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Analytical and Bioanalytical Chemistry (v.387, #5)

Teaching surface characterization to undergraduates by Heather A. Bullen (pp. 1579-1581).

is an assistant professor of chemistry at Northern Kentucky University. She has an active undergraduate research program in bioanalytical and materials science. Her current research interests focus on utilizing surface characterization methods to evaluate interactions of metal oxide surfaces with aqueous environments, biofilm formation, and designing customizable polymers as potential drug delivery systems across the blood brain barrier (for more information see http://www.nku.edu/~bullenh1/ ). Dr. Bullen is active in analytical curriculum development and integrative science course design. She is an associate editor of the analytical sciences digital library, a peer reviewed internet resource for analytical science.

A chemical uncertainty principle challenge by Juris Meija (pp. 1583-1584).

Solution to quality assurance challenge 4 by Manfred Reichenbächer; Jürgen W. Einax (pp. 1585-1586).

Geoffrey B. Cox (Ed.): Preparative enantioselective chromatography by Norbert M. Maier (pp. 1587-1588).

SPEC 2006—Biomedical vibrational spectroscopy by D. Naumann; W. Petrich; J. Schmitt (pp. 1589-1590).

is head of the Biomedical Spectroscopy Group at the Robert Koch Institute in Berlin. His current research interests are the characterization of microorganisms and desease recognition from body fluids by optical spectroscopy and MALDI-TOF-MS, and protein folding/misfolding studies in the context of conformational diseases. He is adjunct Professor in Biophysical Chemistry at the Biology/Chemistry/Pharmacy Faculty of the Free University of Berlin and gives lectures and practical courses on optical spectroscopy of biological molecules. is a research manager and senior project manager at Roche Diagnostics GmbH, Mannheim, Germany. His research interests are biomedical optics with a particular focus on biospectroscopy. In addition, W. Petrich lectures on atomic and molecular spectroscopy as well as biophotonics at the Faculty for Physics and Astronomy at the University of Heidelberg. is founder and CEO of Synthon GmbH (Heidelberg) which specializes in the applications of spectroscopy in industry and academia. His particular research interests cover the development of spectroscopic applications for microbiology, pharmaceutical research and biomedicine where he was over 15 years of experience in the field.

Vibrational microscopy and imaging of skin: from single cells to intact tissue by Guojin Zhang; David J. Moore; Carol R. Flach; Richard Mendelsohn (pp. 1591-1599).

Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications, ranging from studies of isolated single cells (corneocytes) to characterization of endogenous components in intact tissue. Two applications are described to illustrate the power of these techniques for skin research. First, the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated. Two solvents, DMSO and chloroform/methanol, commonly used in dermatological research, are shown to induce large reversible alterations (α-helix to β-sheet) in the secondary structure of keratin in isolated corneocytes. Second, factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 μm in intact pigskin, demonstrates the delineation of specific skin regions. Two particular components that are difficult to identify by other means were observed in the epidermis. One small region was formed from a conformationally ordered lipid phase containing cholesterol. In addition, the presence of nucleated cells in the tissue (most likely keratinocytes) was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA.

Keywords: Confocal Raman; Infrared imaging; Corneocytes; Epidermis; Protein secondary structure

Fourier transform infrared imaging and MR microscopy studies detect compositional and structural changes in cartilage in a rabbit model of osteoarthritis by Xiaohong Bi; Xu Yang; Mathias P. G. Bostrom; Dorota Bartusik; Sharan Ramaswamy; Kenneth W. Fishbein; Richard G. Spencer; Nancy Pleshko Camacho (pp. 1601-1612).

