Abstract 4865: Miniaturized multiplexed highly sensitive protein NanoArray assay for small volume serum and tumor samples
Authors: Gubbins Earl , Amro Nabil A. , Sanedrin Raymond G. , Rozhok Sergey V. , Coen Michael , Delaney Deany , Ouyang YongJie , Ivanov Ivan D. , Aguilera Beatrice , Jamil Haris 
Published: June 2012

Abstract
Advanced development of array technology is needed for ultrasensitive detection of biomarkers in low abundance or small volume samples. Such advanced assays may be essential for understanding and possibly early detection of diseases. Most diseases are easier to treat and control when detected at an early stage of disease progression.
Herein, we report a newly developed ultra-sensitive assay platform based on dip-pen nanolithography (DPN™) using the NLP2000™. This instrument can generate micron- to nano-scale sized features with multiple proteins on a variety surfaces. The proteins are spotted using atomic force microscopy tips in the form of arrays over large areas with exceptional nanometer resolution and accuracy in mass production. This printing technique offers significant advantages over current microarray printing technologies, which suffer from poor spot-to-spot reproducibility in terms of size, shape, and density. Recent patterning experiments of antibodies, as well as screening for their biological activity, yielded high-quality results with excellent uniformity and repeatability of features within and between arrays, allowing for better quantification. Our nano-spots occupy an area approximately 100 times smaller than spots in a conventional microarray, while still generating high signals and low background.
We have developed ultrasensitive nano-scale sandwich ELISA assays for the detection of a variety of cancer biomarkers for angiogenesis and inflammation. Specific antibodies were used to capture their corresponding antigens, which were subsequently bound to specific biotinylated secondary antibodies. Fluorescently labeled streptavidin was used as the detection molecule in conjunction with a high resolution fluorescent scanner (resolution of 0.5 microns). These protein nanoarrays exhibited improved detection levels over conventional microarray technologies in standard and HTS format of 18, 48 and 96 subarrays requiring 2-4 µl of sample with improved sensitivity to detect biomarkers in the 100's of femtograms/ml. This assay platform can be applied to tumor markers or antigens from a variety of other diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. This assay is not limited to testing only serum, but also for detecting biomarkers in small samples such as tissue, saliva, tears or cerebrospinal fluid. We believe that highly sensitive multiplex protein array assays can pave the way for applications such as point-of-care clinical diagnostics as well as drug discovery and drug development.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4865. doi:10.1158/1538-7445.AM2011-4865