Abstract
The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.
Highlights
Microarray technology has emerged as an essential tool for monitoring multiple biomolecular interactions between oligonucleotides, cDNA, proteins, or antibodies immobilized on the surface and their complementary targets in solution [1,2,3]
We have introduced a methodology for checking the quality of the spots on the microarray chips
We have combined a dynamic label-free binding assay with a fluorescence test to investigate the optimal concentration of each bound probe, which results in an optimal fluorescence detection, in terms of specificity, sensitivity and accuracy
Summary
Microarray technology has emerged as an essential tool for monitoring multiple biomolecular interactions between oligonucleotides, cDNA, proteins, or antibodies immobilized on the surface and their complementary targets in solution [1,2,3]. The absence of consistent quality control exacerbates the effects resulting from the variability of print quality. When multiple probes are spotted on the same surface, their attachment density depends on the probe concentration and stability of their functional groups [7]. During the development phase of a microarray assay, it is of utmost importance to establish the probe concentration that saturates the surface binding sites, which represents the density at which crowding effects diminish the mass of the target captured
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