Assessment of subtle changes in proteoglycan (PG) and collagen, the primary macromolecular components of cartilage, which is critical for diagnosis of the early stages of osteoarthritis (OA), has so far remained a challenge. In this study we induced osteoarthritic cartilage changes in a rabbit model by ligament transection and medial meniscectomy and monitored disease progression by infrared fiber optic probe (IFOP) spectroscopy, Fourier transform infrared imaging spectroscopy (FT-IRIS), and magnetic resonance imaging (MRI) microscopy. IFOP studies combined with chemometric partial least-squares analysis enabled us to monitor progressive cartilage surface changes from two to twelve weeks post-surgery. FT-IRIS studies of histological sections of femoral condyle cartilage revealed that compared with control cartilage the OA cartilage had significantly reduced PG content 2 and 4 weeks post-surgery, collagen fibril orientation changes 2 and 4 weeks post-surgery, and changes in collagen integrity 2 and 10 weeks post-surgery, but no significant changes in collagen content at any time. MR microscopy studies revealed reduced fixed charge density (FCD), indicative of reduced PG content, in the OA cartilage, compared with controls, 4 weeks post-surgery. A non-significant trend toward higher apparent MT exchange rate, k_m, was also found in the OA cartilage at this time point, suggesting changes in collagen structural features. These two MR findings for FCD and k_m parallel the FT-IRIS findings of reduced PG content and altered collagen integrity, respectively. MR microscopy studies of the cartilage at the 12-week time point also found a trend toward longer T ₂ values and reduced anisotropy in the deep zone of the OA cartilage, consistent with increased hydration and less ordered collagen. These studies reveal that FT-IRIS and MR microscopy provide complementary data on compositional changes in articular cartilage in the early stages of osteoarthritic degradation.

Keywords: Infrared spectroscopy; Magnetic resonance imaging; Osteoarthritis; Cartilage; Collagen; Proteoglycan

Precision of Raman depolarization and optical attenuation measurements of sound tooth enamel by Michael G. Sowa; Dan P. Popescu; Jeffrey Werner; Mark Hewko; Alex C.-T. Ko; Jeri Payette; Cecilia C. S. Dong; Blaine Cleghorn; Lin-P’ing Choo-Smith (pp. 1613-1619).

The demineralization of enamel that is associated with early caries formation affects the optical properties of the enamel. Polarized Raman spectroscopy and optical coherence tomography have been used to detect these changes and potentially offer a means to detect and monitor early caries development. The total optical attenuation coefficient as measured by optical coherence tomography and the polarization anisotropy of the Raman peak arising from the symmetric ν₁ vibration of $${ ext{PO}}^{{{ ext{3 - }}}}_{{ ext{4}}} $$ at approximately 959 cm⁻¹ have been demonstrated as being sensitive markers of early caries. This ex vivo study on extracted human teeth demonstrates that these measurements can be made with reasonable precision with concomitantly good repeatability and reproducibility in sound enamel. Such reliability is crucial for these techniques to have a practical clinical value.

Keywords: Repeatability; Reproducibility; Variance components; Dental caries; Optical coherence tomography; Raman spectroscopy

Biomolecular profiling of metastatic prostate cancer cells in bone marrow tissue using FTIR microspectroscopy: a pilot study by E. Gazi; J. Dwyer; N. P. Lockyer; P. Gardner; J. H. Shanks; J. Roulson; C. A. Hart; N. W. Clarke; M. D. Brown (pp. 1621-1631).

Prostate cancer (CaP) cells preferentially metastasise to the bone marrow, a microenvironment that plays a substantial role in the sustenance and progression of the CaP tumour. Here we use a combination of FTIR microspectroscopy and histological stains to increase molecular specificity and probe the biochemistry of metastatic CaP cells in bone marrow tissue derived from a limited source of paraffin-embedded biopsies of different patients. This provides distinction between the following dominant metabolic processes driving the proliferation of the metastatic cells in each of these biopsies: glycerophospholipid synthesis from triacylglyceride, available from surrounding adipocytes, in specimen 1, through significantly high (p ≤ 0.05) carbohydrate (8.23 ± 1.44 cm⁻¹), phosphate (6.13 ± 1.5 cm⁻¹) and lipid hydrocarbon (24.14 ± 5.9 cm⁻¹) signals compared with the organ-confined CaP control (OC CaP), together with vacuolation of cell cytoplasm; glycolipid synthesis in specimen 2, through significantly high (p ≤ 0.05) carbohydrate (5.51 ± 0.04 cm⁻¹) and high lipid hydrocarbon (17.91 ± 2.3 cm⁻¹) compared with OC CaP, together with positive diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells; glycolysis in specimen 3, though significantly high (p ≤ 0.05) carbohydrate (8.86 ± 1.78 cm⁻¹) and significantly lower (p ≤ 0.05) lipid hydrocarbon (11.67 ± 0.4 cm⁻¹) than OC CaP, together with negative diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells. Detailed understanding of the biochemistry underpinning the proliferation of tumour cells at metastatic sites may help towards refining chemotherapeutic treatment.

