Transcript PPT - University of Connecticut

Nanoscale Design of Biosensors for
Toxicity Screening and Biomedical
Applications
James F. Rusling
Departments of Chemistry & Pharmacology
University of Connecticut
Storrs, CT, USA
Traditional Electrochemical Biosensors
substrate
product
electrode
Apply voltage
Enzyme, or
Label on Ab/Ag
Or DNA
Measure current prop.
to concentration of substrate
• nanoscale biosensing architecture
• patternable nanomaterials for arrays
Negative
surface
Layer-by-layer
Film assembly
Polycation soln.,
then wash
+ + + + + + + + +
soln. of negative protein
then wash
Lvov, Decher
Protein
layer
Lvov, Y. in Nalwa, R.W.; Ed.;
Handbook Of Surfaces And Interfaces
Of Materials, Vol. 3. Academic, 2001, pp. 170-189.
+ + + + + + + + +
Polycation soln.,
then wash
+ + + + + + + + +
Stable, easily prepared, versatile
Repeat steps for desired
number of layers
Protein
layer
Polycation layers
toxic?
Research
goal
Biomolecular
reporter
transducer
test chemical
• ~ 30 % of drug candidates defeated by toxicity
• Early screening could save drug development costs
In vitro Toxicity Screening
Lipophilic Molecule
Cyt P450, O2
s tyrene
O
Enzyme-activated molecule
+DNA
styrene oxide
Damaged
DNA
Detect by electrochemical sensor
Validate by LC-MS/MS
Collaboration with Prof. John Schenkman,
Pharmacology, Uconn Health Center
Funding from NIH, NIEHS
Films for Toxicity Screening
Enzyme
20-40 nm
ds-DNA
Pyrolytic Graphite
PDDA or
Ru-PVP (catalyst)
(Ru(bpy)22+-PVP)
Mass:
Thickness:
M/A = -DF/1.86 x 108
d = -(0.016) DF
QCM -film growth
6000
5000
-DF, Hz
ST-dsDNA/P450cam
4000
3000
2000
1000
CT-dsDNA/Mb
0
MPA PDDA DNA Mb DNA Mb DNA M b DNA
/P450
/P450
/P450
0
2
4
6
8
Layer No. and Identity
10
Equipment for toxicity biosensors
potentiostat
electrode
material
insulator
reference
N2
inlet
DNA/enzyme film
counter
working electrode
E-t waveform
Square-wave
voltammetry
E, V
Electrochemical cell
I measured,
then subtracted
time
Screening Chemical Toxicity
Enzyme reaction - Incubate:
Reactant + H2O2-->metabolite
Analysis by catalytic SWV or
electrochemiluminescence
RuL2+ = RuL3+ + eRuL3+ + DNA-G --> RuL2+ + DNA-G•
-200
0.2 mM H2O2
(Mb/DNA) films
Enzyme/DNA
films
2
-150
I, A
o
1. incubate 37 C
+ 2% styrene
30 min
15 min
2. SWV, 50 M Ru(bpy)
2+
3
-100
5 min
Controls no styrene
30 min
15 min
0 min
-50
Peak increase
measures damage
of DNA by enzymegenerated
metabolite
Bare PG
0
0.4
0.6
0.8
E, V vs SCE
1
1.2
Cyt P450cam/DNA film + 0.2 M H O
2
30
50 M Ru(bpy)
2
2+
3
I , A
p
20
styrene
10
controls
0
0
10
20
t, min
30
40
Detection of DNA-styrene oxide adducts
after incubations of films + hydrolysis
Nucleobase adducts
LC-UV
LC-MRM-MS/MS
Comparison of toxicity sensors with LC-MS
For DNA damage by methylmethane sulfonate
1.8
60
LC-MS/MS
1.4
20
1.2
1
Sensor
0
10
20
Incubation in MMS, min
0
30
3
Sensor ratio
40
pmol N7-CH G
1.6
electrochemiluminescence
- - - - -- - - - - - - - - - - -
Ru-PVP
[Ru(bpy)2-(PVP)10]2+
DNA
DNA
Pyrolytic Graphite
E=1.15 V
Echemdetection
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 .
Equipment for ECL toxicity sensors
potentiostat
electrode
material
insulator
reference
DNA/enzyme film
N2
inlet
counter
working electrode
E-t waveform
ECL cell
Square-wave
voltammetry
E, V
Monochromator/
PM tube detector
glass
I measured,
then subtracted
optical fiber
time
Incubations with styrene oxide
30
25 min
20
15
3
10
20
ECL
2
5
DNA +
0
1
I, A
0
0
5
-20
-1
0
-10
15
20
-2
10
SWV
-3
25
-30
1.2
1
0.8
E, V vs. SCE
0.6
Rel. ECL Intensity
10
N
N
Ru
N
N
N
N
N
8
Incubation of Ru-PVP/DNA
films with styrene oxide
3
ECL output
a
2.5
ECL Final
ECLInitial
2
8
b
6
I
DNA + Styrene Oxide
DNA + Tolue ne
DNA + Buffer
SWV output
p, final
I
p. inital
4
DNA + Styrene Oxide
DNA + Tolue ne
DNA + Buffer
1.5
2
1
0
0.5
0
20
40
60
t, min
80
100
0
20
40
60
t, mi n
80
100
Direct ECL generation from DNA
RVP-RuL2+ = PVP-RuL3+ + ePVP-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+]*
Arrays: Which Liver Cytochrome P450s generate toxic Benzo[a]pyrene Metabolites?
