Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13–4 nM and 0.56–12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.