Keywords: Prostate cancer; Infrared microspectroscopy; Metastases; Lipid

CH-overtone regions as diagnostic markers for near-infrared spectroscopic diagnosis of primary cancers in human pancreas and colorectal tissue by Venkata Radhakrishna Kondepati; Thomas Oszinda; H. Michael Heise; Klaus Luig; Ralf Mueller; Olaf Schroeder; Michael Keese; Juergen Backhaus (pp. 1633-1641).

We have investigated the application of near-infrared spectroscopy for detection of human primary pancreatic and colorectal cancers. Spectra from cancerous and normal tissue were collected from a total of 37 surgically resected pancreatic and colorectal patient tissue specimens using a fibre-optic probe. Major spectral differences were observed in the CH-stretching first (6,000–5,400 cm⁻¹) and second overtone (9,000–7,900 cm⁻¹) regions. By use of artificial neural networks, linear discriminant analysis, and cluster analysis as pattern-recognition methods the spectra were classified into cancerous and normal tissue groups with accuracy up to 89%. We also explored differences between the spectra obtained from colorectal and pancreatic tissue. Spectral data from cancerous and normal tissue were classified organ-specifically into four groups with accuracy between 80 and 83%. Our results indicate that CH-overtone regions, besides serving as diagnostic markers for NIR spectroscopic diagnosis of primary human pancreas and colorectal cancers, are also useful for elucidating differences between the spectra obtained from colorectal and pancreatic cancerous tissue.

Keywords: Near-infrared spectroscopy; Pancreas cancer; Colorectal cancer; Cancer diagnosis; Pattern recognition

Study of normal colorectal tissue by FT-Raman spectroscopy by P. O. Andrade; R. A. Bitar; K. Yassoyama; H. Martinho; A. M. E. Santo; P. M. Bruno; A. A. Martin (pp. 1643-1648).

FT-Raman spectroscopy was employed to study normal human colorectal tissues in vitro with the aim of evaluating the spectral differences of the complex colon mucous in order to establish a characteristic Raman spectrum. The samples were collected from 39 patients, providing 144 spectra for the statistical analysis. The results enable one to estabilish three well-defined spectroscopic groups of non-altered coloretal tissues that were consistently checked by statistical (clustering) and biological (histopathology) analyses: group 1 is represented by samples with the presence of epithelial layer, connective tissue papillae, and smooth muscle tissue; group 2 comprises tissues with epithelial layer and connective tissue papillae; group 3 presented mostly fatty and slack conjunctive tissue. The study reveals the existence of an intrinsic spectral variability for each patient that must be considered when sampling tissues fragments to build a spectral database. This is the first step for future studies and applications of Raman spectroscopy to optical biopsy and diagnosis of colorectal cancer.

Keywords: FT-Raman spectroscopy; Colorectal; Cancer; Optical biopsy

FTIR and Raman microspectroscopy of normal, benign, and malignant formalin-fixed ovarian tissues by C. Murali Krishna; G. D. Sockalingum; Rani A. Bhat; L. Venteo; Pralhad Kushtagi; M. Pluot; M. Manfait (pp. 1649-1656).

