LRET-Based\nSimultaneous Detection of Dual miRNAs via\nMultitrap Optical Tweezers Assisted Suspension Array Tagged by Two\nDifferent Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification

Nowadays, optical tweezers play a vital role not only\nin optical\nmanipulation but also in bioassay. As principal optical trapping objects,\nmicrobeads can combine optical tweezers with suspension array technology,\nwith amply focused laser beams and adequately concentrated tags contributing\nto highly sensitive detection. In view of the inefficiency of conventional\nsingle-trap optical tweezers, multitrap systems are developed. Here,\ngreen- and blue-emitting core–shell–shell upconversion\nnanoparticles (UCNPs) are adopted to encode microbeads and determine\ndual miRNAs, with the internal shells leading the luminescence process\nto facilitate quenching through luminescence resonance energy transfer\n(LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence\nsignals are recovered and amplified, causing further enhanced detection\nsensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM\nare obtained with excellent specificities verified. Furthermore, dual\nmiRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected,\nwith results consistent with those obtained by PCR. Notably, miR-155\nin MCF-7 and A549 cells is detectable at the single-cell level. Thus,\nthe differences in the measured miRNA levels between MCF-7 and MCF-10A\ncells imply the potential of this method to discriminate breast cancer\ncells from epithelial cells despite the difficulty in distinguishing\ndifferent cancer cells due to similar miRNA levels.