Nanotech 2010

Multicomponent Nanoarrays: Platform for High Sensitive Protein Detections

R. Sanedrin, N. Amro, E. Gubbins, J. Ohayon, D. Delaney, S. Rozhok, M. Coen, K. Rusniak, K. Ouyang, I. Ivanov
NanoInk, Inc., US

Keywords: nanoarrays, nanolithography platform

Abstract:

Detection, identification, and quantification of low abundant proteins in human samples have been of great interest because of their role in early detection of certain human diseases. Various protein detection schemes have been developed over the years and recent advances in the development of printed biological arrays allow for the study of interactions between immobilized protein and other biological systems. Dip-pen nanolithography (DPN) has emerged as a powerful tool in creating array features of various biological molecules such as DNA and proteins on a variety of substrates. Recently, DPN has been shown to produce features of printed biological molecules with dimensions ranging from submicron to nanometer scale. Advantages of using DPN for printing biological samples include high resolution and registration of printed features, simultaneous deposition of multiple proteins, reduced required reagent volumes, and a wide variety of usable substrates. Herein, we show how we use an optimized version of DPN to deposit proteins onto a glass substrate and how the printed biological molecules retain high sensitivity toward their respective antigens. Proteins are spotted onto a glass substrate using NanoInk’s Nanolithography Platform (NLP), an innovative custom designed instrument that utilizes an AFM cantilever to deliver molecules of choice to the substrate surface. The NLP is a new spotting technology which generates micron to nanoscale sized features of biological molecules on solid surfaces. The innovative instrument can generate arrays over millimeter-scale areas with nanometer resolution in mass production. This printing technique offers significant advantages over current microarray printing technologies that 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. Each of our nano-spots occupies only a small fraction of the surface area occupied by a single spot in a conventional microarray, while maintaining a sufficient surface area to bind sufficient numbers of antibodies or antigen. As proof-of-concept study, nanoscale features of multiple cytokine antibodies were simultaneously immobilized onto a glass substrate for the detection of cytokines in a sandwich format similar to that of an ELISA (Enzyme Linked Immunosorbent Assay). The immobilized antibodies were used to capture their corresponding antigens and were subsequently exposed to primary and biotinylated secondary antibodies. Fluorescently labeled streptavidin was used as the detecting molecule in conjunction with a high resolution fluorescent scanner. The protein nanoarrays exhibited improved detection levels over conventional microarray technologies with improved sensitivities. We believe that highly sensitive multiplex protein assays can pave the way for applications such as point-of-care clinical diagnostics and biosensors.
 
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