Sensing biophysical alterations of human lung epithelial cells (A549) in the context of toxicity effects of diesel exhaust particles.

Diesel exhaust particles (DEP) in urban air are associated with numerous respiratory diseases. The role of underlying biomechanics in cytotoxicity of individual lung cells relating to DEP exposure is unclear. In this study, atomic force microscopy (AFM), confocal Raman microspectroscopy (RM), and fluorescence (FL) microscopy were used to monitor alterations of single A549 cells exposed to DEP. Results revealed a significant decrease in membrane surface adhesion force and a significant change in cell elasticity as a function of DEP-cell interaction time, and the dynamic changes in cellular biocomponents which were reflected by changes of characteristic Raman bands: 726 cm(-1) (adenine), 782 cm(-1) (uracil, cytosine, thymine), 788 cm(-1) (O-P-O), 1006 cm(-1) (phenylalanine), and 1320 cm(-1) (guanine) after DEP exposure. These findings suggest that the combination of multi-instruments (e.g., AFM/FL) may offer an exciting platform for investigating the roles of biophysical and biochemical responses to particulate matter-induced cell toxicity.

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy.

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Subcellular spectroscopic markers, topography and nanomechanics of human lung cancer and breast cancer cells examined by combined confocal Raman microspectroscopy and atomic force microscopy.

The nanostructures and hydrophobic properties of cancer cell membranes are important for membrane fusion and cell adhesion. They are directly related to cancer cell biophysical properties, including aggressive growth and migration. Additionally, chemical component analysis of the cancer cell membrane could potentially be applied in clinical diagnosis of cancer by identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined Raman microspectroscopy (RM) and atomic force microscopy (AFM) technique was applied to detect the difference in membrane chemical components and nanomechanics of three cancer cell lines: human lung adenocarcinoma epithelial cells (A549), and human breast cancer cells (MDA-MB-435 with and without BRMS1 metastasis suppressor). Raman spectral analysis indicated similar bands between the A549, 435 and 435/BRMS1 including ~720 cm(-1) (guanine band of DNA), 940 cm(-1) (skeletal mode polysaccharide), 1006 cm(-1) (symmetric ring breathing phenylalanine), and 1451 cm(-1) (CH deformation). The membrane surface adhesion forces for these cancer cells were measured by AFM in culture medium: 0.478 ± 0.091 nN for A549 cells, 0.253 ± 0.070 nN for 435 cells, and 1.114 ± 0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62 ± 0.682 mN m(-1) for A549 cells, 2.105 ± 0.691 mN m(-1) for 435 cells, and 5.448 ± 1.081 mN m(-1) for 435/BRMS1 cells.

BRMS1 expression alters the ultrastructural, biomechanical and biochemical properties of MDA-MB-435 human breast carcinoma cells: an AFM and Raman microspectroscopy study.

Restoring BReast cancer Metastasis Suppressor 1 (BRMS1) expression suppresses metastasis in MDA-MB-435 human breast carcinoma cells at ectopic sites without affecting tumor formation at orthotopic site in the body. BRMS1 expression induces many phenotypic alterations in 435 cells such as cell adhesion, cytoskeleton rearrangement, and the down regulation of epidermal growth factor receptor (EGFR) expression. In order to better understand the role of cellular biomechanics in breast cancer metastasis, the qualitative and quantitative detection of cellular biomechanics and biochemical composition is urgently needed. In the present work, using atomic force microscopy (AFM) and fluorescent microscopy we revealed that BRMS1 expression in 435 cells induced reorganization of F-actin and caused alteration in cytoarchitectures (cell topography and ultrastructure). Results from AFM observed increase in biomechanical properties which include cell adhesion, cellular spring constant, and Young's modulus in 435/BRMS1 cells. Raman microspectroscopy showed weaker vibrational spectroscopic bands in 435/BRMS1 cells, implying decrease in concentration of cellular biochemical components in these cells. This was despite the similar spectral patterns observed between 435 and 435/BRMS1 cells. This work demonstrated the feasibility of applying AFM and Raman techniques for in situ measurements of the cellular biomechanics and biochemical components of breast carcinoma cells. It provides vital clues in understanding of the role of cellular biomechanics in cancer metastasis, and further the development of new techniques for early diagnosis of breast cancer.

Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy.

The structure of a bacterial cell wall may alter during bacterial reproduction. Moreover, these cell wall variations, on a nanoscale resolution, have not yet fully been elucidated. In this work, Raman spectroscopy and atomic force microscopy (AFM) technique are applied to evaluate the culture time-dependent cell wall structure variations of Pseudomonas putida KT2440 at a quorum and single cell level. The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs till 6 h of cultivation time under our experimental conditions. AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time. This work demonstrates the feasibility of utilizing a combined Raman spectroscopy and AFM technique to investigate the cultivation time dependence of bacterial cellular surface biopolymers at single cell level.

Immobilization, hybridization, and oxidation of synthetic DNA on gold surface: electron transfer investigated by electrochemistry and scanning tunneling microscopy.

Fundamental understanding of interfacial electron transfer (ET) among electrolyte/DNA/solid-surface will facilitate the design for electrical detection of DNA molecules. In this report, the electron transfer characteristics of synthetic DNA (sequence from pathogenic Cryptosporidium parvum) self-assembled on a gold surface was electrochemically studied. The effects of immobilization order on the interface ET related parameters such as diffusion coefficient (D0), surface coverage (thetaR), and monolayer thickness (d(i)) were determined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). DNA surface density (Gamma(DNA)) was determined by the integration of the charge of the electro-oxidation current peaks during the initial cyclic voltammetry scans. It was found that the DNA surface densities at different modifications followed the order: Gamma(DNA) (dsS-DNA/Au) > Gamma(DNA) (MCH/dsS-DNA/Au) > Gamma(DNA) (dsS-DNA/MCH/Au). It was also revealed that the electro-oxidation of the DNA modified gold surface would involve the oxidation of nucleotides (guanine and adenine) with a 5.51 electron transfer mechanism and the oxidative desorption of DNA and MCH molecules by a 3 electron transfer mechanism. STM topography and current image analysis indicated that the surface conductivity after each surface modification followed the order: dsS-DNA/Au < MCH/dsS-DNA/Au < oxidized MCH/dsS-DNA/Au < Hoechst/oxidized MCH/dsS-DNA/Au. The results from this study suggested a combination of variations in immobilization order may provide an alternative approach for the optimization of DNA hybridization and the further development for electrical detection of DNA.