Ligand‐Responsive Artificial Protein–Protein Communication for Field‐Deployable Cell‐Free Biosensing

Abstract Natural protein–protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell‐free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour‐level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo‐designed ligand‐responsive artificial protein–protein communication (LIRAC) by redefining the connection between TFs and non‐interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target‐induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogeneous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL‐based cell‐free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi‐protein communication‐based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.