Sensitive Discrimination and Detection of Prion Disease-Associated Isoform with a Dual-Aptamer Strategy by Developing a Sandwich Structure of Magnetic Microparticles and Quantum Dots

摘要

The major challenge of prion disease diagnosis at thenpresymptomatic stage is how to sensitively or selectivelyndiscriminate and detect the minute quantity of diseaseassociatednprion protein isoform (PrPRes) in complexnbiological systems such as serum and brain homogenate.nIn this contribution, we developed a dual-aptamernstrategy by taking the advantages of aptamers, thenexcellent separation ability of magnetic microparticlesn(MMPs), and the high fluorescence emission featuresnof quantum dots (QDs). Two aptamers (Apt1 andnApt2), which can recognize their two correspondingndistinct epitopes of prion proteins (PrP), were couplednto the surfaces of MMPs and QDs, respectively, tonmake MMPs-Apt1 and QDs-Apt2 ready at first, whichnthen could be coassociated together through the specificnrecognitions of the two aptamers with their twoncorresponding distinct epitopes of PrP, forming ansandwich structure of MMPs-Apt1-PrP-Apt2-QDs andndisplaying the strong fluorescence of QDs. Owing tonthe different binding affinities of the two aptamers withnPrPRes and cellular prion protein (PrPC), both of whichnhave distinct denaturing detergent resistance, our dualaptamernstrategy could be applied to discriminatenPrPRes and PrPC successfully in serum. Further identificationsnshowed that the present dual-aptamer assayncould be successfully applied to the detection of PrPnin 0.01% brain homogenate, about 1000-fold lowernthan that of commonly applied antibody-mediatednassays, which can detect PrP just in 10% brain homogenate,nindicating that the present designed dualaptamernassay is highly sensitive and adequate fornclinical diagnosis without isolation of target proteinnprior to assay.