Electrogenerated chemiluminescence aptamer-based method for the determination of thrombin incorporating quenching of tris(2,2′-bipyridine)ruthenium by ferrocene

A novel electrogenerated chemi luminescence (ECL) aptamer-based biosensing method for the determination of thrombin was developed on basis of a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA capture probe, composing of a ss-DNA sequence to adopt two distinct structures-a DNA double strand with a complementary DNA sequence tagged with a ECL signal producer tris(2,2'-bipyridyl)ruthenium derivative and a DNA duplex with a thrombin-binding aptamer tagged with a ECL-quencher ferrocene (FcDNA), was self-assembled onto surface of a gold electrode. In the presence of thrombin, the aptamer sequence prefers to form the aptamer-thrombin complex and the switch of the binding partners occurs in conjunction with the generation of a strong ECL signal owing to the dissociation of FcDNA. The integrated ECL intensity versus the concentration of thrombin was linear in the range from 2.0 x 10(-10) M to 2.0 x 10(-7) M. The detection limit was 6 x 10(-11) M. The relative standard derivation for 2.0 x 10(-9) M was 5.7% (n = 7). This work demonstrates that the sensitivity and specificity of ECL aptamer-based method for proteins can be greatly improved using quenching ECL signal producer by a chemical quencher such as ferrocene. (C) 2008 Elsevier B.V. All rights reserved.