Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E⊕) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography–mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists.

Multicellular tumour spheroids (MTS) are three-dimensional cell cultures that possess their own microenvironments and provide a more meaningful model of tumour biology than monolayer cultures. As a result, MTS are becoming increasingly used as tumor models when measuring the efficiency of therapies. Monitoring the viability of live MTS is complicated by their 3D nature and conventional approaches such as fluorescence often require fixation and sectioning. In this paper we detail the use of Surface Enhanced Raman Spectroscopy (SERS) to measure the viability of MTS grown from prostate cancer (PC3) cells. Our results show that we can monitor loss of viability by measuring pH and redox potential in MTS and furthermore we demonstrate that SERS can be used to measure the effects of fractionation of a dose of radiotherapy in a way that has potential to inform treatment planning.

Redox potential is of key importance in the control and regulation of cellular function and lifecycle, and previous approaches to measuring the biological redox potential noninvasively in real time are limited to areas of hypoxia or normoxia. In this paper, we extend our previous work on nanoparticle-based intracellular nanosensors to cover a much wider redox potential range of −470 to +130 mV vs NHE, which includes the redox potential range occupied by cells in a state of oxidative stress. The nanosensors are rationally designed to target different areas of this redox potential range and are monitored by surface-enhanced Raman spectroscopy, which will permit noninvasive real-time imaging of cells undergoing oxidative stress.

Conventional two dimensional (2D) monolayer cell culture has been considered the ‘gold standard’ technique for in vitro cellular experiments. However, the need for a model that better mimics the three dimensional (3D) architecture of tissue in vivo has led to the development of Multicellular Tumour Spheroids (MTS) as a 3D tissue culture model. To some extent MTS mimic the environment of in vivo tumours where, for example, oxygen and nutrient gradients develop, protein expression changes and cells form a spherical structure with regions of proliferation, senescence and necrosis. This review focuses on the development of techniques for chemical analysis of MTS as a tool for understanding in vivo tumours and a platform for more effective drug and therapy discovery. While traditional monolayer techniques can be translated to 3D models, these often fail to provide the desired spatial resolution and z-penetration for live cell imaging. More recently developed techniques for overcoming these problems will be discussed with particular reference to advances in instrument technology for achieving the increased spatial resolution and imaging depth required.

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.

Hypoxia is considered to be a reductive disorder of cells that is caused either by a lack of oxygen or by the dysregulation of metabolic pathways and is thought to play a role in the pathology of diseases including stroke and cancer. One aspect of hypoxia that remains poorly investigated is the dysregulation of cellular redox potential and its role in controlling biological pathway activation. Since there is currently no way of quantitatively measuring the intracellular redox potential of hypoxic cells, this provided us with the motivation to develop optical nanosensors whose Surface-Enhanced Raman (SER) spectrum provides a quantitative measure of redox potential in hypoxic cells. Our nanosensors are made from organic reporter molecules that show oxidation-state-dependent changes in the Raman spectrum and are chemically adsorbed onto gold nanoshells. These nanosensors can be taken up by cells, and by collecting the SER spectrum we can calculate the localised intracellular redox potential from single hypoxic cells in a non-invasive, reversible way.

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.Keywords: intracellular; nanosensor; redox; SERS

We describe a novel surface enhanced Raman spectroscopy (SERS) sensing approach utilizing modified gold nanoshells and demonstrate its application to analysis of critical redox-potential dependent changes in antigen structure that are implicated in the initiation of a human autoimmune disease. In Goodpasture’s disease, an autoimmune reaction is thought to arise from incomplete proteolysis of the autoantigen, α3(IV)NC167–85 by proteases including Cathepsin D. We have used SERS to study conformational changes in the antigen that correlate with its oxidation state and to show that the antigen must be in the reduced state in order to undergo proteolysis. Our results demonstrate that a redox potential of ∼−200 mV was sufficient for reduction and subsequent productive processing of the antigenic fragment α3(IV)NC167–85. Moreover, we demonstrate that the peptide bonds subsequently cleaved by Cathepsisn D can be identified by comparison with a SERS library of short synthetic peptides.

We demonstrate a strategy for label-free protein detection through monitoring the Surface Enhanced Raman Spectrum of an aptamer probe attached to a gold nanoshell. Low limit of detection and minimal non-specific binding show potential for in vitro and in vivo assays.

Varicella zoster virus (VZV) is a human herpesvirus encoding at least 69 distinct viral proteins which causes chickenpox after primary infection and shingles during reactivation and which is particularly important in pregnancy and immunocompromised patients. Current serodiagnostic tests are either based on whole cell lysates or glycoprotein preparations. In order to investigate the humoral immune response to VZV infection or vaccination in more detail, and to improve the currently available diagnostic assays, we developed a nucleic acid programmable protein array (NAPPA) containing all 69 VZV proteins and performed a detailed analysis of 68 sera from individuals with either no, a previous or an acute VZV infection. In addition to the known reactive glycoprotein antigens (ORF 5, ORF 14, ORF 31, ORF 37, ORF 68), we discovered IgG antibodies against a variety of other membrane (ORF 2, ORF 24), capsid (ORF 20, ORF 23, ORF 43) and tegument (ORF 53, ORF 9, ORF 11) proteins, as well as other proteins involved in virus replication and assembly (ORF 25, ORF 26, ORF 28) and the transactivator proteins ORF 12, ORF 62 and ORF 63. All of these antigens were only reactive in a subset of VZV-positive individuals. A subset of the newly identified VZV antigens was validated by western blot analysis. Using these seroreactive new VZV antigens, more sensitive assays and tests distinguishing between different clinical entities may be developed.

DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.

The application of gold nanoshells (NS) as a surface-enhanced Raman (SER) platform for intracellular sensing in NIH-3T3 fibroblast cells was studied by using a near-infrared Raman system. To show the feasibility of using these 151 ± 5 nm sized solution-stable nanoparticles inside living cells, we investigated the uptake, cellular response, and the health of the cell population. We show that NS are taken up voluntarily and can be found in the cytosol by transmission electron microscopy (TEM), which also provides detailed information about location and immediate surrounding of the NS. The internalization into cells has been found to be independent of active cellular mechanisms, such as endocytosis, and can be suggested to be of passive nature. Uptake of NS into cells can be controlled, and cells show no increase in necrosis or apoptosis as a result; we show that NS-based intracytosolic SER spectra can be measured on biological samples using short acquisition times and low laser powers. We demonstrate its application using 4-mercaptobenzoic acid (4-MBA)-functionalized nanoshells as a pH sensor.Keywords: cell viability; cytotoxicity; nanoparticle uptake; nanosensors; nanoshells (NS); surface-enhanced Raman spectroscopy

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.

We demonstrate a strategy for label-free protein detection through monitoring the Surface Enhanced Raman Spectrum of an aptamer probe attached to a gold nanoshell. Low limit of detection and minimal non-specific binding show potential for in vitro and in vivo assays.