Noninvasive Detection of Ischemic Vascular Damage in a Pig Model of Liver Donation After Circulatory Death.

BACKGROUND AND AIMS: Liver graft quality is evaluated by visual inspection prior to transplantation, a process highly dependent on the surgeon's experience. We present an objective, noninvasive, quantitative way of assessing liver quality in real time using Raman spectroscopy, a laser-based tool for analyzing biomolecular composition. APPROACH AND RESULTS: A porcine model of donation after circulatory death (DCD) with normothermic regional perfusion (NRP) allowed assessment of liver quality premortem, during warm ischemia (WI) and post-NRP. Ten percent of circulating blood volume was removed in half of experiments to simulate blood recovery for DCD heart removal. Left median lobe biopsies were obtained before circulatory arrest, after 45 minutes of WI, and after 2 hours of NRP and analyzed using spontaneous Raman spectroscopy, stimulated Raman spectroscopy (SRS), and staining. Measurements were also taken in situ from the porcine liver using a handheld Raman spectrometer at these time points from left median and right lateral lobes. Raman microspectroscopy detected congestion during WI by measurement of the intrinsic Raman signal of hemoglobin in red blood cells (RBCs), eliminating the need for exogenous labels. Critically, this microvascular damage was not observed during WI when 10% of circulating blood was removed before cardiac arrest. Two hours of NRP effectively cleared RBCs from congested livers. Intact RBCs were visualized rapidly at high resolution using SRS. Optical properties of ischemic livers were significantly different from preischemic and post-NRP livers as measured using a handheld Raman spectrometer. CONCLUSIONS: Raman spectroscopy is an effective tool for detecting microvascular damage which could assist the decision to use marginal livers for transplantation. Reducing the volume of circulating blood before circulatory arrest in DCD may help reduce microvascular damage.

Intracellular redox potential is correlated with miRNA expression in MCF7 cells under hypoxic conditions.

Hypoxia is a ubiquitous feature of cancers, encouraging glycolytic metabolism, proliferation, and resistance to therapy. Nonetheless, hypoxia is a poorly defined term with confounding features described in the literature. Redox biology provides an important link between the external cellular microenvironment and the cell's response to changing oxygen pressures. In this paper, we demonstrate a correlation between intracellular redox potential (measured using optical nanosensors) and the concentrations of microRNAs (miRNAs) involved in the cell's response to changes in oxygen pressure. The correlations were established using surprisal analysis (an approach derived from thermodynamics and information theory). We found that measured redox potential changes reflect changes in the free energy computed by surprisal analysis of miRNAs. Furthermore, surprisal analysis identified groups of miRNAs, functionally related to changes in proliferation and metastatic potential that played the most significant role in the cell's response to changing oxygen pressure.

Automatic Cocrystal Detection by Raman Spectral Deconvolution-Based Novelty Analysis.

Cocrystals are important molecular adducts that have many advantages as a means of modifying the physicochemical properties of active pharmaceutical ingredients, including taste masking and improved solubility, bioavailability, and stability. As a result, the discovery of new cocrystals is of great interest to commercial drug discovery programs. Time-consuming manual analysis of the large volumes of data that emerge from large-scale cocrystal screening programs of up to 1000s of preparations poses a challenge. Raman spectroscopy has been shown to discriminate between cocrystals and physical mixtures and is easy to automate, allowing rapid screening of large numbers of potential cocrystals, but the spectral features that encode the information are often subtle (e.g., slight changes in peak positions or intensities). We have employed an automated signal processing routine based on a sparse decomposition algorithm to speed up the data processing steps while maintaining the accuracy of a trained spectroscopist. We used our algorithm to screen 31 potential cocrystal preparations and found that through the use of a computationally generated threshold, we could achieve a clear classification of cocrystals and physical mixtures in less than a minute, compared to several hours manually.

A SERS-Active Electrospun Polymer Mesh for Spatially Localized pH Measurements of the Cellular Microenvironment.

Extracellular pH (pHe) is an important chemical factor in many cellular processes and disease pathologies. The routine sampling of pHe in vitro could lead to innovative advances in therapeutics. To this end, we have fabricated a novel gold-coated polymer mesh, which facilitates the real-time measurement of pHe via surface-enhanced Raman scattering (SERS). In this proof of concept study, we apply our SERS sensor to measure metabolically induced changes in the pHe of carcinoma-derived cell line HepG2/C3A. We demonstrate that gold-coated polyurethane electrospun nanofibers (AuNF) have strong and reproducible SERS spectra of surface-adsorbed analytes. By functionalizing AuNF with pH-responsive reporter 4-mercaptobenzoic acid (MBA), we have developed an accurate pH SERS sensor for the extracellular microenvironment. We cultured HepG2/C3A on the surface of MBA-AuNF and measured an acidic shift in pHe at the cell-fiber interface. Following exposure to staurosporine, an apoptosis-inducing drug, we observed changes in the HepG2/C3A cellular morphology indicative of controlled cell death, and detected an increase in the pHe of HepG2/C3A. These results demonstrate how subtle changes in pHe, induced by the metabolic activity of cells, can be measured with our novel SERS sensor MBA-AuNF. The excellent pH measurement performance of MBA-AuNF provides a unique platform to study extracellular pH on the microscale and will help to deepen our understanding of pHe in disease pathology.

