nanoscale design of biosensors for toxicity screening and biomedical applications james f. rusling
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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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