Detection of heavy metal ions using protein-functionalized microcantilever sensors

Abstract

Microcantilevers functionalized with metal-binding protein, AgNt84-6, are demonstrated to be sensors for the detection of heavy metal ions like Hg²⁺ and Zn²⁺. AgNt84-6, a protein that has the ability to bind multiple atoms of Ni²⁺, Zn²⁺, Co²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ was attached to the gold-coated side of silicon nitride cantilevers via linker groups. Upon exposure to 0.1 mM HgCl₂ and 0.1 mM ZnCl₂ solutions, the microcantilevers underwent bending corresponding to an expanding gold side. Exposure to a 0.1 mM solution of MnCl₂ solution did not result in a similar bending indicating a weak or no interaction of Mn²⁺ ions with the AgNt84-6 protein. The microcantilever bending data were consistent with data from electrophoresis carried out on SDS-PAGE gels containing metal ions that showed protein interaction with Zn²⁺ ions but not with Mn²⁺ ions. Thus, we demonstrate that microcantilever bending can be used to discriminate between metal ions that bind and do not bind to AgNt84-6 protein in real time.

Introduction

Pollution of ground water and soil with toxic heavy metals like mercury, cadmium, lead, etc. pose a serious health risk. Because metals are non-degradable they tend to bioaccumulate as they move up the food chain. The main sources of soil and ground water pollution are improper waste dumping, agricultural chemicals, and industrial effluents (Chen et al., 1998, Abollino et al., 2002). The toxicity of these heavy metals depends on their concentration and also on their speciation. There exists a need for rapid and wide scale monitoring of heavy metals in the environment. Simple, sensitive sensors that can measure multiple elements simultaneously would be of great significance for wide scale monitoring.

A new class of MEMS-based mechanical transducers are currently being developed using micocantilevers (Gimzewski et al., 1994, Thundat et al., 1994, Raiteri and Butt, 1995). Microcantilevers are mechanical stress sensors that are being developed as physical (Barnes et al., 1994, Datskos et al., 1996, Abedinov et al., 2001) chemical, (Thundat et al., 1995a, Thundat et al., 1995b, Lang et al., 1999, Raiteri et al., 2000, Tipple et al., 2002) and biological (Raiteri et al., 1999, Moulin et al., 2000, Hansen et al., 2001, Grogan et al., 2002) sensors. When molecular binding occurs preferentially on one side of a cantilever, it undergoes bending corresponding to the differential stress occurring on either side of the cantilever. Moreover, the fundamental resonance frequency of the cantilever changes depending on changes in its spring constant and effective mass (Chen et al., 1995). By appropriately modifying the cantilever surface, binding-induced changes can be monitored.

Several proteins and peptides are known to bind heavy metals (Kabata-Pendia and Pendias, 1992, Payne et al., 1999, Hershfinkel et al., 2001, Memon et al., 2001, DeSilva et al., 2002) and many of these are synthesized in cells as a detoxification response to the presence of specific metals (Memon et al., 2001) or to serve transport or regulatory functions (Hershfinkel et al., 2001).

In this paper we report the use of a new class of plant metal-binding proteins as biosensors for heavy metal detection. These proteins, called metallohistins are typified by AgNt84-6 a small glycine- and histidine-rich protein expressed in actinorhizal plants in response to infection by the nitrogen-fixing actinomycete Frankia (Pawlowski et al., 1997, Gupta et al., 2002). AgNt84-6 has been shown by equilibrium dialysis and mass spectral analysis to bind multiple atoms of Zn²⁺, Ni²⁺, Co²⁺, Cd²⁺, Hg²⁺ and Cu²⁺, but not Ca²⁺, Mg²⁺ and Mn²⁺) with average apparent K_ds of from 1.7 to 30.8 μM and β_maxs of from 3 to 11 mol of metal/mol of monomer (Gupta et al., 2002). Using nuclear magnetic resonance (NMR) spectroscopy it was shown that metal binding involves the Cε1 and Cδ2 protons of histidines and that binding is reversible. Here we present initial results that demonstrate that the binding selectivity observed using classical methods of analysis is preserved and can be observed in real time using protein-functionalized microcantilevers. Studies are ongoing to determine the limit of detection for selected heavy metal ions.