Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution.

Caroline Bonazza,Rosa Mastrogiacomo,Paolo Pelosi,Jiao Zhu,Wolfgang Knoll,Roger Hasler

doi:10.3390/s21020499

Abstract

An electronic biosensor for odors was assembled by immobilizing the silk moth Bombyx mori pheromone binding protein (BmorPBP1) on a reduced graphene oxide surface of a field-effect transistor. At physiological pH, the sensor detects the B. mori pheromones, bombykol and bombykal, with good affinity and specificity. Among the other odorants tested, only eugenol elicited a strong signal, while terpenoids and other odorants (linalool, geraniol, isoamyl acetate, and 2-isobutyl-3-methoxypyrazine) produced only very weak responses. Parallel binding assays were performed with the same protein and the same ligands, using the common fluorescence approach adopted for similar proteins. The results are in good agreement with the sensor’s responses: bombykol and bombykal, together with eugenol, proved to be strong ligands, while the other compounds showed only poor affinity. When tested at pH 4, the protein failed to bind bombykol both in solution and when immobilized on the sensor. This result further indicates that the BmorPBP1 retains its full activity when immobilized on a surface, including the conformational change observed in acidic conditions. The good agreement between fluorescence assays and sensor responses suggests that ligand-binding assays in solution can be used to screen mutants of a binding protein when selecting the best form to be immobilized on a biosensor.

Highlights

Odorant-binding proteins (OBPs), both from insects [1,2,3] and from mammals [4,5], have received increasing attention as biosensing elements for detecting odorants and pheromones in the environment [6,7,8]
We focus on the use of graphene-based field-effect transistors, as they have been shown to give excellent sensing performance parameters in terms of high specific surface area, excellent electronic properties, and high flexibility [29]
The aim of this work was to verify whether a protein such as an odorant-binding proteins (OBPs) retained its specificity of binding when covalently immobilized on the biosensor surface

Summary

Introduction

Odorant-binding proteins (OBPs), both from insects [1,2,3] and from mammals [4,5], have received increasing attention as biosensing elements for detecting odorants and pheromones in the environment [6,7,8]. Their exceptional stability to temperature, up to around 70 ◦ C, allows for their use in demanding environmental conditions. They can be denatured by chemicals and refolded back into their active conformations after removing the denaturing agent [9,10].

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