Beating Background Fluorescence on Nitrocellulose Film-Slides

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Near Infrared Detection with Porous Nitrocellulose Films Improves Sensitivity for Protein Arrays.

Despite some clear performance advantages of porous nitrocellulose Film-Slides for protein arrays, one objection to their use is the inherent fluorescence background detected on most scanners when using popular organic dyes with visible emission wavelengths (500-650 nm). However, fluorescence detection in near infrared (NIR) wavelength (>650 nm) nearly eliminates the inherent fluorescence from porous nitrocellulose, and background levels approach that of glass (Figure 1).

nitrocellulose fluorescence detection in near infrared (NIR) wavelength Though NIR dyes have been available for some time, until now available scanners were not tuned to optimally detect the appropriate wavelengths.

We recently compared results of reverse phase protein arrays on porous nitrocellulose with detection in the visible (635nm) and near infrared (670nm and 785nm) wavelengths using the GenePix® 4400 (Molecular Devices) or the Innoscan 710-IR (Innopsys). At scanner settings giving comparable fluorescent signal from all fluorophores either on an Innoscan710-IR or a GenePix® 4400, signal-to-noise in the near-infrared was 1.5-3 times higher than in the visible range, results driven by reduced background fluorescence. 

Experiments and Results:

Serial dilutions of cell lysates were  printed on ONCYTE® SuperNOVA Film-Slides probed for phosho-EGFR (Y1173) and assayed under identical conditions with either Alexa-Fluor®647 (Life Technologies), IRDye®680, or IRDye®800 (LI-COR Biosciences) (Figure 2). Scanner settings were set to obtain comparable signal with all scanner/dye combinations.  Results show that the fluorescence background detected by the InnoScan in the NIR range was 2.5-3-fold lower than with the GenePix at 635nm (Figure 2B), and the resulting signal-to-noise ratio for Alexa-Fluor®647 was higher in the InnoScan compared to in the GenePix (Figure 3). Note that the InnoScan 710-IR showed superior detection of the 647 dye  in spite of using a 670nm excitation laser. Thus the InnoScan 710-IR shows great performance  for detection of all  three dyes tested with a two-laser system.

 

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