关键词: 适配体, 微磁珠分离, 实时定量聚合酶链式反应, 凝血酶, ATP

Abstract:

Aptamers, as recognition molecules, have been applied in a variety of fields due to their well-known advantages. However, there is still a big difficulty to establish a universal aptameric assay in the field of analytical chemistry. In this research, benefited from the specific recognition and the amplification capability of aptamer, a magnetic separation-based aptameric real-time quantitative PCR detection approach was demonstrated. A complementary sequence immobilized onto magnetic beads was applied to effectively remove the free aptamers which did not interact with target molecules in solution by virtue of base-pair binding capability between the complementary sequence and the aptamer sequence. After being separated under magnetic field, the concentrations of aptamers corresponding to various concentrations of target molecules were collected from supernatant solution, and determined by real-time quantitative PCR analysis. To demonstrate the universality of such an approach, thrombin and ATP were chosen as detection targets for representing biomacromolecules and organic small molecules, respectively. The experimental results indicated that the ultra low concentrations of thrombin and ATP spiked in binding buffer could be successfully quantified only after a simple optimization of complementary sequences and other conditions, their limit of detections(LODs) were down to 50 pmol/L and 5 μmol/L, respectively. Currently, this approach was one of the few aptameric assays which could be applied to determine biomacromolecules and organic small molecules with high specificity and sensitivity.

Key words: Aptamer, Magnetic separation, Real-time quantitative PCR, Thrombin, ATP