An enzymatic kinetics investigation into the significantly enhanced activity of functionalized gold nanoparticlesw

Nanoparticles are attracting substantial interest in the rapidly developing area of nanobiotechnology. Gold nanoparticles (AuNPs), which have high affinity for biomolecules, have been used as biosensors, immunoassays, therapeutic agents, and vectors for drug delivery; thus, the conjugation of AuNPs and biomolecules has become a major area of research for advancing the use of nanotechnology in biomedical applications. Indeed, proteins, enzymes, DNA, and oligonucleotides have all been immobilized onto AuNPs; the physicochemical characteristics of these functionalized AuNPs have been investigated in a variety of academic studies. Several techniques have been used to immobilize enzymes on various nanostructures in attempts to improve the enzymatic activity and stability. Although some enzyme-functionalized AuNPs exhibit enhanced catalytic activity, which has been supposed to favorable conformational changes and electrostatic interactions, there have been no detailed studies aimed at quantifying the differences between the enzymatic behavior of AuNPsimmobilized and free enzymes. Besides, it ought to evolve the regulable enzyme-functionalized nanoparticle with optimal efficiency and indicate the ability of the AuNPs to act as a factor for enhancing activity. Enzymes immobilized onto AuNPs in the absence of a linker, using rapid and uncomplicated processes, generally possess higher activity bound to the surfaces through chemical modification. In previous reports, catalytic activity of enzymefunctionalized AuNPs has been investigated with the surface modification of linkers. However, most of these kinetic investigations need steps such as modified biomolecules onto the AuNPs surface and separating the modified AuNPs from the unmodified AuNPs or surplus molecules. These steps, firstly, led to complication and relatively high cost of the experiments. In addition, long-time course (covalent bond) also led to activation loss of enzyme. What’s more, the target binding sites and conformational changes of the enzyme after binding were not all known precisely, so labeling sites were not only difficult to design, but also could weaken the affinity between the reactant and the enzyme. Therefore, developing modification-free AuNPs kinetic assays to simplify the detection process would be important and attractive. In this work, we have systematically investigated the interactions between the nanoparticle monolayer and the affected substrates by quantifying the kinetic parameters to understand the enhanced catalytic action of the AuNPs–lipase complex. We think that such fundamental research will be beneficial for the development of new nanobiotechnological applications. Lipases are used industrially as detergent enzymes, in paper and food technology, in the preparation of specialty fats, and as biocatalysts for the synthesis of organic intermediates, and in various clinical studies and drug delivery. The kinetic model of lipases is based on the so-called ping-pong mechanism, which also applies to many other enzymes, such as glucose oxidase, horseradish peroxidase, and alkaline phosphatase. As a representative esterase, lipase is an excellent model for studying the enhanced activity of the AuNPs-bound enzymes because of its well-defined structure, properties, and applications. Scheme 1 provides a schematic mechanism of lipase-catalysed reactions (see the ESI for a detailed discussionw). As shown in Fig. 1, by exploiting interactions between the AuNPs and lipase, colorimetric changes of the AuNPs could sensitively differentiate the enzyme concentration after titrating with the salt solution. We monitored the stability of the AuNPs solution by its color and the absorbance spectra. If the enzyme did not cap the AuNPs completely, we obtained spectra with the aggregation of the AuNPs. As long as the solution changed dramatically with addition of electrolyte from pink red to violet