Primer exchange reaction-coupled transcription isothermal amplification as a sensitive biomolecular assay.

We devised a novel strategy that relies on a combination of the primer exchange reaction (PER) with transcription isothermal amplification, termed PER-Trap, for a sensitive biomolecular assay. Its design allowed light-up RNA aptamers to be produced as the final product, leading to the generation of an amplified fluorescence signal. The utility of PER-Trap was successfully demonstrated by the detection of exosomes.

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell-Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent.

Colorectal cancer (CRC) as the second leading cause of global cancer deaths poses critical challenges in clinical settings. Cancer-derived small extracellular vesicles (sEVs), which are secreted by cancer cells, have been shown to mediate tumor development, invasion, and even metastasis, and have thus received increasing attention for the development of cancer diagnostic or therapeutic platforms. In the present study, the sEV-targeted systematic evolution of ligands by exponential enrichment (E-SELEX) is developed to generate a high-quality aptamer (CCE-10F) that recognizes and binds to CRC-derived sEVs. Via an in-depth investigation, it is confirmed that this novel aptamer possesses high affinity (Kd = 3.41 nm) for CRC-derived sEVs and exhibits a wide linear range (2.0 × 104 -1.0 × 106 particles µL-1 ) with a limit of detection (LOD) of 1.0 × 103 particles µL-1 . Furthermore, the aptamer discriminates CRC cell-derived sEVs from those derived from normal colon cell, human serum, and other cancer cells, showing high specificity for CRC cell-derived sEVs and significantly suppresses the critical processes of metastasis, including cellular migration, invasion, and angiogenesis, which are originally induced by sEVs themselves. These findings are highly encouraging for the potential use of the aptamer in sEV-based diagnostic and therapeutic applications.

Low-Temperature Loop-Mediated Isothermal Amplification Operating at Physiological Temperature.

Loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal amplification technologies in molecular diagnostics. However, LAMP operates at a high temperature of 65 °C; thus, operating LAMP at a lower temperature is desirable to maximize its usefulness for on-site diagnosis. In this study, we propose a new version of LAMP, termed low-temperature LAMP, which operates at the physiological temperature of 37 °C. Low-temperature LAMP differs from conventional LAMP operating at 65 °C in terms of the concentrations of MgSO4 and deoxyribonucleoside triphosphates (dNTPs), as well as the lengths of DNA probes, which are crucial for the execution of low-temperature LAMP. Under the optimal conditions, the amplification efficiency of low-temperature LAMP is comparable to that of conventional LAMP. In addition, the ligation reaction at 37 °C, which is necessary to detect actual target nucleic acids, is combined without altering the temperature, enabling the identification of miR-21, a cancer-promoting oncogenic miRNA, with high sensitivity and selectivity. The method described in this paper does not require expensive DNA modifications or special additives and would facilitate the widespread application of LAMP in facility-limited or point-of-care settings, paving the way to improvements in other isothermal-amplification-based techniques.