Ovarian cancer is the sixth most common cancer among women worldwide, and mortality rates from this cancer are higher than for other gynecological cancers. This is attributed to a lack of reliable screening methods and the inadequacy of treatment modalities for the advanced stages of the disease. FTIR and Raman spectroscopic studies of formalin-fixed normal, benign, and malignant ovarian tissues have been undertaken in order to investigate and attempt to understand the underlying biochemical changes associated with the disease, and to explore the feasibility of discriminating between these different tissue types. Raman spectra of normal tissues indicate the dominance of proteins and lower contents of DNA and lipids compared to malignant tissues. Among the pathological tissues studied, spectra from benign tissues seem to contain more proteins and less DNA and lipids compared to malignant tissue spectra. FTIR studies corroborate these findings. FTIR and Raman spectra of both normal and benign tissues showed more similarities than those of malignant tissues. Cluster analysis of first-derivative Raman spectra in the 700–1700 cm⁻¹ range gave two clear groups, one corresponding to malignant and the other to normal+benign tissues. At a lower heterogeneity level, the normal+benign cluster gave three nonoverlapping subclusters, one corresponding to normal and two for benign tissues. Cluster analysis of second-derivative FTIR spectra in the combined spectral regions of 1540–1680 and 1720–1780 cm⁻¹ resulted into two clear clusters corresponding to malignant and normal+benign tissues. The cluster corresponding to normal+benign tissues produced nonoverlapping subclusters for normal and benign tissues at a lower heterogeneity level. The findings of this study demonstrate the feasibility of Raman and FTIR microspectroscopic discrimination of formalin-fixed normal, benign, and malignant ovarian tissues.

Keywords: Ovarian cancers; Optical diagnosis; Raman; FTIR; Formalin-fixed tissues; Cluster analysis

The use of Raman spectroscopy to provide an estimation of the gross biochemistry associated with urological pathologies by Nicholas Stone; Maria Consuelo Hart Prieto; Paul Crow; Jeremy Uff; Alistair William Ritchie (pp. 1657-1668).

Near-infrared Raman spectroscopy, an optical technique that is able to interrogate biological tissues, has been used to study bladder and prostate tissues, with the objective being to provide a first approximation of gross biochemical changes associated with the process of carcinogenesis. Prostate samples for this study were obtained by taking a chip at TURP, and bladder samples from a biopsy taken at TURBT and TURP, following ethical approval. Spectra were taken from purchased biochemical constituents and different pathologies within the bladder and the prostate. We were then able to determine the biochemical basis for these pathologies by utilising an ordinary least-squares fit. We have shown for the first time that we are able to utilise Raman spectroscopy in determining the biochemical basis for the different pathologies within the bladder and prostate gland. In this way we can achieve a better understanding of disease processes such as carcinogenesis. This could have major implications in the future of the diagnosis of disease within the bladder and the prostate gland.

Keywords: Raman spectroscopy; Prostate; Bladder; Biochemistry; Carcinogenesis

Classification of malignant gliomas by infrared spectroscopic imaging and linear discriminant analysis by Christoph Krafft; Stephan B. Sobottka; Kathrin D. Geiger; Gabriele Schackert; Reiner Salzer (pp. 1669-1677).

Infrared (IR) spectroscopy provides a sensitive molecular fingerprint for tissue without external markers. Supervised classification models can be trained to identify the tissue type based on the spectroscopic fingerprint. Infrared imaging spectrometers equipped with multi-channel detectors combine the spectral and spatial information. Tissue areas of 4 × 4 mm² can be analyzed within a few minutes in the macroscopic imaging mode. An approach is described to apply this methodology to human astrocytic gliomas, which are graded according to their malignancy from one to four. Multiple IR images of three tissue sections from one patient with a malignant glioma are acquired and assigned to the six classes normal brain tissue, astrocytoma grade II, astrocytoma grade III, glioblastoma multiforme grade IV, hemorrhage, and other tissue by a linear discriminant analysis model which was trained by data from a single-channel detector. Before the model is applied here, the spectra are shown to be virtually identical. The first specimen contained approximately 95% malignant glioma regions, that means astrocytoma grade III or glioblastoma. The smaller percentage of 12–34% malignant glioma in the second specimen is consistent with its location at the tumor periphery. The detection of less than 0.2% malignant glioma in the third specimen points to a location outside the tumor. The results were correlated with the cellularity of the tissue which was obtained from the histopathologic gold standard. Potential applications of IR spectroscopic imaging as a rapid tool to complement established diagnostic methods are discussed.

