Chapter 2 Nanosensors for Intracellular Raman Studies

Raman spectroscopy is an increasingly exploited tool for the study of cell biology, and the focus of this chapter is surface-enhanced Raman spectroscopy (SERS). Using SERS, noble metal nanoparticles can be interrogated with wavelengths of light to which cells exhibit minimal autofluorescence and return useful information about their immediate chemical environment. Using established gold– thiol surface chemistry, gold nanoparticles can be functionalised with suitable reporter molecules used to make nanosensors, which can be chosen to be sensitive to intracellular variables such as pH, protein concentration/activity or redox potential. This chapter presents the general concept of SERS for use in the monitoring of intracellular variables, and we will describe the preparation, use and functions of the nanosensors. 2.1 Background: Intracellular Nanosensors The chemical environment inside cells is of great interest, and many methods are known which can destructively assay properties such as redox potential [1] or the concentration of arbitrary metabolites [2]. However, if such information is desired about the progress of individual cells over time, or living tissues, then nondestructive methods of obtaining information are needed. Fluorescent sensors are widely used [3, 4], with fluorescent proteins and fluorescent nanoparticles being particularly popular because they can be engineered to respond to an environmental stimulus and report it as a measurable change in fluorescence (which is a wellestablished modality in cellular imaging). Nanoparticles are well suited since they are readily taken up by cells, and techniques to engineer the architecture and P.I. T. Thomson • C.J. Campbell ( ) EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, UK e-mail: patrick.thomson@gmail.com; colin.campbell@ed.ac. uk S. Li et al. (eds.), Nanoscale Sensors, Lecture Notes in Nanoscale Science and Technology 19, DOI 10.1007/978-3-319-02772-2__2, © Springer International Publishing Switzerland 2013 35 36 P.I. T. Thomson and C.J. Campbell composition of nanoparticles are well established. Fluorescent proteins exhibit a complementary set of benefits; cells can be engineered to stably express them and genetic engineering techniques can be used to modify their function. However, fluorescence-based methods have limitations, such as background fluorescence of the cell and medium limiting the lower limits of detection, and the requirement of a photoactivatable electronic transition which is sensitive to environmental conditions. Raman spectroscopy and imaging offers an alternative to fluorescence, and in this chapter, we provide some examples where the features of Raman spectroscopy can be used to the benefit of the experimentalist. 2.2 Background: Raman and SERS When photons are scattered by an atom or molecule, most of the light is reemitted unchanged (elastic or Rayleigh scattering). These resultant photons have the same kinetic energy as the incident ones, and are responsible for such phenomena as the colour of the sky [5]. However, approximately one in ten million reemitted photons have a different (usually lower) kinetic energy to the one absorbed (inelastic or Stokes Raman scattering)—indicating that the target molecule has undergone a transition in vibrational energy levels (Fig. 2.1) [6]. The frequency shift in these photons can be directly correlated to vibrational energy levels and thus gives us information about the vibrational modes of the target molecule in a similar fashion to infrared spectroscopy. The phenomenon was first observed and described by Sir Venkata Rāman, for which he was knighted and subsequently awarded the Nobel Prize in Physics in 1930 [7]. Raman and infrared spectroscopy can both probe the vibrational modes of target molecules, but the two techniques have the potential to return complementary information due the differing selection rules that govern which vibrational modes are IR Fig. 2.1 Scattering modes for the interaction of photons with matter 2 Nanosensors for Intracellular Raman Studies 37 active and which are Raman active. The main drawback of Raman spectroscopy is its low sensitivity—and the complex techniques thus required to separate out the Raman-scattered photons from the much more intense Rayleigh background [6]. The utility of Raman spectroscopy received a boost in 1974, when a phenomenon called surface-enhanced Raman spectroscopy (SERS) was first observed by Fleischmann [8, 9]. Specifically, he observed an anomalous enhancement of the Raman spectrum of pyridine adsorbed onto the surface of a roughened silver electrode. Theories to explain the phenomenon were proposed concurrently by Van Duyne [10] and Creighton [11], with the predominant theory today being Van Duyne’s proposed involvement of surface plasmon resonance. Incident light on a metallic surface can excite localised surface plasmons, which increases the local electric field and can enhance the Raman scattering of adsorbed molecules by a factor ranging from 4 to 14 orders of magnitude [12–15]. Silver and gold surfaces and nanoparticles are particularly useful substrates for SERS studies of biological systems since they are relatively inert and the wavelength required to excite their surface plasmons falls in the visible/near-IR range, to which biological systems are permeable [16]. For spherical homogeneous nanoparticles, the SERS signal is greatest when they are clustered together; the nanoparticle–nanoparticle junction experiences a localised enhancement in plasmon intensity, and this dominates signals from single nanoparticles. Various strategies have been developed in order to use single nanoparticles as effective SERS sensors without relying on aggregation, such as dielectric-cored gold nanoshells [17], gold nanorods or gold nanostars [18]. Exploiting these features, the SERS enhancement in some cases can permit the detection of single molecules [13, 19]. 2.3 SERS Measurements in Cells As a result of the high sensitivity, SERS is a very useful technique for measuring the Raman spectra of small numbers of molecules [19] or nanoparticles, and as a result it has been used to explore the environment inside individual cells. Even a single gold nanoparticle can return useful spectral information about analytes close to its surface, and this has been used to probe the internal environments of both bacterial [20] and eukaryotic [21, 22] cells directly. To use SERS in this way, it is necessary to know where in the cell the nanoparticle locates and whether its presence has any detrimental effect on the cell (e.g., does it cause stress or induce cell death? ). Gold nanoparticles delivered to cells in this manner do not typically affect cell viability, and we have demonstrated that they do not induce cell death (either apoptotic or necrotic) or cause oxidative stress. Cellular uptake of gold nanoshells [22] is dependent on a variety of factors. We tested various culture conditions in order to probe different endocytosis mechanisms (e.g., by depleting clathrin, cholesterol or ATP from the cells), but we found that the greatest level of cellular delivery is when using cell culture media containing no calf serum (Fig. 2.2). We attribute this finding to the fact that serum proteins are known to coat nanoparticles, 38 P.I. T. Thomson and C.J. Campbell