Multiplexed protein arrays offer significant advantages for diagnostics over monoplex assays such as ELISA’s. For multiplex detection, fluorescence is the label of choice, offering rapid and sensitive detection and large dynamic range in addition to the ability to separate signals from multiple fluors.While porous nitrocellulose (NC) film slides are an excellent substrate for protein arrays due to their high protein binding capacity, they have inherent (auto) fluorescence in the shorter wavelengths (488-594 nm emission), which can contribute to background signal. Therefore we have recently explored options for labeling proteins in the longer (NIR) wavelengths. One option for NIR labels is quantum dots, which are very bright, highly stable fluorescent labels with narrow emission bands and little emission overlap, thus retaining sensitive detection for multiplex assays.
Most scanners for microarrays are tuned to excite organic fluors with excitation wavelengths of 532 and 635. Our investigation of Quantum Dot chemistry presented here utilizes excitation from a 488 nm laser on a GenePix 4400A scanner (Molecular Devices, Sunnyvale, CA). The 488 nm wavelength effectively excites all Quantum Dot nanocrystals, which are available in different species that emit various wavelengths, including far red and infrared emission channels. Here we present data from multiplexed RPPA analysis using the VIVID® series QDot®655- and QDot®800-labeled secondary antibodies (Life Technologies, Carlsbad, CA). The data show effective multiplexing with no significant difference in signal generated with RPPA assays for Akt and P-EGFR (Y1173) performed in monoplex or multiplex fashion (Figure 1 and 2) or detectable cross-talk between the channels.
Our results show great promise for use of quantum dots with emission in the NIR spectrum for sensitive detection of proteins in multiplex assays on nitrocellulose film slides.
Singleplex and Multiplex QDot RPPA Assays for Detection of P-Akt and P-EGFR
Figure 1. Detection of Akt and P-EGFR (Y1173) from increasing depositions of cell lysates (1 to 10 depositions, 400 pL each, ~500 µg/ml protein concentration) assayed under singleplex and multiplex conditions. Cell lysates included are CL1 from A431 cells and CL2 from A431 cells treated with hEGF. Cell lysates were deposited on SuperNOVA Film-Slides and blocked with Super G Blocking Buffer. Incubations were performed with mouse anti-P-EGFR and rabbit anti-Akt (individually or simultaneously) followed by detection with anti-mouseIgG-QDot®800 and anti-rabbitIgG-QDot®655 (individually or simultaneously). Data are the mean ± S.D. assay signal from replicate arrays (n=4) obtained with a GenePix 4400A (Molecular Devices, Sunnyvael, CA) with emission filters for detection of QDot655 and QDot800.
Fluorescence detection of RPPAs with QDot655 and QDot800
Figure 2. Representative scans of RPPAs after detection for p-EGFR (Y1173) with QDot800 and Akt with QDot655 described in Figure 1. Top two panel rows show detection of either biomarker after singleplex assays and the bottom panel row shows images for detection of both markers simultaneously in the multiplexed assay.