Keywords: Brain tumors; Astrocytic gliomas; FTIR imaging; Tissue classification

Synchrotron FTIR microspectroscopic analysis of the effects of anti-inflammatory therapeutics on wound healing in laminectomized rats by Richard Wiens; Margaret Rak; Nicole Cox; Suraj Abraham; Bernhard H. J. Juurlink; William M. Kulyk; Kathleen M. Gough (pp. 1679-1689).

Peridural scarring, or the excessive formation of scar tissue following spinal surgery, is one of the important contributing factors that result in persistent pain and disability in many individuals who have undergone elective back surgery. Treatment with anti-inflammatory agents following surgery may reduce oxidative stress and scarring, leading to a reduction in post-operative pain. We are using a surgical rat model to test the hypothesis that post-surgical inflammation and oxidative stress following laminectomy can be reduced by systemic administration of L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin. OTC is a cysteine precursor required for the synthesis of glutathione, an important antioxidant. Quercetin is a flavenoid with anti-oxidant properties, found in fruits and vegetables. Synchrotron FTIR microspectroscopy data has been collected on OTC, quercetin and saline (control)-treated post-surgery animals, sacrificed at 3 and 21 days (n = 6 per age and treatment group). This paper presents preliminary IR results, supported by immunocytochemistry, on the heterogenous distribution of biological components present in the healing tissue. The data collected on animals sacrificed at 3 and 21 days post-surgery will be combined in the future with data from animals sacrificed 63 days after surgery (representing a third time point) to evaluate the efficacy of the different treatments. Initial statistical analysis of ED1 immunohistochemistry results indicates a decrease in the number of activated macrophages 21 days post-surgery in the OTC-treated animals compared with the saline controls.

Keywords: Oxidative stress; Wound healing; FTIR microspectroscopy; L-2-Oxo-thiazolidine-4-carboxylate; Quercetin; Peridural scarring

Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation by Bayden R. Wood; Peter Caspers; Gerwin J. Puppels; Shveta Pandiancherri; Don McNaughton (pp. 1691-1703).

Resonance Raman spectra of oxygenated and deoxygenated functional erythrocytes recorded using 785 nm laser excitation are presented. The high-quality spectra show a mixture of enhanced A_1g, A_2g, B_1g, B_2g, E_u and vinyl modes. The high sensitivity of the Raman system enabled spectra from four oxygenation and deoxygenation cycles to be recorded with only 18 mW of power at the sample over a 60-minute period. This low power prevented photo-/thermal degradation and negated protein denaturation leading to heme aggregation. The large database consisting of 210 spectra from the four cycles was analyzed with principal components analysis (PCA). The PC1 loadings plot provided exquisite detail on bands associated with the oxygenated and deoxygenated states. The enhancement of a band at 567 cm⁻¹, observed in the spectra of oxygenated cells and the corresponding PC1 loadings plot, was assigned to the Fe–O₂ stretching mode, while a band appearing at 419 cm⁻¹ was assigned to the Fe–O–O bending mode based on previous studies. For deoxygenated cells, the enhancement of B_1g modes at 785 nm excitation is consistent with vibronic coupling between band III and the Soret transition. In the case of oxygenated cells, the enhancement of iron-axial out-of-plane modes and non-totally symmetric modes is consistent with enhancement into the y,z-polarized transition $${ ext{a}}_{{{ ext{iu}}}} {left( { ext{ $ pi $ }} ight)} o { ext{d}}_{{{ ext{xz}}}} + { ext{O}}_{{ ext{2}}} {left( {{ ext{ $ pi $ }}_{{ ext{g}}} } ight)}$$ centered at 785 nm. The enhancement of non-totally symmetric B_1g modes in oxygenated cells suggests vibronic coupling between band IV and the Soret band. This study provides new insights into the vibrational dynamics, electronic structure and resonant enhancement of heme moieties within functional erythrocytes at near-IR excitation wavelengths.

