Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer–target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1 × 10−10 M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol.

The ATP recognition probe (S1) was immobilized on the surface of MB-AuNPs and hybridized with its partly complementary sequence (S2) to form a duplex. When the target ATP was introduced, S1 changed its conformation to combine with ATP (step Ia) and S2 was released (step Ib) to trigger the MB-assisted strand displacement amplification (step II). In cycle 1, S2 hybridized with the single-stranded toehold domain of S3 and the single-stranded DNA (S4) was dissociated from the MB-AuNPs by branch migration, enabling S3 to possess a newly exposed single-stranded toehold. Then the biotin-tagged single-stranded fuel (S6) was hybridized with the newly exposed toehold, releasing S5 and S2. At the same time, the double-stranded product and the biotin-tagged MB complexes were formed. The released S5 would further participate in the target recycling amplification reaction (cycle 2). S2 was used as catalyst to implement another circulation of the MB-assisted strand displacement amplification. In cycle 1, the released S4 could hybridize with the recognition probe (S1) by DNA displacement reaction, making more S2 to be dissociated to participate in cycle 1. The second amplification process (cycle 2) was initiated by S5, which could hybridize with the extension part in S1/ATP complex and be used as the primer of a polymerization reaction. In the presence of Klenow polymerase and dNTPs, the primer extended and formed a duplex with the ATP recognition probe. Thus the target ATP was displaced, which could bind with another recognition probe and trigger new amplification cycles. Finally, a large amount of biotin-tagged MB complexes were produced. As the fuel (S6) was modified with biotin, the S3/S6 double-stranded product on MB-AuNPs could incorporate with streptavidin-HRP to perform the enzyme-amplification CL detection.Figure optionsDownload as PowerPoint slideHighlights
► An aptasensor is fabricated by strand displacement amplification and target recycling.
► The reaction efficiency is greatly improved by cross-circular amplification.
► Sensitive detection of other targets can be achieved by using different aptamers.