Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum Original Research Article

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg2+ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1 h at 4 °C in the assay buffer containing 5 mM Mg2+ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3 kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01 μg mL−1 of aptamer and 1.205 × 10−7 M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0 × 10−12 M and four orders (1.0 × 10−7 M to 5.0 × 10−11 M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient (R2 = 0.9872): Abs. (in serum) = 0.9612 × Abs. (in buffer) − 0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.