Transcript Slide 1

Development of a Simple Inexpensive Bulk Acoustic Wave (BAW)
Nanosensor for Cancer Biomarkers: Detection of Secreted Sonic Hedgehog
from Prostate Cancer Cells
Christopher
1
Corso , Anthony
2
Dickherber ,
3
Shah , Alexandra
Payal
3
3
2
Datta , Sumana Datta , and William Hunt
3
Migdal ,
Milton W.
1Department
of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332
2Department of Elecrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA 30332
3Departments of Pathology and Urology, Winship Cancer Institute, Emory University, Atlanta, GA 30322
Methodology (Continued)
Principle of a piezoelectric acoustic wave immunosensor
QCM Detection Plot
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0
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Injection Start
Injection End
Freq.
Shift
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2400
2600
2800
Time (sec)
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ELISA Testing
• ELISA plates were coated with anti-SHH antibody (clone N-19) at 1:1,000 in PBS. Either purified SHH diluted in
PBS or conditioned medium was incubated, and subsequently the plated were washed. A second anti-SHH
antibody (clone H-160) was added at 1:20 and incubated and subsequently washed. A final anti-mouse HRP
antibody was added and the plate developed after color activation. Plates were subsequently read for absorbance
(ABS).
DB(|S[1,1]|)
Coated With Antibodies
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4.
0
4.0
5.0
10.0
10.0
2
Average Negative Frequency Shift
(MHz)
0.6
0.4
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•The Anti-SHH devices showed an average
frequency shift of 1.27 MHz (n=32) or approx. 0.3%
of the average resonant frequency of 405 MHz
•The Anti-FITC devices showed an avg. frequency
shift of 0.48 MHz (n=24) which is approx. 0.12% of
the avg. resonant frequency.
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0.8
0
5.0
DB(|S[1,1]|)
After 5ul LNCap
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0.305
3.
3.0
DB(|S[1,1]|)
Uncoated
1
0
S[1,1]
After 5ul LNCap
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0
S[1,1]
Coated With Antibodies
Freq. Shift
SHH Sensor
FITC Sensor
0.315
0.325
0.335
Frequency (GHz)
0.345
0.35
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QCM Testing (Proof of concept)
• A reference sensor was coated with Anti-FITC antibodies (5 µl/ml) using a Self-Assembled Monolayer (SAM) as a
cross-linking mechanism. The target sensor was coated with anti-SHH (5 µl/ml) antibodies.
• The sensors were driven at their resonant frequency while LNCap conditioned medium was injected at a flow rate
of 0.2 ml/min. The injection was stopped and the volume of 70 µl of conditioned medium was static in the flow
chamber incident with the two sensors. A buffer wash provided the washing of unbound non-specific particles
from the surface.
• The transient resonant responses were collected and analyzed
S[1,1]
Uncoated
2.
0
1.2
Swp Max
0.95GHz
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Methodology
Average Sensor Frequency Shifts In Response to LNCap Conditioned Medium
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Resonator Smith Chart
Device Frequency Response
1.0
Frequency shift
1.0
Surface property
change
Δf
-1.0
Device surface
Δρ
Δμ
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Quartz Crystal Microbalance
V


V
f 

0.8
Crosslinker
• The BAW Array design and fabrication was successful and the array is pictured
below. The average frequency shift for SHH sensors is 1.27 MHz as compared to a
shift of 0.48 MHz of the reference sensor. The results were collected from 56
separate devices. The SHH-based change of 0.8 MHz is an approximately 10,000
fold increase in sensor sensitivity.
0.6
• The wavelength () is fixed by lithography process.
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(Not drawn to scale)
Antibody
6
5.1 mm
0.
Gold electrode
(100 nm thick)
shear stiffness and density of the quartz crystal
0.4
13.67 mm
ρ=mass density, μ=stiffness, f= frequency, Δm: mass
loading, hf: film thickness, A: sensing area, μq and ρq :
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Specific target
(antigen)
Hunt equation [2]
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2 fu2 h f 
 
