Citation: HE Xiao-Qin, LUO Li, GUO Lei, XIE Jian-Wei. An Aptameric Biolayer Interferometric Assay for Detection of Recombinant Human Erythropoietin-α[J]. Chinese Journal of Analytical Chemistry, ;2020, 48(5): 670-675. doi: 10.19756/j.issn.0253-3820.191676 shu

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

HE Xiao-Qin ^1,2, ,
LUO Li ^1,3 ,
GUO Lei ¹ ,
XIE Jian-Wei ¹

1.
State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Toxicology and Pharmacology, Academy of Military Medical Sciences, Beijing 100850, China;
2.
Department of Spine Surgery and Institute for Orthopaedic Research, the 2^nd Clinical Medical College of Jinan University and the 1^st Affiliated Hospital of Southern University of Science and Technology(Shenzhen People's Hospital), Shenzhen 518020, China;
3.
School of Public Health, Hebei Medical University, Shijiazhuang 050017, China

Received Date: 15 November 2019
Revised Date: 24 March 2020

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 21175152 and 21974152).

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-α (EPO-α). In this work, a new aptameric BLI assay of EPO-α 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 for 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-α and aptamer 39 nt for ca. 10 times was maintained by optimizing the regeneration condition. Furthermore, a signal amplification approach of a sandwiched format for detection of EPO-αvia 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-α 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.
- Aptamer,
- Recombinant human erythropoietin-α,
- Biolayer interferometry,
- Wheat germ agglutinin,
- Signal amplification
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