Biosensor Array Strategies for Cancer Biomarker Proteins

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Biosensor Array Strategies for Cancer Biomarker
Proteins
James F. Rusling Professor of Chemistry and Cell
Biology University of Connecticut and University
Health Center Collaboration with F.
Papadimitrakopoulos (IMS) and Drs. Gutkind and
Patel (NIH, Bethesda)
Website http//web2.uconn.edu/rusling/ Email
James.Rusling_at_uconn.edu
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Cancer Biomarkers National Institutes of Health
molecules that can be objectively measured and
evaluated as indicators of normal or disease
processes and pharmacologic responses to
therapeutic intervention Cancer Biomarker
Proteins increase in serum concentration At the
onset of cancer, even before tumor develops
Multiple proteins must be measured for reliable
predictions Excellent hope for early detection
and treatment monitoring May also be able to
monitor inflammation May facilitate new
therapies
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Key aspects and needs

• Ultrasensitive detection of multiple cancer
  biomarkers
• Long term objectives
• 1. early cancer detection and monitoring
• 2. tools for cancer research and surgical
  decisions
• point-of-care (POC) clinical assays need to
  be cheap, simple, fast, accurate, multiplexed
• Expensive, and complex methodologies such as
  LC-MS/MS, some automated optical-based methods
  are currently not competitive for POC

Reviews
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Biomarker Targets 1. Prostate Specific Antigen

• PSA - Single chain glycoprotein , MW 33 kDa
  Sensitive, specific biomarker for detection of
  prostate cancer years before clinical signs of
  disease
• Detection of PSA in serum clinical detection of
  prostate cancer 4-10 ng/mL
• Led to less invasive treatment protocols, avoid
  surgery

Adapted From Brookhaven Protein Databank
2. Interleukin 6 (IL-6) - prostate and oral
cancer biomarker - human plasma conc. normal lt 6
pg/mL cancer 20-1000 pg/mL
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ELISA- enzyme linked immuno-sorbent assay
96-well plate
Antigen Protein, pathogen
Well in plate
Secondary antibody and labels
enzyme label
Primary antibody
Antibodies capture The antigen
Detection by optical absorbance plate reader
after running enzyme reaction that gives a
colored product
Reliable method for over 30 years Best DL 3
pg/mL in serum many commercial assay kits for
single proteins limitations in sample size,
speed, multiplexing
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Possible approaches fluorescence labels
surface plasmon resonance, SPR
Electrochemiluminescence (ECL) Ru(bpy)32
labels bead based assays, ECL or Fluorescence
S
Slope sensitivity
Detection limit blank signal 3x avg. noise
Conc. protein
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Multilabel Strategies high sensitivity
detection by fluorescence, amperometry, ECL
non-specific binding must be minimized in any
immunoassay
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Possible multilabel strategies
Dissolve, measure Mn
Also used on particles
Up to 400,000 labels
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SPR arrays
measure refractive index at interface
potentially label free -More susceptible to
Non-specific binding (NSB)
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Bead-based protein assays

