Spherical Nucleic Acid Stabilized Cage Type Three-Dimensional Electrochemiluminescence Probe for Sensitive and Amplification-Free Diagnosis of Infectious Diseases.

Accurate nucleic acid-based pathogen diagnosis is critical for clinical treatment and epidemic control. However, present gold-standard testing methods based on nucleic acid amplification are limited by their time and labor-intensive nature. Hence, we reported an amplification-free assay that facilitates rapid, sensitive, and point-of-care detection of infectious diseases. Herein, spherical nucleic acid (SNA)-stabilized cage-type three-dimensional electrochemiluminescence (ECL) reporter probe, constructed utilizing Ru(dcbpy)3Cl2-doped NH2-MIL-101 (Fe) metal-organic framework, was combined with a paper-based bipolar electrode ECL sensor. Furthermore, surface modifications were performed using polydopamine and SNAs to further improve the stability. Notably, compared with the ordinary Ru(dcbpy)3Cl2, the modified ECL probe attained 103-fold enhancement in luminous efficiency. The developed ECL-based nucleic acid biosensing system offers the advantages of simplicity, portability, low cost, and user-friendly nucleic acid detection, with a sample-to-answer time of approximately 15 min. Additionally, the effectiveness of the developed assay in detecting pathogenic nucleic acid was assessed using Mpox and SARS-Cov-2, exhibiting a wide dynamic range from 33 to 1010 aM and limits of detection as low as 3 copies/µL. Furthermore, the assay exhibited 100% sensitivity and specificity when validated against quantitative polymerase chain reaction-based detection using clinical samples. Altogether, the findings of this study show the efficacy of the novel developed SNA-stabilized cage-type 3D ECL probe-enhanced nucleic acid assay, along with its potential as a promising paradigm for point-of-care diagnosis of infectious pathogens.