Time-correlated single photon Raman spectroscopy at megahertz repetition rates.

Significant improvements in time-correlated single photon counting (TCSPC) Raman spectroscopy acquisition times can be achieved through exploitation of megahertz (MHz) laser repetition rates. We have developed a TCSPC Raman spectroscopy system based on a high peak power (>40W) pulsed laser, a high pulse repetition rate (40 MHz), a custom f/1.5 spectrometer, and a 512 spectral channel × 16 time bin single photon avalanche diode line sensor. We report millisecond Raman spectrum acquisition times, a peak Raman count rate of 104 kcps, and a linewidth aggregated count rate of 440 kcps with a diamond sample. This represents a three-order-of-magnitude increase in measured Raman count rate in comparison with a 104 kHz pulsed laser operating at 300 W and a four-order-of-magnitude increase over a 0.1 W pulsed laser operating at 40 MHz. A Raman-to-fluorescence ratio of 4.76 is achieved with a sesame oil sample at a 20 MHz repetition rate. Achieving high count rates and Raman-to-fluorescence ratios unlocks the potential of combined Raman/fluorescence lifetime spectroscopy for imaging and other short acquisition time applications.

Untargeted Metabolite Mapping in 3D Cell Culture Models Using High Spectral Resolution FT-ICR Mass Spectrometry Imaging.

Multicellular tumor spheroids (MTS) are a well-established model system for drug development and are a valuable in vitro research tool for use prior to employing animal models. These 3D-cell cultures are thought to display chemical gradients of oxygen and nutrients throughout their structure, giving rise to distinct microenvironments in radial layers, thus, mimicking the pathophysiological environment of a tumor. Little is known about the localized distributions of metabolites within these microenvironments. To address this, here we utilize high spectral resolution Fourier-transform ion cyclotron resonance (FT-ICR), MALDI mass spectrometry imaging (MSI) to image the distribution of endogenous metabolites in breast cancer MCF-7 spheroids. We show that known specific metabolite markers (adenosine phosphates and glutathione) indicate that the central region of these cell culture models experiences increased hypoxic and oxidative stress. By using discriminatory analysis, we have identified which m/z values localize toward the outer proliferative or central hypoxic regions of an MTS. Elemental formulae were assigned with sub-ppm mass accuracy, allowing metabolite assignment. Using this information, we have mapped these metabolites back to distinct pathways to improve our understanding of the molecular environment and biochemistry of these tumor models.

MALDI Matrix Application Utilizing a Modified 3D Printer for Accessible High Resolution Mass Spectrometry Imaging.

Successful matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) relies on the selection of the most appropriate matrix and optimization of the matrix application parameters. In order to achieve reproducible high spatial-resolution imaging data, several commercially available automated matrix application platforms have become available. However, the high cost of these commercial matrix sprayers is restricting access into this emerging research field. Here, we report an automated platform for matrix deposition, employing a converted commercially available 3D printer ($300) and other parts commonly found in an analytical chemistry lab as a low-cost alternative to commercial sprayers. Using printed fluorescent rhodamine B microarrays and employing experimental design, the matrix deposition parameters were optimized to minimize surface analyte diffusion. Finally, the optimized matrix application method was applied to image three-dimensional MCF-7 cell culture spheroid sections (ca. 500 µm diameter tissue samples) and sections of mouse brain. Using this system, we demonstrate robust and reproducible observations of endogenous metabolite and steroid distributions with a high spatial resolution.

Dual purpose fibre - SERS pH sensing and bacterial analysis.

The exploitation of fibre based Raman probes has been challenged by often complicated fabrication procedures and difficulties in reproducibility. Here, we have demonstrated a simple and cost-effective approach for sensing pH through an optical fibre, by employing a wax patterned filter paper-based substrate for surface enhanced Raman spectroscopy (SERS). Through this method, high reproducibility between fibres was achieved. In addition to sensing pH, it was possible to extract fluid samples containing P. aeruginosa for further analysis. This dual purpose fibre is bronchoscope deployable, and is able to gather information about both the host and pathogen, which may lead to an improved treatment plan in future in vivo applications.

