Building on the well-established ability of nitrocellulose to bind macromolecules in gel transfer applications, Grace Bio-Labs invented a method to cast thin porous nitrocellulose films directly onto glass microscope slides. These “Film-Slides” or “Films” were initially designed for a new platform for immunocytochemistry, called Cytocoherent Transfer (1, 2). Since that time, Film-Slides have become an established surface for protein microarrays. Due to the unique physiochemical properties of these Films they are well suited for protein microarrays. Specifically, they bind orders of magnitude more protein than 2D surfaces (functionalized glass or hydrogels), they bind protein by weak intermolecular forces rather than covalent attachment, thereby preserving the authentic structure of bound proteins (3) ; and, the crystalline porous structure of these Films creates a coherent backscatter of light that augments sensitivity for fluorescence detection. (4).
De Facto realization of these unique Film attributes however, requires the use of specialized reagents and procedures that don’t always translate well from 2D surfaces (5). Recent developments in Film-Slide technology enhances their value in antibody capture (ELISA) and antigen capture (RPPA) protein microarrays, and also extends these assays into new applications. We review here recent methods and new instrumentation to improve fluorescent assay sensitivity on Film-Slides. Subsequent topics will focus on new spotting and blocking reagents specialized for Film applications, a new reagent for preserving immunogenicity of labile proteins on Films long-term, and new casting methods that allow deposition of nitrocellulose in miniature configurations especially suited for low volume flow-type assays and electrochemical detection.
Improved Fluorescence Detection on Films
Of the various methods that can be used to detect proteins bound to Film, fluorescence is often the method of choice. Fluorescent assays are faster than other detection methods and multiple fluor colors enable multiplexing, which delivers more data per sample. A number of fluorescent dyes with blue, yellow, green and red fluorescence emission spectra are commercially available and relatively inexpensive ( 6, 7). Instruments that are highly tuned to the excitation/emission bands of visible fluorophores are widely used to scan arrays, and accompanying software provides qualitative and quantitative data (8). The cost of these instruments can be a barrier to entry for some researchers interested in casual use of arrays, and so colorimetric assays may be conducted using standard scanners used for print documents.
Nitrocellulose has an inherent (auto) fluorescence, particularly at shorter wavelengths in the green/yellow spectra, which to some degree compromises the use of Film Slides in fluorescence applications. To address this issue, Grace has developed SuperNOVA Film with 1/3 the autofluorescence of other Film slides in the visible spectrum. The SuperNOVA formula from Grace provides unsurpassed signal-to-noise ratios in fluorescence applications when optimized reagents are employed (10).
Two observations promise improved performance of Film for fluorescent applications. First, inherent autofluorescence is extremely low when using (NIR) excitation and emission wavelengths in the range of 600nm to 900nm. Second, as mentioned earlier, signal amplification through coherent backscatter leads to highly sensitive detection of labeled protein. With these observations in mind, Grace Bio-Labs is developing new technology that can significantly improve results with protein microarrays.
Fluors with emission spectra in the NIR range are available as both organic dyes (11) and in the form of nanocrystal technology such as Quantum Dots (12, 13). These nanocrystals have several advantages over organic dyes: they are much brighter; they have a relatively broad excitation curve (several hundreds of nanometers) but narrow emission curves, facilitating multiplexed analysis with sensitive detection. In addition, quantum dots are highly stable, so do not saturate or “bleach” with increasing excitation light intensity. The bleaching characteristic (14, 15) of conventional fluorophore dyes limits the signal amplification effect in porous Film, while quantum dots offer full advantage of this effect.
Most commercially available fluorescent scanners are tuned to fluorescent dyes in the green and red spectra, as these are the most commonly used dyes for microarrays. Two scanners that are tuned to NIR wavelengths are also available (Innopsys’ Innoscan and LiCor’s Odyssey). In our experience, the Innopsys scanner has several advantages over the LiCor related to speed and resolution. Both instruments are relatively expensive with excitation and emission parameters designed for conventional fluorophores. At present, no scanner for protein arrays is designed to take full advantage of the performance value of quantum dots.
Grace is currently developing an inexpensive, compact fluorescence imager (patent pending) that is specifically tuned for quantum dot nanocrystal excitation. This instrument provides the advantage of multiplex signal detection using NIR fluorescence on Film-based arrays with a dynamic range and sensitivity comparable and in some cases exceeding that of other scanners. The combination of a Film platform for high protein binding, optimized reagents and more sensitive detection will push performance of protein arrays to new levels for both the bench scientist and point-of-care diagnostics.