Competition-Mediated Pyrene-Switching Aptasensor: Probing Lysozyme in Human Serum with a Monomer-Excimer Fluorescence Switch

摘要

Lysozyme (Lys) plays crucial roles in the innate immunensystem, and the detection of Lys in urine and serum hasnconsiderable clinical importance. Traditionally, the presencenof Lys has been detected by immunoassays; however,nthese assays are limited by the availability of commercialnantibodies and tedious protein modification and priornsample purification. To address these limitations, wenreport here the design, synthesis, and application of ancompetition-mediated pyrene-switching aptasensor fornselective detection of Lys in buffer and human serum. Thendetection strategy is based on the attachment of pyrenenmolecules to both ends of a hairpin DNA strand, whichnbecomes the partially complementary competitor to annanti-Lys aptamer. In the presence of target Lys, thenaptamer hybridizes with part of the competitor, whichnopens the hairpin such that both pyrene molecules arenspatially separated. In the presence of target Lys, however,nthe competitor is displaced from the aptamer by the target,nsubsequently forming an initial hairpin structure. Thisnbrings the two pyrene moieties into close proximity tongenerate an excimer, which, in turn, results in a shift ofnfluorescence emission from ca. 400 nm (pyrene monomer)nto 495 nm (pyrene excimer). The proposed methodnfor Lys detection showed sensitivity as low as 200 pM andnhigh selectivity in buffer. When measured by a steadystatenfluorescence spectrum, the detection of Lys innhuman serum showed a strong fluorescent background,nwhich obscured detection of the excimer signal. However,ntime-resolved emission measurement (TREM) supportednthe potential of the method in complex environments withnbackground fluorescence by demonstrating the temporalnseparation of probe fluorescence emission decay from thenintense background signal. We have also demonstratednthat the same strategy can be applied to the detection ofnsmall biomolecules such as adenosine triphosphate (ATP),nshowing the generality of our approach. Therefore, thencompetition-mediated pyrene-switching aptasensor is promisingnto have potential for clinical and forensic applications.