The devices were tested for their frequency response: Uncoated

1. l/2 (wavelength) Ta2O5 SiO2 Ta2O5 Electrodes BAW Development of a Simple Inexpensive Bulk Acoustic Wave (BAW) Nanosensor for Cancer Biomarkers: Detection of Secreted Sonic Hedgehog from Prostate Cancer Cells Christopher Corso1, Anthony Dickherber2, Payal Shah3, Alexandra Migdal3, Milton W. Datta3, Sumana Datta3, and William Hunt2 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 Abstract #8866 • Methodology (Continued) • BAW Array Fabrication and Testing • The metallic electrode arrays were fabricated on a 3-layer, piezoelectric Ta2O5 and SiO2 stack on a Si wafer by RF Sputtering Abstract 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. 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.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. 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. 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 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. • The devices were tested for their frequency response: • 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. • Results • 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. Injection Start Injection End Freq. Shift • 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. Principle of a piezoelectric acoustic wave immunosensor Governing equations Target input Molecular recognition Surface perturbation Output quantity (electrical) • 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] Bio-molecules, chemicals Immuno-reaction (binding events) Frequency, phase, V, I, etc. Surface property changes (mass, stiffness, etc.) Hunt equation [2] Non-specific target AT-cut quartz plate (0.17mm thick) ρ=mass density, μ=stiffness, f= frequency, Δm: mass loading, hf: film thickness, A: sensing area, μq and ρq : shear stiffness and density of the quartz crystal Specific target (antigen) 13.67 mm (Not drawn to scale) Gold electrode (100 nm thick) 5.1 mm Antibody • 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. • The wavelength () is fixed by lithography process. Cross-linker Δρ Δμ Δ f Quartz Crystal Microbalance Device surface Surface property change Frequency shift • Methodology • 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 • 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). Freq. Shift • 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. • 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.