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

The biolayer interferometry (BLI) , one promising biosensing technique for real-time, high-sensitive detection of analytes , is a new surface-sensitive spectrometric technique in which the change of biolayer thickness is reflected by measuring the interferometric phase deflections. In our previous research, we selected an ssDNA aptamer, 39 nt, with definite interaction characteristics for recombinant human erythropoietin-alpha (EPO-alpha) . In this work , a new aptameric BLI assay of EPO-alpha based on this aptamer 39 nt was established. Firstly, the biotinylated aptamer 39 nt was immobilized onto the optical fiber streptavidin biosensor tip , and the analytes in microliter volume were quickly diffused via high-speed oscillation at 2200 r/min. Then , the key parameters such as the orientation of aptamer sequence , the concentration 14 immobilization , and the overcome of nonspecific adsorption were investigated and optimized to exhibit the affinity recognition of aptamers , and it was found that the 3 -end biotinylated aptamer 39 nt with 50 nmol/L provided the best sensitivity , and the addition of Tween 20 and bovine serum albumin ( BSA) could efficiently avoid nonspecific adsorption effect. Besides , towards the original designed disposable biosensor tip , the affinity between EPO-alpha and aptamer 39 nt 14 ca. 10 times was maintained by optimizing the regeneration condition. Furthermore , a signal amplification approach of a sandwiched format for detection of EPO-alpha via wheat germ agglutinin (WGA) was established, with the linear range of 10 -200 nmol/L, and a limit of detection of 5 nmol/L. This assay was further applied to the measurement of EPO-alpha in 50% human plasma with recoveries of 86.7% -104.2% and RSD less than 11% , providing a satisfied result. The BLI sensing system developed here provides a helpful way for practical applications for label-free protein interaction and detection.