Keywords: Raman spectroscopy; Red blood cells; Near-infrared excitation; Ligand modes; Vibronic coupling

A new sample substrate for imaging and correlating organic and trace metal composition in biological cells and tissues by Lisa M. Miller; Qi Wang; Randy J. Smith; Hui Zhong; Donald Elliott; John Warren (pp. 1705-1715).

Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and X-ray fluorescence (XRF) microscopes are becoming increasingly popular tools for imaging the organic and trace metal compositions of biological materials, respectively, without the need for extrinsic labels or stains. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2–10 μm in the mid-infrared region. The synchrotron X-ray fluorescence (SXRF) microprobe is a complementary technique used to probe trace element content in the same systems with a similar spatial resolution. However to be most beneficial, it is important to combine the results from both imaging techniques on a single sample, which requires precise overlap of the IR and X-ray images. In this work, we have developed a sample substrate containing a gold grid pattern on its surface, which can be imaged with both the IR and X-ray microscopes. The substrate consists of a low trace element glass slide that has a gold grid patterned on its surface, where the major and minor parts of the grid contain 25 and 12 nm gold, respectively. This grid pattern can be imaged with the IR microscope because the reflectivity of gold differs as a function of thickness. The pattern can also be imaged with the SXRF microprobe because the Au fluorescence intensity changes with gold thickness. The tissue sample is placed on top of the patterned substrate. The grid pattern’s IR reflectivity image and the gold SXRF image are used as fiducial markers for spatially overlapping the IR and SXRF images from the tissue. Results show that IR and X-ray images can be correlated precisely, with a spatial resolution of less than one pixel (i.e., 2–3 microns). The development of this new tool will be presented along with applications to paraffin-embedded metalloprotein crystals, Alzheimer’s disease, and hair composition.

Keywords: Infrared microspectroscopy; X-ray fluorescence microprobe; Nanopatterning; Metals; Tissues

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes by Aidan D. Meade; Fiona M. Lyng; Peter Knief; Hugh J. Byrne (pp. 1717-1728).

Non-invasive measurements of cellular function in in vitro cultured cell lines using vibrational spectroscopy require the use of spectroscopic substrates such as quartz, ZnSe and MirrIR etc. These substrates are generally dissimilar to the original in vivo extracellular environment of a given cell line and are often tolerated poorly by cultured cell lines resulting in morphological and functional changes in the cell. The present study demonstrates various correlations between vibrational spectroscopic analyses and biochemical analyses in the evaluation of the interaction of a normal human epithelial keratinocyte cell line (HaCaT) with MirrIR and quartz substrates coated with fibronectin, laminin and gelatin. The findings of this study suggest that there is a correlation between quantitative measurements of cellular proliferative capacity and viability and peak area ratios in FTIR spectra, with replicated differences in similar areas of the observed Raman spectra. Differences in the physiology of cells were observed between the two spectroscopic substrates coated in fibronectin and laminin, but little differences were observed when the cells were attached to gelatin-coated quartz and MirrIR slides. The correlations demonstrate the sensitivity of the spectroscopic techniques to evaluate the physiology of the system. Furthermore the study suggests that gelatin is a suitable coating for use in spectroscopic measurements of cellular function in human keratinocytes, as it provides a material that normalises the effect of substrate attachment on cellular physiology. This effect is likely to be cell-line dependent, and it is recommended that similar evaluations of this effect are performed for those combinations of spectroscopic substrate and cell lines that are to be used in individual experiments.

Keywords: Raman/FTIR spectroscopy; HaCaT keratinocytes; Adhesion effects; Spectroscopic substrates

FTIR spectroscopy in medical mycology: applications to the differentiation and typing of Candida by Dominique Toubas; Mohammed Essendoubi; Isabelle Adt; Jean-Michel Pinon; Michel Manfait; Ganesh D Sockalingum (pp. 1729-1737).

The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.

Keywords: FTIR spectroscopy; Candida species; Microcolonies; Discrimination; Typing

Fourier transform infrared (FT-IR) spectroscopy in bacteriology: towards a reference method for bacteria discrimination by Ornella Preisner; João Almeida Lopes; Raquel Guiomar; Jorge Machado; José C. Menezes (pp. 1739-1748).