Catalytic Current
Benzo[a]pyrene
Benzo[a]pyrene diolepoxideĞDNA (in film)
Cyt P450
cam
Mb/DNA film
ds-DNA
PDDA
Pyrolytic Graphite
Cyt P450cam/DNA film
PDDA/DNA Control
Cyt P4501A2/ DNA film
Electrode array
Arrays detect in-vitro DNA damage from metabolites of
different enzymes in DNA/enzyme films
1.5
1.4
1.3
I p,f / I p,i
Rel. turnover rate,
1/min (nmol E)
Mb
cyt P450cam
cyt P4501A2
Control
Mb
0.9
1.2
P450cam
3.0
1.1
P450 1A2 3.5
1
Drug discovery
applications
0.9
0.8
0
5
10
15
20
25
30
35
Incubation time, min
Figure 7. Influence of incubation time with 50 M benzo[a]pyrene and 1 mM H2O2 on the peak
current ratios from SWV of PDDA/DNA/(enzyme/DNA)2 films Control is
PDDA/DNA/(Mb/DNA)2 film in 50 M benzo[a]pyrene alone.
Sensors for oxidative stress via oxidized DNA
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 reage nt
12
Os-PVP
PSS
c oxidized DNA
10
Ru-PVP
pH 7
8
Pyrolytic Graphite
ds-DNA
b
a
I, A
no DNA
6
ECL detection in films:
4
Lynn Dennany, Robert J.
Forster, Blanaid White,
Malcolm Smyth and James F.
Rusling, Am. Chem. Soc., 2004,
126, 8835-8841.
2
0
0
0.2
0.4
0.6
0.8
1
E, V vs SCE
Amos Mugweru, Bingquan Wang , and James F. Rusling “Voltammetric Detection of Oxidized
DNA 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
Single-walled carbon nanotube forests as a
basis for immunosensors
James F. Rusling and Xin Yu,
Depts. of Chemistry and Pharmacology, Univ. Connecticut
Maire O’Connor, Anthony Killard, Malcolm Smyth
NCSR, Dublin City University
Sang Nyon Kim, Fotis Papadimitrakopoulos
Institute of Materials Science, University of Connecticut
Carbon Nanotubes
• Single walled (1.4 nm o.d.)
and multi-walled
• Highly conductive,
flexible, strong,
patternable
• Commercially Available
Single-Walled Carbon Nanotube
Forests: Antigen-Antibody Sensing
~1.4 nm diameter, high conductivity
SPAN or
Nafion
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
Experimental Procedure for SWNT Forest Assembly
Acid + U-sound
Negatively
Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.
Covalently Binding Protein to SWNT
Protein
Protein
Protein
NH
NH
O
NH
O
O
NH
NH
NH
g r a p h i t e s u r f a c e / S W N T / HRP
H
O
O
H
O
H
O
O
O
Ends ofgraphite
nanotubes -COOH:
surface/SWNT
water-soluble carbodiimide (1-(3-(dimethylamino) propyl)-3ethylcarbodiimide hydrochloride, EDC, or EDC + NHSS
AFM of SWNT forest with and without antibiotin attached
(a) SWNT
(b)
SWNT + antibody
(EDC coupling)
(a) SWNT forest on smooth silicon and (b) Anti-biotin antibody functionalized SWNT on
smooth silicon
Electrochemical Response of Peroxidases
PFe III
+e-
-e
O2
PFe II
H2O2
•PFeIV=O
active oxida nt
PFe II-O2
2e-, 2H+
H2 O2 + PFeII
H2O2
PFeIII + H2 O + O2
Possible reduced species in red
HRP on electrodes: + H2O2 = current signal
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.
Competitive Immunoassay
Figure B.1.
Competitive enzyme-tagged antigen assay
H2O2
H2O + 1/2 O2
HRP
HRP
Ab HRP
HRP
SWNT forest
Ab
antigen
apply E
measure I
Catalytic current should be inversely proportional to the amount of non-labeled
Ag, depending on binding constant, Ag was pre-bound on Ab
Anti-biotin/biotin-HRP test system
(H2O2 present)
0.37
No mediator
With
with mediator
mediator
a
I, A
0.368
25
LOD ~1 pmol/mL
0.366
SWNT/Ab/BSA
30 pmol/mL
(nM) Ag-HRP
3
Near LOD
0.364
0
20
40
60
80
100
120
t, s
not all the HRP label was communicating
with the measuring circuit - soluble mediator shuttles
electrons from HRP label more efficiently
Sandwich Assay for Human
Serum Albumin
H2O2
HRP
HRP
HRP
HRP
HRP
Ag
Ag
Ab2
Ab1
Ab1
HPR
H
R
P
SWNT forest
Conductive polymer
(SPAN)
Apply E
measure I
Detection of Human Serum albumin in
10 L drops on SWNT forest immunosensor
pmol/mL
30
a
3000
I, nA
1500
20
LOD ~ 10 pmol/mL,
~50-fold better
w/ SPAN
750
10
150
75
30
15
0
0
100
200
300
400
t, s
500
600
700
Design approaches to future arrays
1. Layer-by-layer approach general, simple
2. Stable films, complex architecture, any surface
3. Sensors for toxicity, oxidative stress
4. Ambient T solution processable
5. SWNT forests patterned by solution process
6. Excellent LOD and sensitivity using
conductive polymer bed (SPAN)
7. Possibility of automated array formation
8. 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 a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Thanks to NIH, NSF and ARO for funding!
Thanks to all our coworkers and collaborators
http://web.uconn.edu/rusling/
Thanks to YOU for listening!
Thanks to intangible creative factors
+