Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay.

A sensitive and specific sandwich assay for the detection of thrombin is described. Two affiliative aptamers were used to increase the assay specificity through sandwich recognition. Recognition DNA loaded on gold nanoparticles (AuNPs) partially hybridized with the initiator DNA, which was displaced by surviving DNA. After the initiator DNA was released into the solution, one hairpin structure was opened, which in turn opened another hairpin structure. The initiator DNA was displaced and released into the solution again by another hairpin structure because of the hybridized reaction. Then the released initiator DNA initiated another autocatalytic strand displacement reaction. A sophisticated network of three such duplex formation cycles was designed to amplify the fluorescence signal. Other proteins, such as bovine serum albumin and lysozyme, did not interfere with the detection of thrombin. This approach enables rapid and specific thrombin detection with reduced costs and minimized material consumption compared with traditional assay processes. The detection limit of thrombin was as low as 4.3 × 10⁻¹³ M based on the AuNP amplification and the autocatalytic strand displacement cycle reaction. This method could be used in biological samples with excellent selectivity.

Nicking endonuclease and target recycles signal amplification assisted quantum dots for fluorescence detection of DNA.

An ultrasensitive fluorescence detection method for DNA based on nicking endonuclease (NEase) and target recycles assisted with CdTe quantum dots (QDs) is reported. In the detection system, when the target DNA is present, it hybridizes with a linker strand to from a duplex, in which the NEase recognizes specific nucleotide sequences and cleaves the linker strand. After nicking, the fragments of the linker strand spontaneously dissociate from the target DNA and another linker strand hybridizes to the target to trigger another strand-scission cycle. On the other hand, when the target was absent, no duplex is formed and no fragment of linker strand is produced. Then CdTe QDs and magnetic beads (MBs), which were all modified with DNA sequences complementary to that of the linker strands are added to the solution to detect the presence of a target DNA. The signal was generated through the difference in Förster resonance energy transfer (FRET) between the MB and CdTe QDs. This method indicates that one target DNA leads to cleavage of hundreds of linker DNA, increasing detection sensitivity by nearly three orders of magnitude. This method should be applicable whenever there is a requirement to detect a specific DNA sequence and can also be used for multicomponent detection.