Rapid and reliable discrimination among clinically relevant pathogenic organisms is a crucial task in microbiology. Microorganism resistance to antimicrobial agents increases prevalence of infections. The possibility of Fourier transform infrared (FT-IR) spectroscopy to assess the overall molecular composition of microbial cells in a non-destructive manner is reflected in the specific spectral fingerprints highly typical for different microorganisms. With the objective of using FT-IR spectroscopy for discrimination between diverse microbial species and strains on a routine basis, a wide range of chemometrics techniques need to be applied. Still a major issue in using FT-IR for successful bacteria characterization is the method for spectra pre-processing. We analyzed different spectra pre-processing methods and their impact on the reduction of spectral variability and on the increase of robustness of chemometrics models. Different types of the Enterococcus faecium bacterial strain were classified according to chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis (PFGE). Samples were collected from human patients. Collected FT-IR spectra were used to verify if the same classification was obtained. In order to further optimize bacteria classification we investigated whether a selected combination of the most discriminative spectral regions could improve results. Two different variable selection methods (genetic algorithms (GAs) and bootstrapping) were investigated and their relative merit for bacteria classification is reported by comparing with results obtained using the entire spectra. Discriminant partial least-squares (Di-PLS) models based on corrected spectra showed improved predictive ability up to 40% when compared to equivalent models using the entire spectral range. The uncertainty in estimating scores was reduced by about 50% when compared to models with all wavelengths. Spectral ranges with relevant chemical information for Enterococcus faecium bacteria discrimination were outlined.

Keywords: PFGE typing; FT-IR spectroscopy; Partial least-squares (PLS); Genetic algorithm (GA); PLS-bootstrapping

Raman spectroscopic investigation of the antimalarial agent mefloquine by Torsten Frosch; Michael Schmitt; Jürgen Popp (pp. 1749-1757).

The antimalarial agent mefloquine was investigated using Fourier transform near-infrared (FT NIR) Raman and FT IR spectroscopy. The IR and Raman spectra were calculated with the help of density functional theory (DFT) and a very good agreement with the experimental spectra was achieved. These DFT calculations were applied to unambiguously assign the prominent features in the experimental vibrational spectra. The calculation of the potential energy distribution (PED) and the atomic displacements provide further valuable insight into the molecular vibrations. The most prominent NIR Raman bands at 1,363 cm⁻¹ and 1,434 cm⁻¹ are due to C=C stretching (in the quinoline part of mefloquine) and CH₂ wagging vibrations, while the most intense IR peaks at 1,314 cm⁻¹; 1,147 cm⁻¹; and 1,109 cm⁻¹ mainly consist of ring breathings and δCH (quinoline); C–F stretchings; and asymmetric ring breathings, C–O stretching as well as CH₂ twisting/rockings located at the piperidine moiety. Since the active agent (mefloquine) is usually present in very low concentrations within the biological samples, UV resonance Raman spectra of physiological solutions of mefloquine were recorded. By employing the detailed non-resonant mode assignment it was also possible to unambiguously identify the resonantly enhanced modes at 1,619 cm⁻¹, 1,603 cm⁻¹ and 1,586 cm⁻¹ in the UV Raman spectra as high symmetric C=C stretching vibrations in the quinoline part of mefloquine. These spectroscopic results are important for the interpretation of upcoming in vitro and in vivo mefloquine target interaction experiments.

Keywords: UV resonance Raman; Raman spectroscopy; Malaria; Mefloquine; Density functional theory calculation (DFT)

Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques by Diego Serra; Alejandra Bosch; Daniela M. Russo; María E. Rodríguez; Ángeles Zorreguieta; Juergen Schmitt; Dieter Naumann; Osvaldo Yantorno (pp. 1759-1767).

This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-μm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg⁺) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 μm in diameter and 24 μm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.

Keywords: Bordetella pertussis ; Biofilm structure; Image analysis; Quantification; Fourier transform IR spectroscopy

Investigating the heterogeneity of cell growth in microbial colonies by FTIR microspectroscopy by N. A. Ngo Thi; D. Naumann (pp. 1769-1777).

