Colorimetric Microarray Detection System for Ghrelin Using Aptamer‐Technology

Abstract

The aim of this research was to compare the efficiency in term of miniaturization, multiplexing, reproducibility and sensitivity of colorimetric microarray versus an enzyme linked oligonucleotide assay (ELONA). A competition assay was performed to explore the complex formation between 5′‐biotin NOX‐B11, a special aptamer called spiegelmer, and ghrelin, a growth hormone releasing acylated peptide from stomach. The aptamer technology is a very powerful method suitable for large‐scale synthesis and overcoming the disadvantages of antibodies (unstable, expensive to produce, and so on) while remaining as efficient as antibodies in binding to the target. The aptamer used in this work was based on spiegelmer oligonucleotides approach that has been considered as one of the simplest, most straightforward, and cost‐effective method to develop artificial receptors. The efforts were concentrated on the development of the microarray process which includes the study of the label binding condition and all analytical parameters like concentration, time and temperature involved in the interaction between the target and the ligand. The analytical signal was acquired using a flatbed scanner and elaborated with an image software that coupled with the microarray equipments led to an extremely low cost analytical device. A sigmoidal dose‐response curve was obtained using the microarray system with an EC50 of 6.1 ng/ml in a huge linear range from 0.2 up to 245.5 ng/ml with a detection limit of 0.2 ng/ml and an averaged relative standard deviation (RSD) across all the points of the curve of 6.9%. In the ELONA assay we found an EC50 of 40.0 ng/ml, a detection limit of 2.6 ng/ml with a RSD of 9.9% in a linear range relatively narrow extending between 15.0 and 130.5 ng/ml, with the lower limit almost 45 times higher than that obtained using microarray system. The undoubtedly better performance of the microarray system developed was demonstrated. The normal blood ghrelin level in adults (∼0.337 ng/ml) was within the linear range of the new system proposed making it suitable for clinical application.