Protein microarray technology has progressed significantly and, although new microarray surface chemistries continue to be developed and marketed, porous nitrocellulose has remained the predominant immobilization surface for protein
microarray applications due to its many functional advantages. The 3-dimensional structure of nitrocellulose film slides offers a considerably higher surface area for protein binding compared to conventional 2-dimensional surfaces.Increased surface area for binding translates into increased binding capacity with up to 500 times the binding capacity of conventional 2-dimensional surfaces and is related to pore size, pore structure, pore density and film thickness. This increased binding capacity translates to increased microarray assay sensitivity. Additionally, due to the non-covalent yet robust interaction of proteins with this surface retention of proteins’ native conformation and biological activity is promoted. Grace Bio-Labs’ ONCYTE® AVID, NOVA, and SuperNOVA microporous nitrocellulose film slides are manufactured to increase protein microarray assay sensitivity and dynamic range.
Figure 1. Porous Nitrocellulose Structure Allows for Increased Surface Area for Protein Binding
Figure 1. Figure 1. Right: SEM of an ONCYTE AVID nitrocellulose slide showing the microporous nature of this 3-D matrix. Left: A 3-D rendition of the microporous surface of an AVID slide conceptualizing the interactions between a spotted protein and the binding surface. Due to the heterogeneity of the nitrocellulose surface, surface topography allow for 3-dimesional binding of proteins promoting the retention of proteins native conformation.
Figure 2. Dynamic Range of Protein Binding Compared to 2-D Surfaces
Figure 2. (A) Dynamic range of protein binding of ONCYTE film slides (Blue line) ranges nearly 7 orders magnitude compared to two conventional 2-dimentional surfaces (Red and Purple lines) which typically are truncated at 4 – 5 orders of magnitude. Data are normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3 (N = 4 film slides per slide type, 20 technical replicates per IgG concentration per slide) (B) Increased binding capacity of ONCYTE microporous film slides translates to better assay sensitivity over conventional 2-D surfaces and even over other 3-D surfaces. Right: Colorimetric data are normalized and background-subtracted, collected after HRP/AEC staining from spotted goat IgG assayed with anti-goat IgG on ONCYTE SuperNOVA slides compared to a competing 3-D surface. Right: Fluorescence data are normalized and background-subtracted, collected at 532 nm from spotted rabbit serum assayed with goat anti-rabbit IgG on ONCYTE SuperNOVA slides compared to a competing 3-D surface. All data obtained from spotting concentrations within the linear dynamic range of detection methods employed (N = 4 film slides per slide type, 20 technical replicates per slide).
