This work presents the development and validation of a kinetic model describing the enzymatic hydrolysis of a specifically designed fluorogenic probe for free Penicillin-G Acylase (PGA). The model construction involved tracking reaction kinetics through UV–Vis spectroscopy, identifying product-induced inhibitory effects, and employing initial velocity analysis alongside parameter estimation techniques. The kinetic model was structured around a simple ordered uni-bi mechanism comprising three reversible reaction steps. Validation of the model was performed through spectrofluorometric measurements, successfully predicting the fluorescence intensity progression resulting from the enzymatic cleavage of the probe.

Numerous metabolic processes in nature are governed by extrinsic stimuli such as light and pH variations, which afford opportunities for synthetic and biological applications. In developing a multisensor apparatus, we have integrated submicrometer purple membrane patches, each harboring bacteriorhodopsin, onto a surface. Bacteriorhodopsin is a light-driven proton pump. We conducted monitoring of the interactions between this system and a pH-responsive supramolecular hydrogel to evaluate fibrous matrix growth. Initial photostimulation induced localized reductions in pH at the membrane surface, thereby catalyzing fibrogenesis within the hydrogel. Utilizing liquid atomic force microscopy alongside confocal laser scanning microscopy, we observed the hydrogel’s morphogenesis and structural adaptations in real time. The system adeptly modulated microscale pH environments, fostering targeted fibrous development within the hydrogel matrix. This elucidates the potential for engineering responsive materials that emulate natural bioprocesses.

We present an approach for detecting thiol analytes through a self-propagating amplification cycle that triggers the macroscopic degradation of a hydrogel scaffold. The amplification system consists of an allylic phosphonium salt that upon reaction with the thiol analyte releases a phosphine, which reduces a disulfide to form two thiols, closing the cycle and ultimately resulting in exponential amplification of the thiol input. When integrated in a disulfide cross-linked hydrogel, the amplification process leads to physical degradation of the hydrogel in response to thiol analytes. We developed a numerical model to predict the behavior of the amplification cycle in response to varying concentrations of thiol triggers and validated it with experimental data. Using this system, we were able to detect multiple thiol analytes, including a small molecule probe, glutathione, DNA, and a protein, at concentrations ranging from 132 to 0.132 μM. In addition, we discovered that the self-propagating amplification cycle could be initiated by force-generated molecular scission, enabling damage-triggered hydrogel destruction.