Elizabeth Vargis

Nanoparticle Properties and Synthesis Effects on Surface-Enhanced Raman Scattering Enhancement Factor: An Introduction

Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface-enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.

Sensitivity of Raman spectroscopy to normal patient variability

Many groups have used Raman spectroscopy for diagnosing cervical dysplasia; however, there have been few studies looking at the effect of normal physiological variations on Raman spectra. We assess four patient variables that may affect normal Raman spectra: Race/ethnicity, body mass index (BMI), parity, and socioeconomic status. Raman spectra were acquired from a diverse population of 75 patients undergoing routine screening for cervical dysplasia. Classification of Raman spectra from patients with a normal cervix is performed using sparse multinomial logistic regression (SMLR) to determine if any of these variables has a significant effect. Results suggest that BMI and parity have the greatest impact, whereas race/ethnicity and socioeconomic status have a limited effect. Incorporating BMI and obstetric history into classification algorithms may increase sensitivity and specificity rates of disease classification using Raman spectroscopy. Studies are underway to assess the effect of these variables on disease.

Detecting Biochemical Changes in the Rodent Cervix During Pregnancy Using Raman Spectroscopy

The goal of this research is to determine whether Raman spectroscopy (RS), an optical method that probes the vibrational modes of tissue components, can be used in vivo to study changes in the mouse cervix during pregnancy. If successful, such a tool could be used to detect cervical changes due to pregnancy, both normal and abnormal, in animal models and humans. For this study, Raman spectra were acquired before, during and after a 19-day mouse gestational period. In some cases, after Raman data was obtained, cervices were excised for structural testing and histological staining for collagen and smooth muscle. Various peaks of the Raman spectra, such as the areas corresponding to fatty acid content and collagen organization, changed as the cervix became softer in preparation for labor and delivery. These findings correspond to the increase in compliance of the tissue and the collagen disorganization visualized with the histological staining. The results of this study suggest that non-invasive RS can be used to study cervical changes during pregnancy, labor and delivery and can possibly predict preterm delivery before overt clinical manifestations, potentially lead to more effective preventive and therapeutic interventions.

Assessing Variability of In Vivo Tissue Raman Spectra

Raman spectroscopy (RS) has received increasing attention as a potential tool for clinical diagnostics. However, the unknown comparability of multiple tissue RS systems remains a major issue for technique standardization and future multisystem trials. In this study, we evaluated potential factors affecting data collection and interpretation, utilizing the skin as an example tissue. The effects of contact pressure and probe angle were characterized as potential user-induced variability sources. Similarly, instrumentation-induced variability sources of system stability and system-dependent response were also analyzed on skin and a nonvolatile biological tissue analog. Physiologically induced variations were studied on multiple tissue locations and patients. The effect of variability sources on spectral line shape and dispersion was analyzed with analysis-of-variance methods, and a new metric for comparing spectral dispersion was defined. In this study, in vivo measurements were made on multiple sites of skin from five healthy volunteers, with four stand-alone fiber optic probe–based tissue RS systems. System stability and controlled user-induced variables had no effects on obtained spectra. By contrast, instrumentation and anatomical location of measurement were significant sources of variability. These findings establish the comparability of tissue Raman spectra obtained by unique systems. Furthermore, we suggest steps for further procedural and instrumentation standardization prior to broad clinical applications of the technique.

Methods for Culturing Retinal Pigment Epithelial Cells: a Review of Current Protocols and Future Recommendations

The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

Raman spectroscopy provides a noninvasive approach for determining biochemical composition of the pregnant cervix in vivo

The molecular changes that occur with cervical remodelling during pregnancy are not completely understood. This study reviews Raman spectroscopy, an optical technique for detecting changes in the pregnant cervix, and reports preliminary studies on cervical remodelling in mice that suggest that the technique provides advantages over other methods.

