Mycotoxins are small (MW ~ 700 g/mol), toxic chemical products formed as secondary metabolites by a few fungal species that readily contaminate crops in the field or after harvest. These compounds pose a potential threat to human and animal health through the ingestion of food products prepared from contaminated commodities.
At this time, the great diversity of toxins represents a challenge; detection methods are currently specific for individual toxins or groups of similar toxins. Because each toxin each requires a different method, standardization of techniques to detect all mycotoxins remains elusive. Likewise, practical requirements for high-sensitivity detection and the need for a specialized laboratory setting create challenges for routine analysis. Therefore, depending on the physical and chemical properties of the toxins, procedures have been developed around existing analytical techniques, which offer flexible and broad-based methods of detecting compounds (Turner et al. 2009
Traditionally, thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) have been employed for toxin detection. However, the tedious sample preparation and cleanup often lead to inconsistent results and poor sensitivity (Daly et al. 2000
). Various research groups have employed Surface Plasmon Resonance (SPR) - based sensors for applications such as inhibition immunoassays (Stubenrauch et al. 2009
) and antibody affinity analysis (Reid et al. 2007
). SPR analyzes changes in the interfacial optical properties of modified electrodes induced by the binding of biomolecules on the surface. Although the SPR platform is capable of label-free, real time monitoring of molecules as small as 200 Da, this requires highly sophisticated and expensive equipment (Skottrup et al. 2008
In their 2002 study, Schnerr et al. (Schnerr et al. 2002
) developed an inhibition immunoassay for the rapid quantification of the trichothecene mycotoxin deoxynivalenol using the SPR-based Biacore system. Despite its versatility, the complexity and the cost of the Biacore instrumentation remain very high (Mullett et al. 1998
). The low molecular weight of mycotoxins is often not enough to induce significant change upon binding to the sensor surface. Consequently, an alternative assay strategy is required for mycotoxin detection using SPR. One of the most established laboratory-based biochemical assays for pathogen detection is ELISA, which is based on the detection of pathogen-specific surface epitopes using antibodies (Cunningham 2000
). With its very high specificity and exceptional sensitivity, ELISA is often referred to as the gold standard of toxin detection. Nevertheless, current assays typically involve reporter molecules or labels conjugated to enzymes or fluorescent markers, which makes ELISA restricted to advanced laboratory settings with specialized read-out equipment (Skottrup et al. 2008
). Accurate and rapid read-out on site would provide vital efficiency in toxin detection, reducing potential risks of further unnecessary foodborne pathogen contamination. However, implementing ELISA into a point-of-use test remains challenging due to the sheer complexity of the instrumentation involved. In 2009, Valdes et al. reviewed the application of nanotechnology-based platforms for the detection of mycotoxins (Valdés et al. 2009
More recently, our lab employed a magnetic nanotag (MNT) detection platform for multiplexed mycotoxin detection (Mak et al. 2010
). Real-time measurements were conducted upon the addition of MNTs onto the spin-valve sensor surface immobilized with capture antibodies for mycotoxins (aflatoxin-B1, zearalenone and HT-2). The MNT technology demonstrated detection limits for mycotoxins in the pg/mL level.
Here we describe a new technique, Signal Transduction by Ion Nano Gating (STING), which uses a functionalized quartz nanopipette as an electrochemical biosensor. A key feature of this technology is that it doesn’t require any nanofabrication facility; each nanopipette can be easily, reproducibly, and inexpensively fabricated and tailored at the bench, thus reducing the cost and the turnaround time. The electrochemical sensitivity of the device is maximized at the nanopipette tip, essentially an elongated cone, making the dimension and geometry of the tip orifice crucial for biosensor performance (Umehara et al. 2009
). Permanent blockade, or gating, from binding events at the nanoscale-sized tip opening cause distinctive changes to the nanopipette electrical signature. The electrical changes are then detected with simple electrochemical measurements in real time without any need for labeling. For a more detailed explanation of the ion nano gating mechanism and the electrochemical characteristic of nanopipette electrodes, see our recent review, (Actis et al. 2010
). The selectivity of the nanopipette sensor can be customized for many different targets by introducing highly specific bio-recognition agents such as antibodies (Umehara et al. 2009
), DNA (Fu et al. 2009
), and aptamers (Ding et al. 2009
). The quartz pipettes also provide an ideal interface to append such bio-receptors using established surface-modification chemistry. Nanopipette-based platforms have been used to investigate single molecule biophysics (Clarke et al. 2005
), for the controlled delivery of molecules inside a single cell (Laforge et al. 2007
), and to image cells at the nanoscale (Klenerman and Korchev 2006
). We have recently demonstrated that STING technology can selectively detect interactions such as biotin-streptavidin and protein-protein binding (Umehara et al. 2009
In this paper, we discuss application of the STING platform for the ultrasensitive detection of a mycotoxin belonging to the species Fusarium, HT-2 toxin. The detection of HT-2 toxins presents unique challenges due to their low molecular weight (< 500 Da) and their insolubility. We examine the sensor’s limit of detection and linear range and compare the STING capabilities with respect to conventional sandwich assay techniques.