A sensitive biomolecules detection device with catalytic hairpin assembly and cationic conjugated polymer-assisted dual signal amplification strategy.

A simple, sensitive, selective, and enzyme-free homogeneous fluorescent biosensing device for DNA and protein detection is fabricated based on catalytic hairpin assembly (CHA), cationic conjugated polymer (CCP), and graphene oxide (GO). In this biosensing device, CCP together with CHA, provides dual signal amplification, and GO suppresses the background when the target is absent. Thus, this CHA/CCP/GO-based biosensor shows improved sensitivity compared with conventional CHA-based biosensors. In the biosensor, two 6-carboxyfluorescein (FAM)-labeled hairpin DNA probes (H1 and H2) are designed, and in the initial state, they could absorb on the surface of GO, leading the system to produce a low background fluorescence signal. When the target DNA appears, it continually catalyzes the formation of H1-H2 double-stranded DNA (dsDNA) complex by CHA reaction, which could be regarded as the first-step amplification. At the same time, the H1-H2 dsDNA complex departures from the surface of GO and interacts with CCP through electrostatic interaction. Then, CCP provides the second-step amplification due to its high fluorescence resonance energy transfer (FRET) efficiency from CCP to FAM. The limit of detection (LOD) and the limit of quantification (LOQ) for the target DNA could reach 32 pM and 1 nM, respectively. The linear range was from 0.1 to 40 nM, and relative standard deviation (RSD) for the points on the calibration curve ranged from 2.8% to 13.9%. This strategy could also be applied to protein detection potentially by integrating the aptamer of the target protein into the hairpin DNA. As proof of concept, thrombin was detected, and the LOD and LOQ was 11 pM and 33 pM, respectively. The linear range was from 3 to 54 nM, and RSD ranged from 3.3% to 10.4%. It showed good selectivity for thrombin compared to equal concentrations of interferences. It was also applied to quantify the thrombin (5, 10, 20 nM) in 1% spiked human serum, which showed satisfying recovery in the range of 94.7 ± 5.3 to 103.7 ± 4.9%.