An interesting paper in (Analytical Chemistry) describes the use of confocal fluorescence section imaging to analyze protein distribution in microarray spots on nitrocellulose film slides. By scanning and imaging through the Z dimension of a printed spot of fluor labeled protein, a 3-dimensional quantitation of fluorophore density in the films was obtained, showing that commercial nitrocellulose slides from different sources have very different behaviors towards materials applied to their surfaces.
In the study, 3 commercially sourced Nitrocellulose microarray slides were tested: GE Whatman FAST, Sartorius-Stedim Biotech Unisart, and Grace Bio-Labs ONCYTE AVID. Of the 3 types tested, 2 (FAST and Unisart) were measured to have a distribution of biomolecules that demonstrated higher protein density in the outer region of absorption, while one type (ONCYTE AVID) showed a more homogenous distribution throughout the measurement area, suggesting that the interaction of liquid with the membrane is highly dependent on the formulation and chemistry of the nitrocellulose membrane (Figure 1).
Figure 1. (A) CLSM images of IgG-FITC spots on various NC membrane slides. (B) “Z” stack data for distribution of IgG-FITC spot down into various NC membrane slides. (C) CLSM images of BSA-Alexa spots on various NC membrane slides. (D) “Z” stack data for distribution of BSAAlexa spot down into various NC membrane slides (reprinted with permission from Ref 1. Copyright 2013 American Chemical Society).
Since nitrocellulose slides are marketed as having permeable 3-D matrices providing a scaffold for printed biomolecules to adhere, one might expect that proteins arrayed in a small spot would “soak in” and distribute homogenously through the matrix, but that is apparently not the case. Rather, different film slides show different distributions throughout the “cylinder of analysis”, suggesting that the nitrocellulose formulation drives the way the sample is absorbed into the matrix. The end result can have important effects on the actual assay being performed, as homogenous distribution would be expected to provide more consistent (lower variability) signals than an un-evenly distributed capture analyte.
Figure 2. Change in contact angle monitored by a “side-view” HSC for a droplet of IgG-FITC in PBS (pH7.4), (reprinted with permission from Ref 1. Copyright 2013 American Chemical Society).
Evidence for the mechanical action of the biomolecule distribution is presented by an analysis of droplet absorption into membranes executed by taking a series of High Speed Camera side view images of liquid drops on membranes in the seconds following deposition (Figure 2). These pictures show very different behaviors by membrane types, with an apparent correlation between maintenance of contact angle during absorption and homogenous distribution through the membrane. This is supported by other studies that show a spreading of droplets in contact with a surface (decreasing contact angle) forces movement of biomolecules in solution towards the air/liquid/surface interface, resulting in heterogeneous biomolecule concentrations in a drying spot, and development of irregular material deposition on the surface (the “doughnut effect”)2,3. In contrast, if a formulation of a surface membrane is such that spots don’t spread (ie contact angle remains high) biomolecule concentration in the drying spot remains relatively homogenous, resulting in more even distribution through the membrane. The ONCYTE AVID slides’ contact angle remained comparatively high during the absorption period, consistent with the observation that the protein was more evenly distributed in this formulation.
Another factor at play that is mentioned but not addressed is the effect of print buffer components on spot drying and biomolecule distribution. Various additives to printing solutions, such as detergents, polymers, salts, or neutral proteins can alter the liquid/surface interaction and change the rate at which water evaporates, causing dramatic differences in the way microarray spots deposit content onto a surface or into a nitrocellulose membrane. Further, these components can have a very large effect on the capture efficiency of bound biomolecular ligands. All these factors add up the conclusion that there is no “one size fits all” solution to protein microarray development, and researchers must carefully examine and test all components of their assay systems to determine the optimally performing methods for their applications.
Additional information on how to select the optimal nitrocellulose substrate for your application can be found in our Protein Microarray Slide Selection guide. Please feel free to contact a Grace BioLabs technical support representative for any microarray assay optimization or reagent support at any of our contact options.
1) Mujawar, L.H., Maan, A.A., Khan, M.K.I., Norde, W., van Amerongen, A., Anal. Chem. 2013, 85, 3723-3729.
2) Ressine, A.; Marko-Varga, G.; Laurell, T.; El-Gewely, M. R. In Biotechnology Annual Review; Elsevier: New York, 2007; Vol. 13, pp 149−200.
3) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827−829.