| Alzheimer's disease(AD),considered as the “third health killer”,seriously threats the health of the elderly.However,the modern diagnostic strategies of AD still present several disadvantages: the low accuracy and low specificity resulting in some falsenegative diagnosis and the poor sensitivity leading to a delayed treatment.In view of this situation,a novelty enzyme-free and target-triggered signal amplification strategy based on graphene oxide(GO)and entropy-driven strand displacement reaction(ESDR)principle was proposed.In this strategy,the skillfully devised hairpin structure probes(H)would open when it specifically binds with A?42 oligomers,causing the bases complementary to FAM-labeled replacement probes R(R1 and R2)exposed.At this time,R1 and R2 will hybridize with H and the bound A?42 oligomers will release.The released A?42 oligomers participate in next circular reaction,resulting the signal amplified.As quencher,GO could absorb the free single-stranded DNA R1 and R2 and quench their fluorescence;however,the DNA duplex still exists free and keeps its signal-on.As exosomes can cross the blood-brain barrier to reflect the true situation of the brain disease earlier.In addition,exosomes exist in a variety of body fluids and they are easy to extract.Therefore,this method could be used to detect A?42 oligomers in exosomes,and the early,high sensitive and noninvasive detection of AD could be achieved.This study was mainly researched at the following three levels.(1)Establishment and method validation in vitro of A?42-oligomer's ESDR signal amplification detection strategy.A?42-oligomer's ESDR signal amplification detection strategy was constructed in phosphate buffered saline(PBS)and its methodology was validated.In order to make this detection system under ideal conditions,its detection conditions,such as the hairpin probe's structure,chain replacement reaction time,GO concentration and GO incubation time were optimized.In addition,the methodological of this detection method was studied,these results indicated that the detection system could be applied for the detection of A?42 oligomers,and it behaved excellent specificity and accuracy.(2)Detection of A?42 oligomers in simulated serum.The A?42-oligomer's ESDR signal amplification detection assay was used to detect A?42 oligomers in dilute fetal bovine serum(simulated serum),and its detection limit as low as 20 pM.In simulated serum,the detection method showed a higher detection ability,because of favorable ion conditions and suitable acid-base environment of serum,which are favorable for the conformational change of aptamer when it binds to the target,thus,the sensitivity of this A?42-oligomer's signal amplification detection assay in diluted serum was greatly enhanced.In simulated serum,the application of this method has been further investigated.The results showed that the A?42-oligomer's ESDR signal amplification detection method was more suitable for the detection of biological samples.(3)Detection of A?42 oligomers in exosomes.Exosomes were extracted from mouse serum and the extracted exosomes were characterized by laser nanometer particle size analyzer and transmission electron microscopy(TEM).A?42 oligomers were encapsulated in these exosomes,the obtained exosomes containing A?42 oligomer(Exo-A?42)was used to mimic the exosomes in serum of AD patients,and the size and morphology of Exo-A?42 were characterized.A?42 oligomers in ExoA?42 were detected by A?42-oligomer's ESDR signal amplification detection strategy.The results showed that the detection method could detect AD's biomarker in exosomes efficiently and sensitively.In summary,the novel ESDR fluorescence signal amplification assay had good specificity and high sensitivity,and could detect A?42 oligomers quickly and easily,and it showed great potential in AD's highly sensitive,non-invasive and early diagnosis. |
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