Dual-Color, Multiplex Analysis of Protein Microarrays for Precision Medicine.

Generating molecular information in a clinically relevant time frame is the first hurdle to truly integrating precision medicine in health care. Reverse phase protein microarrays are being utilized in clinical trials for quantifying posttranslationally modified signal transduction proteins and cellular signaling pathways, allowing direct comparison of the activation state of proteins from multiple specimens, or individual patient specimens, within the same array. This technology provides diagnostic and therapeutic information critical to precision medicine. To enhance accessibility of this technology, two hurdles must be overcome: data normalization and data acquisition. Herein we describe an unamplified, dual-color signal detection methodology for reverse phase protein microarrays that allows multiplex, within spot data normalization, reduces data acquisition time, simplifies automated spot detection, and provides a stable signal output. This method utilizes Quantum Nanocrystal fluorophore labels (Qdot) substituted for organic fluorophores coupled with an imager (ArrayCAM) that captures images of the microarray rather than sequentially scanning the array. Streamlining and standardizing the data analysis steps with ArrayCAM high-resolution, dual mode chromogenic/fluorescent array imaging overcomes the data acquisition hurdle. The spot location and analysis algorithm provides certain parameter settings that can be tailored to the particular microarray type (fluorescent vs. colorimetric), resulting in greater than 99 % spot location sensitivity. The described method demonstrates equivalent sensitivity for a non-amplified Qdot immunoassay when using automated vs. manual immunostaining procedures.

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.