Versatile multi-functionalization of protein nanofibrils for biosensor applications

Abstract

Protein nanofibrils offer advantages over other nanostructures due to the ease in their self-assembly and the versatility of surface chemistry available. Yet, an efficient and general methodology for their post-assembly functionalization remains a significant challenge. We introduce a generic approach, based on biotinylation and thiolation, for the multi-functionalization of protein nanofibrils self-assembled from whey proteins. Biochemical characterization shows the effects of the functionalization onto the nanofibrils' surface, giving insights into the changes in surface chemistry of the nanostructures. We show how these methods can be used to decorate whey protein nanofibrils with several components such as fluorescent quantum dots, enzymes, and metal nanoparticles. A multi-functionalization approach is used, as a proof of principle, for the development of a glucose biosensor platform, where the protein nanofibrils act as nanoscaffolds for glucose oxidase. Biotinylation is used for enzyme attachment and thiolation for nanoscaffold anchoring onto a gold electrode surface. Characterization via cyclic voltammetry shows an increase in glucose-oxidase mediated current response due to thiol-metal interactions with the gold electrode. The presented approach for protein nanofibril multi-functionalization is novel and has the potential of being applied to other protein nanostructures with similar surface chemistry.

A Compact Microelectrode Array Chip with Multiple Measuring Sites for Electrochemical Applications

“…Although there are some issues with the electrode surface properties in this particular experiment, there is a clear difference between the control and the glucose oxidase modified nanofibrils. These results are comparable to similar PNF-based glucose biosensor systems that utilize commercially available electrodes [15]. …”

“…PNFs formed from whey protein isolate were utilized as a nanoscaffold for glucose oxidase (GOx) immobilization on the interdigitated WEs. The PNFs were functionalized with GOx for biosensing and with thiol moieties for gold surface attachment using a multifunctionalization approach [15]. After PNF-GOx immobilization onto the electrodes, cyclic voltammetry was used as detection method.…”

“…Protein nanofibrils (PNFs) from whey protein isolate were fabricated and functionalized with glucose oxidase and thiol moieties using a previously reported multifunctionalization approach [15]. Deposition of the functionalized PNFs onto electrodes was done by pipetting 200 µL of a PNF solution (2 mg/mL) onto the microchip and allowing the PNFs to sediment onto the electrodes for 30 min.…”

Protein nanorings organized by poly(styrene-block-ethylene oxide) self-assembled thin films

“…24 The main challenge facing the field of protein nanotechnology is the ability to control proteinprotein interactions to build up higher order structures, and in particular to order these structures. Previous work on creating protein assemblies has been largely centered around amyloid fibers [25][26][27][28][29] but has recently also involved native protein structures [30][31][32] with ring shaped proteins emerging as a commonly used self-assembling feature. [33][34][35][36][37] Recent work has established methods to post functionalize assembled structures, 38,39 an important step towards applications based on protein nanostructures.…”

Design of whey protein nanostructures for incorporation and release of nutraceutical compounds in food

“…Recently, other potential applications have attracted a wide interest from food and biomedical industries, such as enzyme immobilization, microencapsulation of bioactive ingredients, or even development of biosensors (Loveday et al, 2012a;Sasso et al, 2014).…”