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

Abstract

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) (PDAD-MAC) 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 51fold 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.

Coacervate-Based Plexcitonic Assembly toward Peroxidase-like Activity and Ultrasensitive Glucose Sensing

“…Next, we studied the effect of aging of colloidal Au@QD-Ds on the peroxidase activity for a period of 28 days. The activity of natural peroxidase enzymes such as HRP is known to degrade over time which limits their applicability . Notably, no appreciable changes in the LSPR peak for Au@QD-Ds were observed over a period of 28 days (Figure S15).…”

“…The initial rates of the reactions were plotted against substrate concentrations by considering the molar extinction coefficient of ox-TMB at 652 nm as 39,000 M −1 cm −1 . 38 The obtained kinetic data were fitted to Michaelis−Menten and double reciprocal Lineweaver−Burk plots to obtain the Michaelis− Menten constant, K m , and the maximum initial velocity V max . The kinetic parameters were evaluated using the equation Similarly, the pH-dependent peroxidase-like activity of Au@QD-Ds was studied in the pH range of 2−11.…”

“…All the absorbance measurements were recorded at 652 nm corresponding to the formation of ox-TMB. The initial rates of the reactions were plotted against substrate concentrations by considering the molar extinction coefficient of ox-TMB at 652 nm as 39,000 M –1 cm –1 . The obtained kinetic data were fitted to Michaelis–Menten and double reciprocal Lineweaver–Burk plots to obtain the Michaelis–Menten constant, K m , and the maximum initial velocity V max .…”

“…The activity of natural peroxidase enzymes such as HRP is known to degrade over time which limits their applicability. 38 Notably, no appreciable changes in the LSPR peak for Au@QD-Ds were observed over a period of 28 days (Figure S15). Furthermore, it has been observed that these Au@QD-Ds can retain more than 94% of their initial peroxidase-like activity over a period of 28 days, suggesting an excellent storage stability for long durations (Figure 5E).…”

“…Coacervates are the liquid-like membraneless condensates of the macromolecular rich aqueous phase formed via liquid–liquid phase separation between oppositely charged polycations and polyanions in aqueous medium. Coacervates find particular importance in artificial protocell research due to their bio-mimicking environment and membraneless architecture. , In the past decade, various functional coacervates have been designed using a wide range of polyelectrolytes. ,, Earlier, coacervates fabricated from organic molecules have been utilized as a scaffold to encapsulate various enzymes to study their enzymatic activity in a confined environment. − Recently, our group has fabricated a new class of hybrid coacervates via simple mixing of oppositely charged polyelectrolyte and inorganic NPs in aqueous medium. − These highly stable, robust, and optically active droplets have earlier been utilized for theranostic applications, , environmental remediation, and catalytic and photocatalytic nanoreactors. , In the present work, we have utilized quantum dot (QD)-embedded coacervate droplets (QD-Ds) as a scaffold for the fabrication of a new class of coacervate-based plexcitonic assembly via in situ generation of homogeneously dispersed Au NPs at room temperature without any reducing agent. The fabricated Au@QD-Ds have been found to possess intrinsic peroxidase-like activity with excellent storage ability, high recyclability as well as high structural stability at a wide pH and temperature range.…”