f  
   2 
V 
 q q 
Non-specific
target
AT-cut quartz plate
(0.17mm thick)
A   
BAW
• The QCM sensor detection (80-Hz change to detect less than 200 picograms SHH
protein) demonstrates the sensitivity of the system. While non-specific binding
could compound the results, a control FITC antibody was used to subtract
background binding. Subsequent array studies using multiple different SHH
antibodies will allow us to confirm the specificity of the frequency shifts and
calculate more accurate sensitivities.
Sauerbrey equation [1]
0.2
Frequency, phase, V, I, etc.
f 
2  f 02  m
0
Surface property changes
(mass, stiffness, etc.)
Governing equations
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Output quantity
(electrical)
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Immuno-reaction
(binding events)
Surface
perturbation
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Bio-molecules,
chemicals
Molecular
recognition
• The QCM-SHH biosensors were capable of detecting SHH in undiluted conditioned
medium in a repeatable manner (n=5). In the undiluted samples, the average
resonant frequency shift was 80 Hz for a conditioned medium sample of 70
microliters.
• ELISA assays for purified SHH demonstrated a detection sensitivity to less than
0.2 ng of purified SHH (blue curve). When SHH was added to conditioned
medium detection was reduced, but ELISA assays detected less than 0.3 ng. Of
note, when the LNCAP conditioned medium used in the BAW studies was assayed,
the SHH was noted to be present at less than 0.2 ng/70 microliter sample, with a
detected total concentration of less than 6 ng SHH for the conditioned medium
used in the sensor studies.
0
Target
input
Electrodes
Results
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Conclusions: Here we propose the first use of a bulk acoustic wave sensor chip for the detection of a
cancer biomarker in complex media. We have created an inexpensive BAW sensor chip whose
production cost is a few cents per chip. These chips can be rapidly and efficiently conjugated to
antibodies and used for the detection of circulating antigens in complex solutions such as blood or
serum. The proof of concept was shown with a quartz crystal microbalance device. This is
demonstrated through the conjugation of anti-SHH antibodies and the subsequent detection of SHH in
conditioned medium from prostate cancer cells. While the sensitivity of the quartz crystal microbalance
is high (1x10-9 g per 1 Hz shift), theoretical calculations show that the bulk acoustic wave sensor chips
will have a much higher sensitivity than the QCM devices (1x10-15 g per 1 Hz shift). Additionally, the
array formation of our devices allows for immediate and efficient repeatability of the test as well as the
possibility for statistical analysis of the results. The ability to detect low levels of Sonic Hedgehog in
serum, whether combined with or independent of serum PSA levels, could be used to diagnose
aggressive prostate cancers or monitor response to treatment.
Ta2O5
SiO2
Ta2O5
(wavelength)
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Results: SHH antibodies were efficiently coupled to the BAW sensor through a self-assembled
monolayer (SAM) alkane-thiol crosslinker. This immobilization procedure is a simple 7-step process
that takes less than 10 hours. The QCM-SHH biosensors were capable of detecting SHH in undiluted
conditioned medium in a repeatable manner. Sensor reaction curves were notable for detection above
the noise background for the undiluted sample and will be further tested with serially diluted samples
until the detection limit is reached. In the undiluted samples, the average resonant frequency shift was
80 Hz which corresponds to roughly 100 ng of SHH bound to the device.
• The metallic electrode arrays were fabricated on a 3-layer, piezoelectric Ta2O5 and SiO2 stack
on a Si wafer by RF Sputtering
• The devices were tested for their frequency response:
/2
• Uncoated
• Coated only with antibodies Anti-SHH and Anti-FITC (pre-LNCap conditioned
medium exposure)
• After being exposed to 5 ul of LNCap conditioned medium for 20 minutes
followed by a buffer wash.
• The frequency responses at each stage were recorded and analyzed.
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Methods: We have developed a standardized BAW sensor chip platform that can be used as a
biosensor in complex solutions such as blood or serum. While it is not yet ready for testing, progress
towards the finished sensor chip is moving rapidly and a testable product is expected soon. A Quartz
Crystal Microbalance (QCM) is a BAW device with an identical mode of physical operation to our chip,
and has been implemented by us to prove the viability of the approach. The QCM platform was
conjugated with anti-Sonic Hedgehog antibodies, and the resulting BAW-SHH biosensor was used to
assay for the presence of SHH in conditioned medium from LNCaP prostate cancer cells. Resultant
sensitivities and detection range were calculated for the BAW-SHH biosensor.
Acoustic wave device technology and standard
photolithographic processes can be employed to
produce small inexpensive sensors as disposable
assay platforms for cancer biomarkers. We have
developed a Bulk Acoustic Wave (BAW)
piezoelectric sensor chip containing an array of 8
independent sensors that can be conjugated with
antibodies and used as a biosensor in complex
solutions such as serum or blood. Advanced
prostate cancers have increased levels of Sonic
Hedgehog (SHH) protein production and signaling.
Therapeutics that target the SHH pathway have
been shown to stop prostate cancer cell
proliferation. A biosensor that could detect SHH
could be used towards prostate cancer diagnosis
and treatment.
BAW Array Fabrication and Testing
0.2
Introduction: Acoustic wave device technology and standard photolithographic processes can be
employed to produce small inexpensive sensors as disposable assay platforms for cancer
biomarkers. We are developing a Bulk Acoustic Wave (BAW) sensor chip containing 16 independent
sensors that can be conjugated with antibodies and used as a biosensor in complex solutions such as
serum or blood. Advanced prostate cancers have increased levels of Sonic Hedgehog (SHH)
production and signaling, and therapeutics that target the SHH pathway stop prostate cancer cell
proliferation. A biosensor that could detect SHH could be used in prostate cancer diagnosis and
treatment.
Differential Frequency Shift (Hz)
Introduction
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Abstract
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Abstract #8866
Swp Min
0.245GHz
Conclusions
• Here we demonstrate the first use of a bulk acoustic wave sensor chip for the detection of a cancer biomarker in
complex media. We have created an easily fabricated, inexpensive BAW sensor chip whose production cost is a
few cents per chip. These chips can be rapidly and efficiently conjugated to antibodies and used for the detection
of circulating antigens in complex solutions such as blood or serum. The array formation of our devices allows
for immediate and efficient repeatability of the test as well as the possibility for statistical analysis of the results.
The ability to detect low levels of sonic hedgehog in serum, whether combined with or independent of serum PSA
levels, could be used to diagnose aggressive prostate cancers or monitor response to treatment.