Blood Screening\nof Femtomole Level Multiple Alzheimer’s\nDisease Biomarkers by Metal Isotopic DNA Walkers

Aging is a critical global issue that contributes to\nthe high incidence\nof Alzheimer’s disease (AD). Blood screening emerges as the\nmost promising measure for early diagnosis and intervention of AD\ndue to its noninvasive and low cost. However, the practical application\nof AD blood screening confronts two significant challenges. First,\ndue to the blood–brain barrier, the concentration of AD biomarkers\nin blood is much lower than that in cerebrospinal fluid. Second, simultaneous\nquantitative analysis of multiple biomarkers is necessary due to the\nlow specificity of individual biomarkers. Herein, we propose DNAzyme-based\n3D DNA walkers for the sensitive and multiplex detection of five AD-associated\nmiRNA biomarkers: hsa-miR-125b, hsa-miR-342-3p, hsa-miR-29b, hsa-miR-191-5p,\nand hsa-miR-7d-5. The DNAzyme-based 3D DNA walkers provide highly\nefficient and autonomous amplification of the minimal biomarkers’\nquantities. The walking-released metal isotopes <sup>89</sup>Y, <sup>165</sup>Ho, <sup>139</sup>La, <sup>140</sup>Ce, and <sup>159</sup>Tb can be sensitively detected by elemental mass spectrometry without\nany spectral overlap. The detection limit was achieved to be as low\nas 1.0 fmol. The proposed method was successfully applied to human\nserum samples with satisfactory spiked recoveries. With its high sensitivity\nand multiplexity capabilities, this metal isotope strategy may contribute\nto the early diagnosis and intervention of AD.