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Serum miR-501-3p and miR-455-3p are potential biomarkers for the diagnosis of Alzheimer’s disease (AD). The combination of both microRNAs (miRNAs) is anticipated to improve AD diagnostic accuracy and provide stage information. In this work, simultaneous determination of both miRNAs was performed using a dual-signal DNA probe based on the fluorescent recovery of carbon quantum dots (CQDs) and the sulfo-cyanine5 (Cy5) dye. Duplex specific nuclease was introduced to amplify the signals via a DNA degradation/RNA re-hybridization cycle. The dynamic ranges of the method were 0.01 to 4 pM for miR-501-3p and 0.01 to 5 pM for miR-455-3p. The accuracy of the method was confirmed by the recoveries of the miRNAs in spiked human serum and high correlation with measurements by the real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR). The concentrations of the miRNAs were determined in serum from healthy donors and AD patients in preclinical and mild stages. Detailed  t -test analysis revealed that despite of the poor correlations of independent miR-501-3p and miR-455-3p with AD progression, the concentration ratio of miR-501-3p to miR-455-3p was significantly correlated with AD diagnosis ( p  &lt; 0.001 between preclinical AD and healthy control) and early progression ( p  &lt; 0.001 between preclinical and mild-stage AD). A simple, sensitive method is reported for the simultaneous determination of the miRNAs in serum with high accuracy and shows that the concentration ratio of miR-501-3p to miR-455-3p is an effective blood index for early and in-stage diagnosis of AD.

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Early and In-Stage Diagnosis of Alzheimer’s Disease by the Simultaneous Fluorescent Determination of the Biomarkers miR-501-3p and miR-455-3p with Carbon Quantum Dots in Serum