ABSTRACT
Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.
Subject(s)
Biosensing Techniques , Extracellular Vesicles , Limit of Detection , Metal-Organic Frameworks , MicroRNAs , Paper , Metal-Organic Frameworks/chemistry , Extracellular Vesicles/chemistry , Humans , Biosensing Techniques/methods , DNA, Catalytic/chemistry , Graphite/chemistry , Gold/chemistry , DNA/chemistry , Metal Nanoparticles/chemistry , Nucleic Acid Hybridization , Electrochemical Techniques/methods , Electrodes , Zirconium/chemistryABSTRACT
There is an urgent need to accurately quantify tumor-derived exosomes, which have emerged as promising non-invasive tumor diagnostic biomarkers. Herein, a bispecific-aptamer sandwich-type gold nanoparticle-modified electrochemical aptasensor was developed based on a four-way junction (4-WJ)-triggered dual rolling circle amplification (RCA)-assisted methylene blue (MB)/G-quadruplex strategy for extremely specific and sensitive exosome detection. This aptamer/exosome/aptamer sandwich-type design contained a CD63-specific aptamer and a cancerous mucin-1 (MUC1) protein-specific aptamer. The CD63 aptamer modified on a gold electrode captured exosomes, and then the sandwich-type aptasensor was formed with the addition of the MUC1 aptamer. The MUC1 aptamer's 3'-end sequence facilitated the formation of 4-WJ, assisted by a molecular beacon probe and a binary DNA probe. Subsequently, a dual-RCA reaction was triggered by binding to two cytosine-rich circle DNA templates at both ends of 4-WJ. Ultimately, dual-RCA products containing multiple G-quadruplex conformations were generated with the assistance of K+ to trap abundant MB indicators and amplify electrochemical signals. The aptasensor exhibited high specificity, sensitivity, repeatability, and stability toward MCF-7-derived exosomes, with a detection limit of 20 particles/mL and a linear range of 1 × 102 to 1 × 107 particles/mL. Moreover, it showed excellent applicability in clinical settings to recover exosomes in normal human serum. Our aptasensor is anticipated to serve as a versatile platform for detecting various specific aptamer-based targets in biomedical and bioanalytical applications.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Exosomes , Metal Nanoparticles , Neoplasms , Humans , Exosomes/metabolism , Gold/chemistry , Aptamers, Nucleotide/chemistry , Limit of Detection , Electrochemical Techniques , DNA/chemistry , Neoplasms/diagnosis , Neoplasms/metabolismABSTRACT
Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus - 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.