CRISPR/Cas9-Assisted Microrobots for Fast and Ultrasensitive "On-The-Fly" Next-Generation DNA Detection

Abstract

Fast, sensitive, and selective nucleic acids detection is essential for applications in clinical diagnostics, food safety, and environmental monitoring. Hence, in this study, we introduce a novel motion-assisted CRISPR/Cas9 microrobots-based biosensor, consisting of CRISPR/Cas9 functionalized on gold microrobots (Au-MRs) forming CRISPR/Cas9@Au-MRs, which exhibit self-propulsion leading to rapid and ultrasensitive detection of target DNA. The proposed system achieved the limit of detection in low fM DNA concentration, enabling detection across a wide dynamic range within only 5 min, which is significantly faster than any previously reported systems. Further, this self-propelled system removes the need for complicated sample preparation, enabling real-time detection by using a minimal amount of sample. Unlike conventional CRISPR biosensors, this platform utilizes "on-the-fly" motion-enhanced signal recovery, ensuring superior specificity and sensitivity in diverse biological environments. Importantly, the presented system shows exceptional analytical performance in biological samples, highlighting its potential for real-world sensing applications. Overall, the motion-assisted CRISPR/Cas9@Au-MRs biosensor represents evolutionary progress in nucleic acids detection, providing a fast, reliable platform that can be easily scalable for applications in disease detection, biosafety and environmental detection.