Research Program
Biomolecular Devices and Analysis

Project #
BDA16

Participating Faculty:	D. Lin, H. Craighead
NBTC Students/Postdocs:	Steve Rodriguez
Other Students/Postdocs:	Christine Tan, Phil Waggoner

Objectives
We proposed to explore the feasibility of using resonator frequency cantilever sensors as the basis of a next-generation microarray platform. These cantilever arrays would potentially have several advantages over conventional approaches. They would be capable of detecting absolute levels of gene expression, would not require amplification or labeling, and would be more sensitive than existing platforms.

Methods
Cantilevers of different geometries were fabricated using a two-step photolithography. The cantilevers were made of silicon nitride, with a sacrificial layer of silicon oxide that was removed during release of devices. A pad of gold was patterned on top of the nitride, at the tip of each cantilever. Thiol-linked oligonucleotide probes were bath-applied and kinetics of assembly monitored by resonant frequency. Biotin-labeled target cDNA was hybridized and bound product detected using streptavidin-nanoparticles. Scanning electron microscopy and resonant frequency measurements were used to confirm binding of target cDNA to cantilever sensors.

Summary
Our goals for the past year were to: 1) develop means to functionalize probes to cantilever sensors; 2) determine if conventional microarray equipment could be used to print oligonucleotides onto cantilevers; and 3) determine the sensitivity of cantilever sensors in detecting dilute target transcripts. We have made significant progress on all three goals, setting the foundation for future experiments aimed at developing massively parallel cantilever biosensor arrays.

Goal 1: We successfully functionalized thiol-linked oligonucleotide probes onto gold surfaces on our cantilever sensors, and characterized conditions for optimal thiol-probe self-assembly. This is a key first step in order to ensure that bound probe will not be removed during hybridization with target samples or during subsequent washing steps.

Goal 2: We successfully used a conventional microarrayer equipped with quill-type pins to spot oligonucleotide probes onto our cantilevers. Ultimately, the development of optimal printing conditions, including print buffer composition, humidity, dwell-time, and other standard parameters, will enable us to develop arrays of cantilever sensors capable of detecting multiple transcripts in a target sample.

Goal 3: We used as our initial test condition cantilevers functionalized with a probe corresponding to the beta-actin gene. We generated purified sense and anti-sense biotinylated cDNA to use as target. Bound cDNA was detected with streptavidin-conjugated nanoparticles, and visualized with scanning electron microscopy. Few nanoparticles were observed when using anti-sense (non-complementary) cDNA as target. However, significant numbers of nanoparticles were seen with the sense (complementary) cDNA target. Significant shifts in resonant frequency measurements were observed with chips hybridized with the sense-strand target but not with the anti-sense target. For these first experiments, we used a concentration of target cDNA that matched that used in standard microarray experiments. The results indicate that cantilever sensors are at least as sensitive as conventional microarray platforms.

Future experiments will include determining the sensitivity limit of cantilever sensors, and to begin developing cantilever sensor arrays capable of detecting multiple transcripts within a target sample.

Accomplishments

• Functionalization of cantilevers with thiol-linked oligonucleotides
• Detection of bound target cDNA with nanoparticles