Acoustically controlled enhancement of molecular sensing to assess oxidative stress in cells

We demonstrate a microfluidic platform for the controlled aggregation of colloidal silver nanoparticles using surface acoustic waves (SAWs), enabling surface enhanced Raman scattering (SERS) analysis of a cell based model for oxidative damage. We show that by varying the frequency and the power of the acoustic energy, it is possible to modulate the aggregation of the colloid within the sample and hence to optimise the SERS analysis

SERS nanosensors for intracellular redox potential measurements

Redox regulation and homeostasis are critically important in the regulation of cell function; however, there are significant challenges in quantitatively measuring and monitoring intracellular redox potentials. The work in this thesis details a novel approach to intracellular redox monitoring. The approach is based on the use of nanosensors, which comprise molecules capable of sensing the local redox potential, assembled on gold nanoshells. Since the Raman spectra of the sensor molecules change depending on their oxidation state, and since the nanoshells allow a large enhancement of the Raman scattering, intracellular potential can be calculated by simple optical measurements. A full description of the design, fabrication and characterisation (spectroscopic and electrochemical) of the nanosensors is provided within. The ability to deliver nanosensors into cells in a controllable fashion was confirmed using electron microscopy. Results from a range of assays are also presented which reveal that introduction of nanosensors does not result in any cytotoxicity. Sensor utility in monitoring redox potentials as cells responded to physiological and superphysiological oxidative and reductive stimuli was investigated. Importantly, the capability of the nanosensors in monitoring intracellular potentials in a reversible, non-invasive manner, and over a previously unattainable potential range, is demonstrated

Multivariate spectral analysis of pH SERS probes for improved sensing capabilities

Appropriate functional groups adsorbed to the surface of plasmonic nanoparticles provides a platform for localised optical sensing. For example, intracellular pH nanosensors based on surface enhanced Raman spectroscopy (SERS) have been developed. However, the measurement methods and analysis of pH-SERS can greatly impact the precision and accuracy of pH calibration. This paper provides several key improvements to the performance and analysis pH nanosensors which thus transforms the performance into a useable intracellular pH sensor. We report the plasmon-induced decarboxylation of para-mercaptobenzoic acid (pMBA) pH-reporters which are bound to the gold nanoparticles, and attribute this to the laser power. This detrimental decarboxylation of pMBA has significant implications for accurate reporting and analysis due to the sensitivity and reliability of the pH sensor. The greatest implication of decarboxylation of pH sensors is inaccurate or false pH reporting, because the decarboxylation spectral signatures map directly onto those that are typically used to record pH changes. Here a unique application of the multivariate statistical technique, principal components analysis (PCA) is presented along with an optimal spectral region for pH calibration. By direct comparisons between the PCA method with the typically employed ratio-metric analysis a significant improvement in generating accurate pH sensing is demonstrated. An application of intracellular pH sensing in macrophage cells using these nanosensors promotes these step-changes in pH measurement methodology

Simultaneous intracellular redox potential and pH measurements in live cells using SERS nanosensors

The authors gratefully acknowledge the School of Chemistry at the University of Edinburgh, a Neil Campbell Travel Award, the Faculty of Chemistry at Jagiellonian University and Jagiellonian Centre for Experimental Therapeutics (JCET). A. J.’s work was supported by National Center of Science (grant PRELUDIUM DEC-2012/05/N/ST4/00218) and by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09).Intracellular redox potential is a highly regulated cellular characteristic and is critically involved in maintaining cellular health and function. The dysregulation of redox potential can result in the initiation and progression of numerous diseases. Redox potential is determined by the balance of oxidants and reductants in the cell and also by pH. For this reason a technique for quantitative measurement of intracellular redox potential and pH is highly desirable. In this paper we demonstrate how surface enhanced Raman scattering (SERS) nanosensors can be used for multiplexed measurement of both pH and redox potential in live single cells.Publisher PDFPeer reviewe

SERS-based monitoring of the intracellular pH in endothelial cells:the influence of the extracellular environment and tumour necrosis factor-alpha

The intracellular pH plays an important role in various cellular processes. In this work, we describe a method for monitoring of the intracellular pH in endothelial cells by using surface enhanced Raman spectroscopy (SERS) and 4-mercaptobenzoic acid (MBA) anchored to gold nanoparticles as pH-sensitive probes. Using the Raman microimaging technique, we analysed changes in intracellular pH induced by buffers with acid or alkaline pH, as well as in endothelial inflammation induced by tumour necrosis factor-alpha (TNF alpha). The targeted nanosensor enabled spatial pH measurements revealing distinct changes of the intracellular pH in endosomal compartments of the endothelium. Altogether, SERS-based analysis of intracellular pH proves to be a promising technique for a better understanding of intracellular pH regulation in various subcellular compartments.This work was supported by the National Center of Science (grant PRELUDIUM DEC-2012/05/N/ST4/00218) and by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, no. POIG.01.01.02-00-069/09). We also thank the University of Edinburgh School of Chemistry for the Neil Campbell Travel Award for supporting LJ. We also thank Joanna Jalmuzna from the Department of Mathematics and Computer Sciences, Jagiellonian University in Krakow for fitting the calibration curve using Gnuplot software

Raman spectroscopy: techniques and applications in the life sciences

Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine. It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration

Monitoring Intracellular Redox Potential Changes Using SERS Nanosensors

Redox homeostasis and signaling are critically important in the regulation of cell function. There are significant challenges in quantitatively measuring intracellular redox potentials, and in this paper, we introduce a new approach. Our approach is based on the use of nanosensors which comprise molecules that sense the local redox potential, assembled on a gold nanoshell. Since the Raman spectrum of the sensor molecule changes depending on its oxidation state and since the nanoshell allows a huge enhancement of the Raman spectrum, intracellular potential can be calculated by a simple optical measurement. The nanosensors can be controllably delivered to the cytoplasm, without any toxic effects, allowing redox potential to be monitored in a reversible, non-invasive manner over a previously unattainable potential range encompassing both superphysiological and physiological oxidative stress