Although major advances have occurred toward understanding the molecular events in various cancers, their translation into therapies has with few exceptions been limited, and early detection remains the cornerstone of successful treatment. Consequently, there is a great deal of interest in identifying sensitive and specific markers for individual cancers, particularly in blood. Two areas of intense investigation are identification and quantification of circulating tumor cells (CTCs), as well as identification of sensitive and specific tumor markers in plasma. We are developing a chip-based RNA sensor platform using derivatized nanowires (NWs) for early detection of CTCs. This sensor platform, which relies on formation of a hybridization """"""""sandwich"""""""", possesses excellent specificity (single nucleotide mismatch discrimination for 2 recognition events) as well as sensitivity (data indicate each single """"""""sandwich"""""""" binding induces a resonance frequency shift of ~ 1 kHz, which is easily measurable). The platform is being constructed in """"""""bottom-up"""""""" fashion, and we have shown that NWs derivatized with antisense oligonucleotides fully retain their functionality after integration on chip. Here we propose to derivatize NWs with modified DNA aptamers, specific for the clinically relevant plasma melanoma tumor markers S100B and MIA, extending the functionality of our chip- based sensor and considerably expanding its versatility while retaining multiplex capabilities and exquisite sensitivity/specificity.
The Specific Aims of the application encompass: 1) Make constructs and express S100B and MIA and purify them in vitro. 2) Perform Aptamer selection using our Primer-Free protocol until ~ 5-10 high-affinity aptamers are identified for each protein, and characterize binding properties. Introduce various modifications to aptamers to enhance stability/nuclease resistance and re-test binding parameters. 3) Perform pair-wise testing of fluorescent """"""""sandwich"""""""" binding of spotted microarrays with purified S100B/MIA to determine the optimal pair of aptamers (Ap1 and Ap2) recognizing distinct epitopes. 4) Use the optimal pairs of Ap1/Ap2 on microarrays to detect various concentrations of S100B and MIA spiked into fractionated control plasma samples, to determine non-specific binding and ~ limits of detection. 5) Derivatize NWs with Ap1, and perform sandwich binding assays with: a) fluorescently-labeled Ap2;and b) Ap2 covalently linked to 50-nm AuNPs, with spiked plasma samples as in 4). 6) Electrofluidically deposit derivatized NWs on chips, perform sandwich binding assays as in 4), and measure shifts in resonance frequency of individual NWs: Benchmark vs. conventional ELISA. 7) Quantify S100B and MIA levels in plasma from melanoma patients using conventional ELISA-based reagents for benchmarking, compare with CTC levels from the same patient samples, and relate to AJCC Stage. Bank the balance of the samples for subsequent testing with the chip-based sensor.
This application, which is focused on detection of tumor markers in blood from melanoma patients, seeks to develop a nanotechnology-based platform with wide-ranging applicability in cancer diagnostics. The methods to be utilized can be applied to any cancer for which suitable markers are available, will be easily adaptable to new markers as they are discovered. The platform is designed to allow simultaneous detection of a number of different tumor markers, and we envision its development as a screening"""""""" tool for early detection of all major cancer types.
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| Cederquist, Kristin B; Keating, Christine D (2010) Hybridization efficiency of molecular beacons bound to gold nanowires: effect of surface coverage and target length. Langmuir 26:18273-80 |
