Hourglass-inspired total analysis system for the sensitive colorimetric detection of exosomal miRNA using the CRISPR-Cas12a system with isothermal amplification

• A novel H-TAS was developed for exosomal miRNA analysis from cell culture media. • H-TAS performed all required processes in one-pot with a simple flipping action. • Combination of nanoprobes/CRIPSR-Cas12a/isothermal amplification realized the H-TAS. • The system analyzed Exo-miR-21 with good sensitivity (LOD = 1.308 fM) in 190 min. • H-TAS successfully analyzed exosomal miR-21 from complex biological samples. The clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, combined with isothermal nucleic acid amplification, offers a powerful sensing mechanism for transducing sequence-specific information. However, analyzing exosomal miRNAs (Exo-miRs) using the CRISPR-Cas system remains difficult and often involves complex steps, such as external exosome isolation/ enrichment or complex signal amplification and detection. Here, we introduce hourglass-inspired total analysis system (H-TAS) for easy and sensitive colorimetric detection of Exo-miR in one-pot using the CRISPR-Cas12a system with isothermal nucleic amplification. In this device, the entire system operates through a successive configuration of two innovative systems: (i) exosome isolation/enrichment and one-pot isothermal Exo-miR amplification via promoter/antibody-enriched gold-satellite-magnetic nanoparticles and (ii) enhanced colorimetric signal analysis using multi-enzyme-encapsulated nanoparticles and the CRISPR-Cas12a system. Moreover, to facilitate the successive combination and ease of use of the two systems, we developed an hourglass-inspired device capable of performing all the analysis steps to detect Exo-miRs without changing the reaction vial. Consequently, the developed H-TAS can readily proceed with all processes required for Exo-miR detection using only a simple flipping action. As a proof-of-concept demonstration, we applied H-TAS to miR-21 and Exo-miR-21 analysis and confirmed adequate specificity and sensitivity (limit of detection = 0.711 fM, 1.308 fM respectively) within 190 min (sample-to-answer time) without needing specialized instruments. Furthermore, the adaptability of the proposed device was verified by analysis of Exo-miR-21 from various breast cell-derived exosomes and clinical samples. Therefore, H-TAS may be a promising platform for detecting Exo-miRs in a highly sensitive, selective, simple, and time-efficient manner.