DNA aptamer folding on magnetic beads for sequential detection of adenosine and cocaine by substrate-resolved chemiluminescence technology

Abstract

We have developed a new aptamer-based chemiluminescence (CL) biosensing platform for the sequential detection of two small molecules as exemplified by adenosine and cocaine. Each biosensing platform comprises NH₂-functionalized capture DNA immobilized on magnetic beads; this can hybridize with one end of the aptamer. A corresponding reporter DNA probe labeled with either digoxigenin or biotin on its 5′-terminus recognizes the other end of the aptamer. The target compounds adenosine or cocaine act as specific competitors to aptamer–reporter DNA binding, and the corresponding aptamers preferentially form target–aptamer complexes. This results in detachment of the reporter DNA probe from the magnetic beads, with more target molecules resulting in less reporter DNA probe remaining on the beads. Those left are sequentially detected by using substrate-resolved anti-digoxigenin-alkaline phosphatase and streptavidin-horseradish peroxidase. Experimental results confirm that this CL immunosensing platform has good sensitivity with detection limits of 5.2 × 10⁻⁹ M and 3.2 × 10⁻⁹ M for adenosine and cocaine, respectively. Because it is straightforward to adapt this strategy to detect a spectrum of small molecules by using different aptamers, this method may offer a new direction in designing high-performance CL aptasensors for sensitive and sequential determination of a limited number of small molecules.