Biosensing based on surface plasmon resonance and living cells
Introduction
Environmental and biomedical research is pushing towards the development of biosensors able to rapidly monitor the biological activity of molecules, such as toxins, chemicals or agonists. Most of the research in these fields is in the development of specific biosensors, where the detector surface is functionalized in order to detect the presence of a specific target molecule (Malhotra and Chaubey, 2003). However, there is a need for broad-range biosensors, where the focus is to detect molecules involved in a wide variety of biological responses (Bousse, 1996). In this branch of sensing lies the concept of a living cell biosensor, where the definition of the sensor is extended to include a living cell population as the sensing element (Pancrazio et al., 1999). The cells therefore act as reporters of their environment, where the detection method must register the cellular signals.
Living cell sensing is already the subject of extensive research and many non-optical sensing methods allow the measurement of cellular metabolism, such as electrochemical sensors and light-addressable potentiometric sensors (Yotter and Wilson, 2004, Xu et al., 2005). The concept of a biosensor, based on cells as the sensing elements to monitor toxicity and cell metabolism, has already been demonstrated using electric cell-substrate impedance sensing (Keese and Giaever, 1994, Arndt et al., 2004). Optical sensing methods, such as nanoprobes and fluorescence microscopy, are also extensively applied to monitor living cell activity (Krishnan et al., 2001, Vo-Dinh et al., 2006). Among the optical detection methods for living cell sensing, surface plasmon resonance (SPR) is one of the most promising candidates for non-invasive detection assays because it does not require labelling agents (Yotter et al., 2004). Here, we demonstrate that SPR can be combined with living cells to monitor the effects of different molecular stimuli on cellular activity.
A surface plasmon is a charge-density oscillation occurring at the interface between a metal such as gold or silver and a dielectric (Homola et al., 1999). The following equation defines the wave vector of a surface plasmon wave (Ksp):where ω is the angular frequency, c the speed of light, ɛM the metal permittivity and nD the dielectric (sensing medium) refractive index. This equation defines a surface plasmon when the real part of ɛM is negative and its absolute value is greater than (Homola, 2003).
In order to match the wavevectors of the incident light and the surface plasmons, as required for surface plasmon resonance, a coupling method (prism or grating) must be used (Zayats and Smolyaninov, 2003). In the Kretschmann geometry, a p-polarized laser beam is reflected off the sample through a high refractive index prism at an angle superior to the angle of total internal reflection (Fig. 1a). The reflection at the metal–dielectric interface creates an evanescent field in both the metal and the dielectric (Homola, 2006). This exponentially decaying field is strongly attenuated in the metal, but propagates a greater distance (∼200 nm) into the dielectric (sensing medium). The evanescent field has the following wave vector (Kev) equation:where np is the refractive index of the prism and θ the angle of incidence.
We see that at specific conditions of incident angle, laser wavelength and refractive indices of the prism and sensing medium, optimal coupling between light and plasmons occurs because the momentum conditions match (Kev = Ksp), giving:
Therefore, when the prism refractive index np and the laser wavelength (affecting ɛM) are kept constant, we see that the conditions for maximum coupling will change with a modification of the sensing medium refractive index nD, caused for example by the adsorption of biomolecules on the surface. The coupling appears as a sharp dip in the measured laser reflectance as a function of incident angle (Fig. 1b).
Recently, SPR has been applied to the real-time monitoring of living cells to probe molecular processes (Hide et al., 2002, Yanase et al., 2007). It has also been used to detect blood coagulation and platelet adhesion under physiological flow conditions (Hansson et al., 2007). Infrared SPR has also been used to monitor the enrichment and depletion of cholesterol in the cell plasma membrane (Ziblat et al., 2006). Changes in cellular morphology, observed through microscopy techniques, are often used as a simple mean to evaluate cellular activity following specific activation (Giuliano et al., 2003, Tencza and Sipe, 2004, Butcher, 2005). These techniques are ultimately limited by the capacity to discriminate changes observed at typical microscopic resolutions. Since cellular activation often results in morphology changes such as cell contraction and spreading, it should affect the basal portion of the cell, which lies within the evanescent field of the surface plasmon (Fig. 1c). Therefore, surface plasmon resonance is an excellent candidate for the real-time monitoring of cellular activation involving morphological remodeling.