Antitumour activity of the novel flavonoid Oncamex in preclinical breast cancer models.

BACKGROUND: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents. METHODS: We studied the effect of a second-generation flavonoid analogue Oncamex in a panel of seven breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy and immunohistochemistry of xenograft tissue to investigate its mechanism of action. RESULTS: Proliferation assays showed that Oncamex treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in the expression of genes controlling cell cycle and apoptosis. Fluorescence microscopy showed the compound's mitochondrial targeting and reactive oxygen species-modulating properties, inducing superoxide production at concentrations associated with antiproliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no signs of untoward effects on the animals. CONCLUSIONS: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent.

Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.

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.

SERS as a tool for in vitro toxicology.

Measuring markers of stress such as pH and redox potential are important when studying toxicology in in vitro models because they are markers of oxidative stress, apoptosis and viability. While surface enhanced Raman spectroscopy is ideally suited to the measurement of redox potential and pH in live cells, the time-intensive nature and perceived difficulty in signal analysis and interpretation can be a barrier to its broad uptake by the biological community. In this paper we detail the development of signal processing and analysis algorithms that allow SERS spectra to be automatically processed so that the output of the processing is a pH or redox potential value. By automating signal processing we were able to carry out a comparative evaluation of the toxicology of silver and zinc oxide nanoparticles and correlate our findings with qPCR analysis. The combination of these two analytical techniques sheds light on the differences in toxicology between these two materials from the perspective of oxidative stress.

Measuring the effects of fractionated radiation therapy in a 3D prostate cancer model system using SERS nanosensors.

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.

Correction: Measuring the effects of fractionated radiation therapy in a 3D prostate cancer model system using SERS nanosensors.

Correction for 'Measuring the effects of fractionated radiation therapy in a 3D prostate cancer model system using SERS nanosensors' by Victoria L. Camus, et al., Analyst, 2016, 141, 5056-5061.

Series of quinone-containing nanosensors for biologically relevant redox potential determination by surface-enhanced Raman spectroscopy.

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.

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

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-α (TNFα). 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.

Ratiometric biological nanosensors.

The measurement of intracellular analytes has been key in understanding cellular processes and function, and the use of biological nanosensors has revealed the spatial and temporal variation in their concentrations. In particular, ratiometric nanosensors allow quantitative measurements of analyte concentrations. The present review focuses on the recent advances in ratiometric intracellular biological nanosensors, with an emphasis on their utility in measuring analytes that are important in cell function.

SERS Microsensors for the Study of pH Regulation in Cystic Fibrosis Patient-Derived Airway Cultures.

Acidification of the airway surface liquid in the respiratory system could play a role in the pathology of Cystic Fibrosis, but its low volume and proximity to the airway epithelium make it a challenging biological environment in which to noninvasively collect pH measurements. To address this challenge, we explored surface enhanced Raman scattering microsensors (SERS-MS), with a 4-mercaptobenzoic acid (MBA) pH reporter molecule, as pH sensors for the airway surface liquid of patient-derived in vitro models of the human airway. Using air-liquid interface (ALI) cultures to model the respiratory epithelium, we show that SERS-MS facilitates the optical measurement of trans-epithelial pH gradients between the airway surface liquid and the basolateral culture medium. SERS-MS also enabled the successful quantification of pH changes in the airway surface liquid following stimulation of the Cystic Fibrosis transmembrane conductance regulator (CFTR, the apical ion channel that is dysfunctional in Cystic Fibrosis airways). Finally, the influence of CFTR mutations on baseline airway surface liquid pH was explored by using SERS-MS to measure the pH in ALIs grown from Cystic Fibrosis and non-Cystic Fibrosis donors.

SERS microsensors for pH measurements in the lumen and ECM of stem cell derived human airway organoids.

Patient derived organoids have the potential to improve the physiological relevance of in vitro disease models. However, the 3D architecture of these self-assembled cellular structures makes probing their biochemistry more complex than in traditional 2D culture. We explore the application of surface enhanced Raman scattering microsensors (SERS-MS) to probe local pH gradients within patient derived airway organoid cultures. SERS-MS consist of solid polymer cores decorated with surface immobilised gold nanoparticles which are functionalised with pH sensitive reporter molecule 4-mercaptobenzoic acid (MBA). We demonstrate that by mixing SERS-MS into the extracellular matrix (ECM) of airway organoid cultures the probes can be engulfed by expanding organoids and report on local pH in the organoid lumen and ECM.

Redox potential dependence of peptide structure studied using surface enhanced Raman spectroscopy.

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)NC1(67-85) 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)NC1(67-85). 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.