Fluorescent proteins as biosensors by quenching resonance energy transfer from endogenous tryptophan: Detection of nitroaromatic explosives

Highlights

• We examined seven fluorescent proteins (FPs) as biosensors for nitrated explosives.

• Utilizing FRET from endogenous tryptophan residues is a novel application.

• Unmodified fluorescent proteins can discriminate between TNT and RDX.

• FPs incorporating peptides from phage display libraries might increase selectivity and sensitivity.

• This method can be applied for detecting any reagents that quench indole fluorescence.

Abstract

Ensuring domestic safety from terrorist attack is a daunting challenge because of the wide array of chemical agents that must be screened. A panel of purified fluorescent protein isoforms (FPs) was screened for the ability to detect various explosives, explosive simulants, and toxic agents. In addition to their commonly used visible excitation wavelengths, essentially all FPs can be excited by UV light at 280 nm. Ultraviolet illumination excites electrons in endogenous tryptophan (W) residues, which then relax by Förster Resonance Energy Transfer (FRET) to the chromophore of the FP, and thus the FPs emit with their typical visible spectra. Taking advantage of the fact that tryptophan excitation can be quenched by numerous agents, including nitroaromatics like TNT and nitramines like RDX, it is demonstrated that quenching of visible fluorescence from UV illumination of FPs can be used as the basis for detecting these explosives and explosive degradation products. This work provides the foundation for production of an array of genetically-modified FPs for in vitro biosensors capable of rapid, simultaneous, sensitive and selective detection of a wide range of explosive or toxic agents.

Introduction

Accurate and rapid identification of explosives, toxins, and poisons is critical for the military, civilian security, first responders, and for humanitarian remediation work. Detection of TNT and other nitroorganic explosives is of particular concern. Fortunately, the past decade has seen a tremendous advancement in both sensitivity and selectivity for TNT detection methodologies. These detection techniques can be generally classified as physical, chemical or biological, depending upon the nature of the sensor utilized (Wang et al., 2012). Techniques amenable to TNT biosensing include colorometric analysis (Smith et al., 2008), fluorometric analysis (Bromage et al., 2007) including FRET (Medintz et al., 2005) and quenching (Abe et al., 2011), chemiluminescence (Maioloni et al., 2009), gravimetric analysis (Wang et al., 2012), electrochemical analysis (Caygill et al., 2013) and surface plasmon resonance (Shankaran et al., 2005) among others. Sensitivities for these methods vary widely, as do the times required to accurately identify and quantify TNT, but concentrations of TNT in the ppm–ppb range can commonly be detected with times ranging from seconds to minutes. Most of the biosensing techniques utilize monoclonal or polyclonal antibodies that recognize TNT (Charles et al., 2004), although TNT binding peptides (Jaworski, 2009), nucleic acid polymers (Ehrentreich-Foerster et al., 2008), and even live cell techniques (Burlage, 2009) have also been utilized. With improvements in antibody production and specificity using better haptens, these techniques should continue to improve detection limits and selectivity (Ramin and Weller, 2012). While antibody stability is acceptable, those from most species except camelids (Anderson and Goldman, 2008) are subject to thermal denaturation at relatively moderate temperatures (Zhang et al., 2012). Antibodies are also sensitive to proteases (Cho et al., 2006) and chemical denaturants, and relatively low concentrations of many organic solvents (Horacek and Skladal, 2000) alter binding. Finally when fluorescence-based biosensing with antibodies utilizes conventional organic dyes, they are subject to bleaching (Lesner, 2012).

In the past two decades, thousands of unique FP isoforms and fusion proteins have been produced with emission maxima in every portion of the visible spectrum (Shaner et al., 2005, Tsien, 2009, Miyawaki, 2011, Suetsugu et al., 2012, Vizcaino-Caston and Wyre, 2012), and the Protein Data Bank contains over 400 entries of various FPs. Of the hundreds of thousands of papers describing FPs and their applications, only a handful mention the UV excitation peak centered near 280 nm (Niwa et al., 1996, van Thor et al., 2002, Sniegowski et al., 2005, Gurskaya et al., 2001, Alvarez et al., 2010), and fewer still discuss or evaluate that peak in any detail (van Thor et al., 1998, Visser et al., 2005, Budisa et al., 2004). Visser et al. (2005) unambiguously demonstrated that FRET from endogenous W residues to the chromophore is genuinely occurring within FPs and is responsible for the 280 nm excitation peaks. Because most applications for FPs have been for in vivo use, the UV excitation has been largely ignored as excessive UV illumination would almost certainly lead to cellular damage in living cells and complicate interpretation of results. However, UV excitation of FPs could be valuable for in vitro biosensing. Importantly, the FPs are superior to dye-labeled fluorescent probes in that they are remarkably stable to proteases and chemical agents (Mazzola et al., 2006), they can tolerate relatively high concentrations of many organic solvents (Samarkina et al., 2009), they are extremely resistant to bleaching (Yoo et al., 2012), and mutations providing hyperthermophilic stability have been identified (Kiss et al., 2009). Herein, we describe a panel of fluorescent proteins used for detection of explosives and toxic agents including TNT, RDX and paraoxon. This is the first report describing the use of endogenous W FRET within FPs for biosensing.