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 0 -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 EC 50 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 EC 50 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.

Electrochemical Immunosensor for the Determination of Total Ghrelin Hormone in Saliva

“…where s is the standard deviation ( n =7) of the zero value (the i p value measured in the absence of GHRL), ±0.24 µA. It is important to remark that the obtained value, 7 pg/mL, is much smaller than the LOD values reported for the commercially available ELISA kits (ranging between 7 and 140 times smaller), as well as with respect to the values obtained with the immunosensors described in the literature, 0.2 ng/mL 18 and 8 ng/mL 19. The reproducibility of the DP voltammetric measurements was evaluated by carrying out assays with different immunosensors for solution containing a 1.0 ng/mL GHRL concentration level on the same day.…”

“…Concerning biosensors, Mascini et al developed a colorimetric microarray detection system for GHRL using aptamers technology, with a linear range between 0.2 and 245.5 ng/mL and a detection limit of 0.2 ng/mL 18. The same authors developed an electrochemical aptasensor where the aptamer was adsorbed on the surface of a screen‐printed electrode and measurement of the decrease in the guanine oxidation signal in the presence of GHRL.…”

Emerging applications of aptamers to micro- and nanoscale biosensing

“…To increase throughput and experimental redundancy of conventional bioanalytical assays, microarrays utilizing aptamer receptors and fluorescence detection have been recently reported (Aberem et al 2006;Bock et al 2004;Brody et al 1999;Cho et al 2006;Collett et al 2005;Kirby et al 2004;Mascini et al 2007;McCauley et al 2003;Stadtherr et al 2005;Wang et al 2006a). Typically, these arrays are fabricated by robotically inking or patterning glass substrates with micrometer-sized spots in a visually addressable matrix.…”

Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes

“…14, 16, 17 Another example used Spiegelmers as affinity probes in electrochemical and colorimetric detection of ghrelin. 18, 19 However, these bioassays involved immobilization of either ghrelin or its Spiegelmer on a solid surface and included multiple washing steps, making the assays time consuming and laborious.…”