Novel Nanozeolitic Imidazolate Framework (ZIF-8)–Luciferase Biocomposite for Nanosensing Applications

Abstract

The identification of new strategies to improve the stability of proteins is of utmost importance for a number of applications, from biosensing to biocatalysis. Metal−organic frameworks (MOFs) have been shown as a versatile host platform for the immobilization of proteins, with the potential to protect proteins in harsh conditions. In this work, a new thermostable luciferase mutant has been selected as a bioluminescent protein model to investigate the suitability of MOFs to improve its stability and prompt its applications in real-world applications, for example, ATP detection in portable systems. The luciferase has been immobilized onto zeolitic imidazolate framework-8 (ZIF-8) to obtain a bioluminescent biocomposite with enhanced performance. The biocomposite ZIF-8@luc has been characterized in harsh conditions (e.g., high temperature, non-native pH, etc.). Bioluminescence properties confirmed that MOF enhanced the luciferase stability at acidic pH, in the presence of organic solvents, and at −20 °C. To assess the feasibility of this approach, the recyclability, storage stability, precision, and Michaelis−Menten constants (K m ) for ATP and D-luciferin have been also evaluated. As a proof of principle, the suitability for ATP detection was investigated and the biocomposite outperformed the free enzyme in the same experimental conditions, achieving a limit of detection for ATP down to 0.2 fmol.

Super-Stable Metal–Organic Framework (MOF)/Luciferase Paper-Sensing Platform for Rapid ATP Detection

“…We recently reported the advantageous combination of ZIF-8 and luciferase for improving the analytical performance and stability of luciferase-based assays [ 32 ]. Encouraged by these results, we explored the applicability of these nanobiocomposites for the development of ready-to-use BL paper biosensing platforms ( Figure 1 ).…”

“…The synthesis of the biocomposite was based on a previously reported procedure [ 32 , 35 ], with minor modifications. Briefly, ZIF-8 was synthesized by adding a solution of 50 mL of MeOH containing 19.76 mmol of HMIM dropwise into 50 mL of a MeOH solution containing 2.47 mmol of Zn(NO 3 ) 2 ×6H 2 O.…”

“…ZIF-8 is constructed from Zn(II) ions and 2-methylimidazole ligands, synthesized under mild conditions in short periods of time. Despite the popularity of this material and its benefits, the combination of ZIF-8 with luciferase has only recently been explored [ 32 ]. We demonstrated that the immobilization of luciferase onto ZIF-8 not only favored enzyme protection towards denaturing conditions (e.g., the presence of organic solvents, an acidic pH and repeated freeze/thawing processes), but also increased the storage stability at room temperature.…”

“…We demonstrated that the immobilization of luciferase onto ZIF-8 not only favored enzyme protection towards denaturing conditions (e.g., the presence of organic solvents, an acidic pH and repeated freeze/thawing processes), but also increased the storage stability at room temperature. Although other MOF supports (e.g., UiO-66 and MIL-101) could be used to host biomolecules, the intrinsic properties of ZIF-8 make it the most suitable candidate for biosensor development [ 32 ].…”

Colorimetric Paper Sensor for Food Spoilage Based on Biogenic Amine Monitoring

“…In this scenario, the reduction of food waste and monitoring of food quality and safety are of the utmost importance. The food industry is always looking for new methodologies to monitor the quality and freshness of food [2], the authenticity and potential adulterations of food [3,4] and toxins and other contaminants [5,6]. With the increase in consumers' desire to actively monitor food quality, the food packaging industry is facing new challenges in monitoring the entire food distribution chain and preventing food spoilage and counterfeited food products.…”