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Proteomics have the potential to transform biomarker discovery, diagnostics, and personalized medicine as it provides functional and actionable information linked to disease phenotypes. However, mass spectrometry (MS) – based proteomics has not been possible at large scale due to limited dynamic range, complex workflow and high experimental cost per sample. Antibody-based methods, although widely used in targeted proteomics for their better sensitivity, have been limited to the profiling of a few hundred selected proteins, partly due to the limited availability of antibody pairs. NanoMosaic has demonstrated antibody-aptamer sandwich assays on the MosaicNeedle࣪ platform. This expands the content size from a few hundred to a few thousand, achievable in one reaction with high sensitivity and specificity and low cross-reactivity. MosaicNeedles࣪ are densely integrated nanoneedle sensors fabricated on a planar substrate. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can also be grouped into 96, 384 or 1536 formats. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture antibodies or aptamers. At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. We have demonstrated that the aptamer-antibody assay on the MosaicNeedle࣪ platform achieves better sensitivity than the well-developed antibody-pair assay. It also achieves larger dynamic range and alleviates the antibody cross-reactivity by refining aptamers during the SELEX process. The scalability in manufacturing nanoneedles combined with the low sample requirement and no fluorescent labeling all help to drive down the cost-per-protein to enable large-scale, unbiased proteomics. We envision researchers will use MosaicNeedle࣪ platform for both the upstream target discovery phase and then transfer seamlessly to the downstream validation phase by building customized and targeted panels for proteins of high interest that emerge from the discovery phase. Citation Format: Qimin Quan, Alaina Kaiser, Joshua Ritchey, Joe Wilkinson, John Geanacopoulos, John Boyce. MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3919. |