Rational design of Raman-labeled nanoparticles for a dual-modality, light scattering immunoassay on a polystyrene substrate

BackgroundSurface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate.ResultsWe have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes.ConclusionSERS nanoparticle probes were fabricated to produce a strong light scattering signal despite substrate interference. The optical extinction and inelastic light scattering of these probes was detected by optical absorbance spectroscopy and Raman spectroscopy, respectively. This immunoassay demonstrates the feasibility of analyzing strongly enhanced Raman signals on polystyrene, which is an inexpensive yet non-ideal Raman substrate. The assay sensitivity, which is in the low pM range, suggests that these SERS probe particles could be used for Raman labeling of cell or tissue samples in a polystyrene tissue culture plate. With continued development, this approach could be used for direct labeling of multiple cell surface biomarkers on strongly interfering substrate platforms.

The Effect of Retinal Pigment Epithelial Cell Patch Size on Growth Factor Expression

The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlled using micropatterning techniques in order to examine the effect of patch size on cell health and differentiation. Understanding this effect is a critical step in the development of multiplexed high throughput fluidic assays and provides a model for replicating disease states associated with the deterioration of retinal tissue during age-related macular degeneration (AMD). Microcontact printing of fibronectin on polystyrene and glass substrates was used to promote cell attachment, forming RPE patches of controlled size and shape. These colonies mimic the effect of atrophy and loss-of-function that occurs in the retina during degenerative diseases such as AMD. After 72 h of cell growth, levels of vascular endothelial growth factor (VEGF), an important biomarker of AMD, were measured. Cells were counted and morphological indicators of cell viability and tight junction formation were assessed via fluorescence microscopy. Up to a twofold increase of VEGF expression per cell was measured as colony size decreased, suggesting that the local microenvironment of, and connections between, RPE cells influences growth factor expression leading to the initiation and progression of diseases such as AMD.

In vitro biophysical, microspectroscopic and cytotoxic evaluation of metastatic and non-metastatic cancer cells in responses to anti-cancer drug.

The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses cancer metastasis without affecting the growth of the primary tumor. Previous work has shown that it decreases the expression of protein mediators involved in chemoresistance. This study measured the biomechanical and biochemical changes in BRMS1 expression and the responses of BRMS1 to drug treatments on cancer cells in vitro. The results show that BRMS1 expression affects biomechanical properties by decreasing the Youngs modulus and adhesion force of breast cancer cells after doxorubicin (DOX) exposure. Raman spectral bands corresponding to DNA/RNA, lipids and proteins were similar for all cells after DOX treatment. The expression of cytokines were similar for cancer cells after DOX exposure, although BRMS1 expression had different effects on the secretion of cytokines for breast cancer cells. The absence of significant changes on apoptosis, reactive oxygen species (ROS) expression and cell viability after BRMS1 expression shows that BRMS1 has little effect on cellular chemoresistance. Analyzing cancer protein expression is critical in evaluating therapeutics. Our study may provide evidence of the benefit of metastatic suppressor expression before chemotherapy.

Bridging the multiscale gap: Identifying cellular parameters from multicellular data

Multiscale models that link sub-cellular, cellular and multicellular components offer powerful insights in disease development. Such models need a realistic set of parameters to represent the physical and chemical mechanisms at the sub-cellular and cellular levels to produce high fidelity multicellular outcomes. However, determining correct values for some of the parameters is often difficult and expensive using high-throughput microfluidic approaches. This work presents an alternative approach that estimates cellular parameters from spatiotemporal data produced from bioengineered multicellular in vitro experiments. Specifically, we apply a search technique to an integrated cellular and multicellular model of retinal pigment epithelial (RPE) cells to estimate the binding rate and auto-regulation rate of vascular endothelial growth factor (VEGF). Understanding VEGF regulation is critical in treating age-related macular degeneration and many other diseases. The method successfully identifies realistic values for autoregulatory cellular parameters that reproduce the spatiotemporal in vitro experimental data.