Label enzyme Fluorophor DNA, RuBPY(ECL)
Protein in sample
multi-enzyme- Magnetic bead-Ab2 Multiple labels
Bead captures protein, Magnetic separation, wash
Detection ECL RuBPY label Fluorescence
Fl. Label 1-10 pg/mL DL 200,000 for machine
Magnet under well in 96-well plate, wash to
remove NSB
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2.
1.
A
conventional
Single electrode AuNP film
Multilabel magnetic particle, on-line capture
B
Electrochemical Immunosensors
Capture Antibody
Ab2-enzyme
Multi-enzyme- Magnetic particle-Ab2
AuNP
Protein analyte
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AuNP-based immunosensors single sensors
nanostructures sensor massive multilabel
strategies
Antibodies on AuNPs
AFM, carboxylated-AuNPs on polycation underlayer
Rotating disk amperometry (A) and calibration
for PSA on AuNP platform
AuNPs DL for PSA 0.5 pg/mL (8 x)
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Accurate PSA detection in cell lysates and
patient serum Using AuNP-based immunosensors
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SWNT array for 4 prostate cancer proteins in
human serum vs. ELISA
8-sensor array
Bhaskara V. Chikkaveeraiah, Ashwin Bhirde,
Ruchika Malhotra, Vyomesh Patel, J. Silvio
Gutkind, and James F. Rusling, Single-Wall Carbon
Nanotube Forest Immunoarrays For electrochemical
measurement of 4 Protein Biomarkers for Prostate
Cancer, Anal. Chem., 2009, 81, 91299134.
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5 nm Au nanoparticle vs. single wall nanotube
electrodes head to head comparison on IL-6
detection show better Detection limits and better
linearity with AuNPs AuNPs are easier to
handle and produce highly reproducible electrodes
Bernard S. Munge, Colleen E. Krause, Ruchika
Malhotra, Vyomesh Patel, J. Silvio Gutkind, and
James F. Rusling, Electrochemical Immunosensors
for Interleukin-6. Comparison of Carbon Nanotube
Forest and Gold Nanoparticle platforms,
Electrochem. Comm., 2009, 11, 10091012
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Off-line capture magnetic particle microfluidic
strategy
A
Conventional, single label
array electrode
Multilabel magnetic Particle, off line capture
B
Electrical contacts
-0.2V H2O2 HQ ? signal
8 electrodes In channel
Capture Antibody
Ab2-enzyme
1 mm multi-enzyme- Magnetic particle-Ab2 7000-400,
000 labels
AuNP
Protein analyte
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8-electrode PDMS microfluidic array
electrodes
Electrical contacts
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Components of microfluidic device made of
micro-machined polymethylmethacrylate), soft
PDMS microfluidic channel screen printed 8
electrode carbon array
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Microfluidic protein assay system
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Microfluidic array detection of PSA in serum
samples
Using off-line capture with 1 ?m multilabel
magnetic particle Detection limit 100 fg/mL PSA
in 10 ?L serum 5-fold better than manual assay
without off-line capture
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Microfluidic array data for mixture of PSA and
IL-6 in serum
Flow rate 100 µL/min H2O2 100 µM Hydroquinone
1 mM
Limit of detection of PSA 225 fg/mL Limit of
detection of IL6 300 fg/mL
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Low cross reactivity of PSA and IL-6
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Immunosensor assay validation on human serum
PSA
IL-6
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Serum Protein Biomarkers for Oral Cancer

• Interleukin-6 IL-6
• Interleukin-8 IL-8
• Vascular Endothelial Growth Factor VEGF
• Vascular Endothelial Growth Factor C VEGF-C

Trikha, M. Corringham, R. Klein, B. Rossi, J.
Clin. Cancer Res. 2003, 9, 4653-4665 Hebert, C.
A. Baker, J. B. Cancer Invest. 1993, 11,
743-750 O-charoenrat, O. Rhys-Evans, P. Eccles,
S. A. Cancer 2001, 92, 556-568

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Microfluidic Immunoarray Oral Cancer Biomarkers

Off-line capture using magnetic particles with
400,000 HRPs
IL-6 DL 10 fg mL-1
IL-8 DL 15 fg mL-1
VEGF-C DL 60 fg mL-1
VEGF DL 8 fg mL-1
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Protein Array using RuBPY ECL label Immunoassays
in 10 mL wells
Carbon (PG) chip (no microelectronics)
SWCNT forest
ECL Electrochemiluminescence
Detection labels are 100 nm d. silica with
internal RuBPY
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Forster and Voss, 1980s (synthesis)
RuPVP
Solid PG chip
Spots contain capture antibody on RuPVP
ECL polymer
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ECL arrays for detection of PSA and IL-6
DL 0.1 pg/mL
2 ng/mL 0.2 ng/mL 0.1 pg/mL 0
Control
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Surface plasmon resonance (SPR) detection of
protein biomarkers using superparamagnetic beads
labels
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SPR response to PSA in Serum with Magnetic and
Silica labels
Using off-line capture with 1 ?m magnetic
particle label Detection limit 10 fg/mL PSA
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Ultrasensitive multiple protein arrays
Combining nanostructured sensors or SPR with
(multi-label) magnetic particles gives ultrahigh
sensitivity in fg/mL range Microfluidics with
off-line analyte protein capture gives very low
S/N, semiautomated May open door to new
ultralow abundance biomarkers ECL provides
simpler array for protein detection, no
microelectronic chip needed
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Cancer Biomaker Protein Measurements as yet,
limited POC or clinical use except for PSA
ELISA, commercial kits, one protein, 3 pg/mL DL
bead based methods, up to 10 proteins, equip. and
kits expensive, 1-10 pg/mL DLs LC-MS, great for
discovery, emerging for routine tests new
experimental methods promise ultrasensitivity,
detection in fg/mL range, multiplexing -
microfluidic amperometric arrays, multilabel -
ECL arrays, simplicity - SPR arrays with
magnetic particle labels - fiber optic
microwell arrays D. Walt (Tufts) - DNA label
bar-codes - C. Mirkin (Northwestern)