Molecular Engineering of a Fluorescent Bioprobe for Sensitive and Selective Detection of Amphibole Asbestos

Takenori Ishida,Takeshi Ikeda,Maxym Alexandrov,Tomoki Nishimura,Ryuichi Hirota,Akio Kuroda,Anna Mitraki

doi:10.1371/journal.pone.0076231

Abstract

Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an Escherichia coli histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe’s affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe’s affinity to asbestos.

Highlights

Interaction between proteins or peptides and inorganic materials is an important research subject in various fields of applied research, from nanotechnology to the development of bioassays, biosensors and biocompatible materials [1]
To improve the specificity of the amphibole probe, we conducted another screening for amphibole asbestos–binding proteins from E. coli lysate under more stringent conditions, and identified histone-like nucleoid-structuring (H-NS) protein using MALDI-TOF analysis (Figure 1)
The probable mechanism of the increase in affinity is by preventing dissociation of the probe after its initial contact with the asbestos surface

Summary

Introduction

Interaction between proteins or peptides and inorganic materials is an important research subject in various fields of applied research, from nanotechnology to the development of bioassays, biosensors and biocompatible materials [1]. One of the emerging applications for the material-binding proteins and peptides is the fluorescence microscopy-based affinity assay for asbestos, a common inorganic pollutant that can cause asbestosis, mesothelioma and lung cancer [2,3,4]. The use of asbestos is prohibited in most developed countries, large amounts of asbestos still remain in many older buildings. Asbestos contamination remains a major problem, with many countries reporting rising incidence of asbestoslinked pleural mesothelioma and lung cancers [6,7]

Objectives

Methods

Results

Conclusion