An Aptameric Biolayer Interferometric Assay for Detection of Recombinant Human Erythropoietin-α

Abstract

The biolayer interferometry (BLI), one promising biosensing technique for real-time, high-sensitive detection of analytes, is a new surface-sensitive spectroscopic technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, an ssDNA aptamer, 39nt, with definite interaction characteristics for recombinant human erythropoietin-α (EPO-α) was screened. In this work, a new aptameric BLI assay of EPO-α based on this aptamer 39nt was established. The biotinylated aptamer 39nt was firstly immobilized onto the optical fiber streptavidin biosensor chip, and the analytes in microliter volume were quickly diffused via high-speed vibration of 2200 r/min. The key parameters such as the orientation of aptamer sequence, the concentration for immobilization, and the overcome of nonspecific adsorption to exhibit the affinity recognition of aptamers were optimized. It was found that the 3'-end biotinylated aptamer 39nt with 50 nmol/L provided the best sensitivity, and the addition of Tween 20 and BSA could efficiently overcome nonspecific adsorption effect. Besides, towards the original designed disposable sensor chip, the affinity between protein EPO-α and ssDNA aptamer 39nt for ca. 10 times was still maintained under optimized regeneration condition, which reflected the operational flexibility of single-channel BLI system. Furthermore, a signal amplification approach of a sandwiched format via wheat germ agglutinin for detection of EPO-α was established, with the linear range of 10–200 nmol/L, and the LOD was 5 nmol/L. This assay was directly applied to the complex matrix such as 50% human plasma with recoveries in the range of 86.7%–104.2% and RSDs less than 15%. The BLI sensing system developed here provided a helpful way for practical applications in label-free protein interaction and detection. A new aptameric biolayer interferometry assay based on single stranded DNA aptamer 39nt was firstly established. Concomitantly with a signal amplification approach of a sandwiched format via wheat germ agglutinin, a sensitive measurement of recombinant human erythropoietin-α in complex matrix such as 50% human plasma were fulfilled.