In this study, we monitored living cells grown on SPR sensing surfaces following the injection of various molecular stimuli. First, we used a bacterial endotoxin, lipopolysaccharides (LPS), known to cause an important cellular response often leading to cell death (Heumann and Roger, 2002). LPS is strongly implicated in the inflammatory response induced by Gram-negative bacteria such as Escherichia coli and Staphylococcus aureus (Heumann and Roger, 2002). Important efforts have been deployed to develop new diagnostic methods to detect in real-time such infections in a clinical environment (Lazcka et al., 2007, Maalouf et al., 2007). Many studies have already described the cellular effects of LPS on various cell types by the activation of its membrane receptor (Raetz and Whitfield, 2002) and have demonstrated that this agent induces cell death, associated with morphological changes such as cell rounding and membrane blebbing (Aliprantis et al., 1999, Chakravortty et al., 2000).
To evaluate the sensitivity to activation by a chemical agent, we injected sodium azide, a chemical toxin known to prevent cellular respiration by inhibiting the Cytochrome c oxidase-respiratory chain complex IV (Leary et al., 2002, Ishikawa et al., 2006). This toxic chemical compound is used for many purposes, such as a preservative for aqueous laboratory reagents as well as a gas-generating agent in automobile airbags (Chang and Lamm, 2003). Sodium azide is noxious for human health and its presence in an increasing variety of products makes it a relevant candidate for toxicity detection by our system.
Finally, to demonstrate the SPR response to specific pharmacological activation, we used thrombin, which is a physiological agonist involved in the modulation of cell layer integrity (Bogatcheva et al., 2002, Coughlin, 2005). Thrombin is well known to be implicated in hemostasis and vascular endothelium permeability (Arnout et al., 2006, Komarova et al., 2007). The activation of protease activated receptors (PAR-1) by thrombin is well documented to lead to cell contraction (Nobe et al., 2005).
Section snippets
Surface plasmon resonance substrates
Standard glass microscope slides were used as base substrates. Prior to metal deposition, the glass slides were cleaned in piranha solution to remove any contaminants; afterwards they were placed under vacuum for metal deposition (BOC Edwards evaporator, model: AUTO 306). A chromium adhesion layer (3 nm) and a gold layer (48 nm) were deposited subsequently without breaking vacuum between evaporation. Prior to cell culture, the gold surfaces were coated with poly-l-lysine (Sigma, Oakville, ON,
Results
To show that SPR can be applied to the monitoring of cell morphology remodeling, we first stimulated the cells with lipopolysaccharides (LPS). Fig. 2a shows two typical SPR angular scans of a confluent HEK-293 cell monolayer, taken before (○) and after a 30 min stimulation (×) with 5 mg/ml LPS. The quasi-linear region of the reflectance curve, between 71° and 74°, is displaced to lower angles after stimulation. As shown in Fig. 2b, at a fixed angle of 72° (see Section 2 for the selection
Discussion
Association of the SPR response with cell morphological changes confirmed by phase contrast microscopy shows that combining surface plasmon resonance with a confluent living cell monolayer can be used to form a broad-range biosensor that allows the monitoring of chemical or biological agents that induce morphological changes in the cells.
Our SPR measurements for LPS stimulation show a rapid decrease of the signal, which present a strong correlation with the microscopic observations. Indeed,
Conclusion
We have shown that surface plasmon resonance has the capacity to determine adhesion and morphological changes in cellular activity in real-time following activation through various agents. Moreover, the cell population can be used as the sensing element and thus can act as a reporter of the cellular environment. The specific outcome of this work is the development of a biosensor with environmental and health care applications to determine toxin presence and their corresponding cellular
Acknowledgements
This work was supported by funds from Canadian Institutes of Health Research (CIHR: E.E., M.G.), the Fonds de la Recherche en Santé du Québec (FRSQ: E.E.), the Natural Sciences and Engineering Research Council of Canada (NSERC: V.A., M.G., P.C.) and the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT: E.E., V.A., M.G., P.C.).
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