Gold nanoparticles as a substrate in bio-analytical near-infrared surface-enhanced Raman spectroscopy.

As biospectroscopy techniques continue to be developed for screening or diagnosis within a point-of-care setting, an important development for this field will be high-throughput optimization. For many of these techniques, it is therefore necessary to adapt and develop parameters to generate a robust yet simple approach delivering high-quality spectra from biological samples. Specifically, this is important for surface-enhanced Raman spectroscopy (SERS) wherein there are multiple variables that can be optimised to achieve an enhancement of the Raman signal from a sample. One hypothesis is that "large" diameter (>100 nm) gold nanoparticles provide a greater enhancement at near-infrared (NIR) and infrared (IR) wavelengths than those <100 nm in diameter. Herein, we examine this notion using examples in which SERS spectra were acquired from MCF-7 breast cancer cells incubated with 150 nm gold nanoparticles. It was found that 150 nm gold nanoparticles are an excellent material for NIR/IR SERS. Larger gold nanoparticles may better satisfy the theoretical restraints for SERS enhancement at NIR/IR wavelengths compared to smaller nanoparticles. Also, larger nanoparticles or their aggregates are more readily observed via optical microscopy (and especially electron microscopy) compared to smaller ones. This allows rapid and straightforward identification of target areas containing a high concentration of nanoparticles and facilitating SERS spectral acquisition. To some extent, these observations appear to extend to biofluids such as blood plasma or (especially) serum; SERS spectra of such biological samples often exhibit a low signal-to-noise ratio in the absence of nanoparticles. With protein-rich biofluids such as serum, a dramatic SERS effect can be observed; although this might facilitate improved spectral biomarker identification in the future, it may not always improve classification between control vs. cancer. Thus, use of "large" gold nanoparticles are a good starting point in order to derive informative NIR/IR SERS analysis of biological samples.

Surface-enhanced Raman spectroscopy of the endothelial cell membrane.

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.

Using Fourier transform IR spectroscopy to analyze biological materials.

IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women: implications for latent stages of breast carcinogenesis.

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States' high risk-posing environment and: (2) marked (> 1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE- cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the "biochemical fingerprint" of 4HNE+ vs. 4HNE- luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy's ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

Vibrational biospectroscopy coupled with multivariate analysis extracts potentially diagnostic features in blood plasma/serum of ovarian cancer patients.

Despite numerous advances in "omics" research, early detection of ovarian cancer still remains a challenge. The aim of this study was to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectroscopy could characterise alterations in the biomolecular signatures of human blood plasma/serum obtained from ovarian cancer patients compared to non-cancer controls. Blood samples isolated from ovarian cancer patients (n = 30) and healthy controls (n = 30) were analysed using ATR-FTIR spectroscopy. For comparison, a smaller cohort of samples (n = 8) were analysed using an InVia Renishaw Raman spectrometer. Resultant spectra were pre-processed prior to being inputted into principal component analysis (PCA) and linear discriminant analysis (LDA). Statistically significant differences (P < 0.001) were observed between spectra of ovarian cancer versus control subjects for both biospectroscopy methods. Using a support vector machine classifier for Raman spectra of blood plasma, a diagnostic accuracy of 74% was achieved, while the same classifier showed 93.3% accuracy for IR spectra of blood plasma. These observations suggest that a biospectroscopy approach could be applied to identify spectral alterations associated with the presence of insidious ovarian cancer.

Dose-related alterations of carbon nanoparticles in mammalian cells detected using biospectroscopy: potential for real-world effects.

Nanotechnologies generate a wide range of engineered nanomaterials that enter into our ecosystem, especially carbon-based nanoparticles (CNPs). As these novel materials acquire ever increasing numbers of applications, they may pose a risk to organisms, including humans. However, our knowledge of nanoparticle-induced effects remains limited. We are yet to understand the interaction between nanoparticles and organisms, and classical toxicology fails to provide models for risk assessment. Biospectroscopy techniques were employed to identify the effects induced by real-world levels of a panel of CNPs. MCF-7 cells concentrated in S-phase or G0/G1-phase were treated for 24 h with short or long multiwalled carbon nanotubes (MWCNTs) or Fullerene (C60) at the following concentrations: 0.0025 mg/L, 0.005 mg/L, 0.01 mg/L, 0.025 mg/L, 0.05 mg/L, and 0.1 mg/L. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy coupled with computational analysis was then applied to interrogate the cells and significant dose-related effects were detected. From derived infrared spectra, distinct spectral biomarkers of cell alteration induced by each CNP type were identified. Additionally, Raman spectroscopy was applied and allowed us to determine that reactive oxygen species (ROS) were generated by CNPs. These observations highlight the potential of biospectroscopy techniques to determine CNP-induced alterations in target mammalian cells at ppb levels.

Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy.

Understanding stem cell (SC) biology remains challenging and one of the few human tissues within which their in situ location is well characterized is the cornea. Individual human corneal epithelial cells were isolated from biopsies of live tissues using fluorescence-activated cell sorting (FACS); these were divided into putative SCs, transit-amplifying (TA) cells and terminally-differentiated (TD) cells. Employing synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy with a focal plane array (FPA), sub-cellular spatial resolution analysis of unstained isolated cells was achieved as a consequence of the brilliance of a 12 collimated beams arrangement allowing rapid spectral acquisition. Infrared (IR) spectra were extracted and pre-processed. Subsequent categorization with multivariate analysis of IR spectra derived from FPA images was used to investigate biomolecular changes between classes. A progressive segregation in cell-specific spectral categories with differentiation from SC to TA cell to TD cell was noted. Multiple different absorption peaks that discriminated putative SCs, TA cells and TD cells across DNA, protein and lipid spectral regions were identified. DNA regions (1080 and 1225 cm(-1)) and some protein regions (1443 cm(-1)) primarily segregated SCs from TA cells and TD cells, whilst amide regions and lipids (1,550, 1650 and 1740 cm(-1)) segregated TA cells and TD cells. Scanning electron microscopy images verified the external phenotypic characteristics of the different isolated cell types. These findings highlight the applicability of SR-FTIR microspectroscopy towards distinguishing SCs, TA cells and TD cells, and suggest that cellular classification via traditional methods of immunolabelling can be greatly aided by the use of spectral biomarkers.