Previously, we have reported data from multiplexed protein microarray assays with quantum dots which show great promise for use of these labels with nitrocellulose film-slides. The use of quantum dots with emission in the red and NIR spectrum allow for sensitive detection on nitrocellulose film slides due to low inherent fluorescence background at longer wavelengths.There have been concerns expressed in the scientific community about the ability of these molecules to diffuse efficiently through the porous nitrocellulose structure due to their larger size compared to organic fluorophores. Additional concerns with early versions of quantum dot nanocrystals were that they varied in size (~ 10 – 20 nm diameters) depending on the emission wavelength, raising the question of steric hindrance and competition in multiplexed assays. Newer quantum dot chemistries (VIVID® Qdot® Series, Life Technologies) are more uniform in size as well as emission intensity, minimizing the concerns over binding competition and promising better performance for comparative expression studies.
We report here experiments to address the performance of quantum dots for protein arrays on nitrocellulose film. Assays were conducted to assess competition for binding to rabbit IgG arrays on SuperNOVA (Grace Bio-Labs) film-slides comparing anti-rabbit IgG labeled with Qdot® 655 or –Qdot®800. At four different ratios of antibody:target, representing variable protein abundance, we saw no difference between singleplex assays compared to duplex assays (where both forms of Qdots were present). These results indicate no significant competition between Qdots of different size, at least for a duplex assay. In addition, we compared binding kinetics of antibody labeled with either Qdot® or Alexa-Fluor and found very similar kinetics (figure 2), indicating that antibodies labeled with the larger quantum dots diffuse as efficiently as fluorophore-conjugated antibodies through the nitrocellulose membrane. Taken together, we see no practical limitations to using quantum dots compared to organic fluorophores for either singleplex or duplex assays on porous nitrocellulose.
Figure 1. Singleplex and Multiplex QDot Assays for rabbit-IgG.
Figure 2. Assay kinetics of anti-rabbit IgG-QDot800 versus anti-rabbit IgG-Alexa647.
Figure 1. Detection of arrayed rabbit IgG on SuperNOVA film-slides with Qdot®655 and Qdot®800 conjugated antibodies. (A) Microarray images from assays at 4 different ratios of Qdot®-labeled antibody versus bound rabbit IgG (1:101, 1:100, 1:10-1, 1:10-2). Rabbit IgG was deposited at either 4000, 400, 40, or 4 pg/spot and incubations were performed with 0.4 nM conjugated antibody in either singleplex (Qdot-655 only or Qdot-800 only) or multiplex configurations (0.4 nM both antibodies). (B) Data are the mean ± S.D. assay signal from 3 replicate arrays (n=9 spots per array) obtained with a GenePix 4400A (Molecular Devices Inc.) with emission filters for detection of 655nm (655WB20) and 800 nm (800WB80).
Figure 2. Timecourse performed at 0.25, 0.5, 1, 2, and 8 hours’ incubation with anti-rabbit IgG-QDot800 or –Alexa647 on arrays spotted with 4000 pg/spot of rabbit IgG. Arrays were probed with 8 nM anti-rabbit IgG conjugate for both fluors, results are from 3 replicate arrays (n=9 spots per array) obtained with a GenePix 4400A (Molecular Devices Inc. ) with emission filters for detection of Alexa647 (675BP20) and QDot800 (800WB80).