Synthetic Protocell as Efficient Bioreactor: Enzymatic Superactivity and Ultrasensitive Glucose Sensing in Urine

It is believed that membraneless cellular condensates play a critical role in accelerating various slow and thermodynamically unfavorable biochemical processes. However, the exact mechanisms behind the enhanced activity within biocondensates remain poorly understood. Here, we report the fabrication of a high-performance integrated cascade bioplatform based on synthetic droplets for ultrasensitive glucose sensing. Using a horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we report an unprecedented enhancement in the catalytic activity of HRP inside the synthetic membraneless droplet. Liquidlike membraneless droplets have been prepared via multivalent electrostatic interactions between adenosine triphosphate (ATP) and poly(diallyldimethylammonium chloride) (PDADMAC) in an aqueous medium. Compartmentalized enzymes (GOx/HRP@Droplet) exhibit high encapsulation efficiency, low leakage, prolong retention of activity, and exceptional stability toward protease digestion. Using an HRP@Droplet composite, we have shown that the enzymatic reaction within the droplet follows the classical Michaelis-Menten model. Our findings reveal remarkable enhancement in the catalytic activity of up to 100- and 51-fold for HRP@Droplet and GOx/HRP@Droplet, respectively. These enhanced activities have been explained on the basis of increased local concentrations of enzymes and substrates, along with altered conformations of sequestered enzymes. Furthermore, we have utilized highly efficient and recyclable GOx/HRP@Droplet composite to demonstrate ultrasensitive glucose sensing with a limit of detection of 228 nM. Finally, the composite platform has been exploited to detect glucose in spiked urine samples in solution and filter paper. Our present study illustrates the unprecedented activity of the compartmentalized enzymes and paves the way for next-generation composite bioreactors for a wide range of applications.