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Integration of Raman microscopy, differential interference contrast microscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in Candida albicans biofilms.

Two spectroscopic techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and Raman microscopy (RM), were used to characterize transport of chlorhexidine digluconate (CHG) in Candida albicans (CA) biofilms. Different (volumetric) regions of the biofilm are sampled by these two vibrational spectroscopies making them complementary techniques. Simple mathematical models were developed to analyze ATR-FTIR and RM data to obtain an effective diffusion coefficient describing transport through CA biofilms. CA biofilms were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of biofilms that had become confluent, (i.e., biofilms that completely covered the germanium (Ge) substratum) were composed primarily of a tangled mass of hyphae with openings between germtubes about 10 to 50 microm across. Quantitative analysis of ATR-FTIR kinetic data curves indicated that the effective diffusion coefficient for transport of CHG through confluent biofilms about 200-microm thick was reduced 0.1 to 0.3 times compared to the diffusion coefficient for CHG in water. Effective diffusion coefficients obtained from analysis of RM data were consistently higher than those indicated by ATR-FTIR data suggesting that transport is more hindered in regions near the base of the biofilm than in the outer layers. Analysis of both ATR-FTIR and RM data obtained from thicker films indicated that adsorption of CHG to biofilm components was responsible for a substantial portion of the transport limitation imposed by the biofilm. Comparison of ATR-FTIR and RM data for both types of biofilms indicated that sites of CHG adsorption were more concentrated in the interfacial region than in the bulk biofilm. Comparison of results for ATR-FTIR and RM measurements suggests that these relatively thick CA biofilms can be modeled, for purposes of predicting transport, approximately as a homogeneous thin planar sheet. Thus, these biofilms offer a relatively tractable model system for initial investigations of the relation between antimicrobial transport and kinetics of antimicrobial action.

Anti-Bacterial Agents↗

Size-exclusion chromatography with on-line ultraviolet, proton nuclear magnetic resonance and mass spectrometric detection and on-line collection for off-line Fourier transform infrared spectroscopy.

The coupling of HPLC with UV detection and on-line NMR spectroscopy and mass spectrometry combined with a dedicated interface for the collection of the chromatographic eluent for subsequent Fourier transform (FT) IR has been investigated using a number of polymer additives as model compounds. Size-exclusion chromatography was performed using deuterated chloroform as eluent with the separation monitored on-line by UV detection at 254 nm and on-flow 1H-NMR and MS. The effluent from the NMR probe was directed to a dedicated HPLC interface where it was deposited on a germanium plate for subsequent FT-IR. NMR and MS spectra were successfully obtained for 2,6-di-tert.-butyl-4-methylphenol, octadecyl-3-(3,5-di-tert.-butyl-4-hydroxyphenyl) propionate (Irganox 1076) and diisooctyl phthalate on-line and FT-IR spectra for all three compounds were obtained off-line. Practical problems encountered with this multiple hyphenation are described.

Chromatography, Gel↗

Light-induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy.

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.

Bacillus subtilis↗

Pressure- and temperature-induced unfolding and aggregation of recombinant human interferon-gamma: a Fourier transform infrared spectroscopy study.

The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-gamma (rhIFN-gamma) and its biologically inactive truncated form rhIFN-Delta C15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the alpha-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5 M MgCl(2) significantly reduces the pressure stability. Releasing the pressure below 300 MPa results in the formation of intermolecular antiparallel beta-sheets, which is seldom observed. This suggests that the intermolecular beta-sheet of rhIFN-gamma is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of alpha-helical structure into intermolecular antiparallel beta-sheet, as is usually observed for most proteins.

Hot Temperature↗

Fourier transform infrared spectroscopy in cancer detection.

The rapid developments in the field of infrared spectroscopy in the past decade have demonstrated a potential for disease diagnosis using noninvasive technologies. Several earlier studies have highlighted the advantage of using infrared spectroscopy both in the near- and mid-infrared regions for diagnostic purposes at clinical levels. The areas of focus have been the distinction of premalignant and malignant cells and tissues from their normal state using specific parameters obtained from Fourier transform infrared spectra, making it a rapid and reagent-free method. While it still requires pilot studies and designed clinical trials to ensure the applicability of such systems for cancer diagnosis, substantial progress has been made in incorporating advances in computational methods into the system to increase the sensitivity of the entire setup, making it an objective and sensitive technique suitable for automation to suit the demands of the medical community. The development of fiber-optics systems for infrared spectroscopy have further opened up new and modern avenues in medical diagnosis at various levels of cells, tissues and organs under laboratory and clinical conditions.

Fiber Optic Technology↗

Selective complex formation of some chromogenic calix[4]arene derivatives detected by Fourier transform infrared spectroscopy 2. Solid experiments.

Two chromogenic calix[4]arene derivatives having identical coordination sphere (consisting of ester groups) but different chromogenic units (pyridinium type and dinitro-phenyl type) were the subject of structural studies with sodium(I) and thallium(I) ions in solid state, both as bulk and surface complexes. Their diffuse reflectance infrared Fourier transform (DRIFTS) spectra were recorded and the characteristic changes of the vibrational spectra due to complex formation were analysed based on curve fitting.

Calcium↗