PeroxiHUB: A Modular Cell-Free Biosensing Platform Using H₂O₂ as Signal Integrator.

Area of Science:

• Synthetic biology
• Biosensor engineering
• Metabolomics

Background:

• Cell-free systems offer potential for field-deployable biosensors.
• Discovery and implementation of transcription factor-based biosensors remain challenging.
• Hydrogen peroxide (H₂O₂) is a central metabolite and byproduct of many enzymatic reactions.

Purpose of the Study:

• To engineer PeroxiHUB, an optimized H₂O₂-centered platform for cell-free metabolite detection.
• To enable rapid reprogramming of sensor specificity using enzymatic transducers.
• To demonstrate modular detection of clinically relevant metabolites.

Main Methods:

• Screening and optimization of transcription factors (OxyR) for H₂O₂ response in cell-free systems.
• Utilizing enzymatic transducers for metabolite-to-H₂O₂ conversion.
• Employing a custom retrosynthesis workflow for transducer discovery.

Main Results:

• Optimized OxyR transcription factor for H₂O₂ detection with preincubation steps for signal-to-noise ratio.
• Demonstrated modular detection of lactate, sarcosine, and choline using PeroxiHUB.
• Confirmed the necessity of transducer expression during preincubation for optimal performance.
• Showcased adaptability with different reporter systems and detection of endogenous lactate in clinical samples.

Conclusions:

• PeroxiHUB provides a modular and scalable platform for cell-free metabolite detection.
• The platform's H₂O₂-centered design allows for rapid reprogramming of specificity.
• PeroxiHUB facilitates the development of field-deployable biosensors for diverse applications, including clinical diagnostics.