Evaluation of quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays.

Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners.

Photoaptamer arrays applied to multiplexed proteomic analysis.

Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.

Processing of photoaptamer microarrays.

Photoaptamers are single-stranded nucleic acids selected for their high affinity to specific proteins of interest. Photoaptamer microarrays capture and quantify proteins from complex samples using a unique protocol that leverages both high-affinity capture with covalent retention of analytes. The initial capture of proteins from solution is similar to the well-known antibody capture, but the "secondary binding event" affected by photoaptamers is a covalent crosslink between the photoaptamer capture agent and the protein analyte. The nature of this specific covalent reaction allows a unique microarray processing that is described in detail in this chapter.