterned by solution process6. Excellent LOD and sensitivity using conductive polymer bed (SPAN)7. Possibility of automated array formation8. Applications to proteins, pathogens, etc.

Future work: pattern SWNT forest arrays onto microchip;collaboration with Univ. of Edinburgh Genomics Inst. (GTI)

Also, screen printed carbon arrays, Lab 901, Edinbugh

Detection of Protein biomarkers for Cancer:• NIH, NIDCR• prostate, squamous cell, and breast cancers

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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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