Mesophilic
Argonaute-Mediated Polydisperse Droplet
Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic
Acid Detection Using Deep Learning
Detection of nucleic acids from a single multiplexed
and amplification-free
test is critical for ensuring food safety, clinical diagnostics, and
environmental monitoring. In this study, we introduced a mesophilic
Argonaute protein from Clostridium butyricum (CbAgo),
which exhibits nucleic acid endonuclease activity, to achieve a programmable,
amplification-free system (PASS) for rapid nucleic acid quantification
at ambient temperatures in one pot. By using CbAgo-mediated binding
with specific guide DNA (gDNA) and subsequent targeted cleavage of
wild-type target DNAs complementary to gDNA, PASS can detect multiple
foodborne pathogen DNA (<102 CFU/mL) simultaneously.
The fluorescence signals were then transferred to polydisperse emulsions
and analyzed by using deep learning. This simplifies the process and
increases the suitability of polydisperse emulsions compared to traditional
digital PCR, which requires homogeneous droplets for accurate detection.
We believe that PASS has the potential to become a next-generation
point-of-care digital nucleic acid detection method.