Microorganisms rarely occur as individual cells in nature and are, instead, organized in complex multicellular communities such as colonies, fruiting bodies, or biofilms. Interest in the natural microbial life-style has increased during the last decade and a whole plethora of techniques has been used to gain insight into the development, structure and composition of diverse microbial communities. We have developed a technique for investigating the spatial heterogeneity of microbial growth in macro-colonies which essentially entails excision of the colonies with the underlying agar, freezing and subsequent cryotoming of the colonies, then FTIR microspectroscopic mapping of the cryosections. Colonies from Legionella, Bacillus, and Candida strains were chosen as model systems of multi-cellular communities to evaluate the technique. The results obtained indicate pronounced cell population heterogeneity even in relatively young colonies cultivated under laboratory conditions. Spectral data obtained from different positions within, e.g., a colony of Legionella bozemanii 120 h old indicated that levels of the storage material poly-β-hydroxybutyric acid were significantly higher in cells at the surface of the colonies than in those growing at the bottom next to the agar surface. Similarly, in a 24-h-old macro-colony of Bacillus megaterium significantly more of the capsular compound polyglutamic acid was detected in upper layers than in deeper layers of the colony. Results demonstrate that FTIR microspectroscopy can be an useful tool for investigation of the spatial heterogeneity of cell growth within microbial macro-colonies. It is suggested that the method also can be adapted to the analysis of more complex multicellular communities, for example fruiting bodies, biofilms, or colonies growing under natural conditions.

Keywords: FTIR microspectroscopy; Microorganisms; Micro-colonies; Macro-colonies; Growth heterogeneity; Poly-β-hydroxybutyric acid

A synchrotron FTIR microspectroscopy investigation of fungal hyphae grown under optimal and stressed conditions by Adriana Szeghalmi; Susan Kaminskyj; Kathleen M. Gough (pp. 1779-1789).

Synchrotron FTIR can provide high spatial resolution (<10 μm pixel size) in situ biochemical analyses of intact biotissues, an area of increasing importance in the post-genomic era, as gene functions and gene networks are coming under direct scrutiny. With this technique, we can simultaneously assess multiple aspects of cell biochemistry and cytoplasmic composition. In this paper, we report the first results of our synchrotron FTIR examination of hyphae of three important fungal model systems, each with sequenced genomes and a wealth of research: Aspergillus, Neurospora, and Rhizopus. We have analyzed the FTIR maps of Aspergillus nidulans cells containing the hypA1 allele, a well-characterized single-gene temperature-sensitive morphogenetic mutation. The hypA1 cells resemble wildtype at 28 °C but have growth defects at 42 °C. We have also investigated Neurospora and Rhizopus cultures grown in media with optimal or elevated pH. Significant differences between the spectra of the three fungi are likely related to differences in composition and structure. In addition, high spatial resolution synchrotron FTIR spectroscopy provides an outstanding method for monitoring subtle subcellular changes that accompany environmental stress. Figure Photomicrographs and FTIR spectra acquired along Rhizopus hyphae grown at pH 6.5 (a) and pH 8.5 (b). Scale bars 50 μm

Keywords: Synchrotron FTIR spectromicroscopy; Aspergillus nidulans ; Neurospora ; Rhizopus ; Fungal tip growth under stress

Detection of preclinical scrapie from serum by infrared spectroscopy and chemometrics by Peter Lasch; Michael Beekes; Jürgen Schmitt; Dieter Naumann (pp. 1791-1800).

In this study we describe a methodology for diagnosing preclinical scrapie infection in hamsters from serum by a combination of Fourier-transform infrared (FT–IR) spectroscopy and chemometrics. Syrian hamsters (Mesocricetus auratus) were orally inoculated with the 263K scrapie agent, or mock-infected, and sera were obtained at 70, 100 and 130 days post infection (dpi) and at the terminal stage of scrapie (160 ± 10 dpi). The analysis of hamster sera by FT–IR spectroscopy and artificial neural networks (ANN) confirmed results from earlier studies which had indicated the existence of disease-related structural and compositional alterations in the sera of infected donors in the terminal stage of scrapie [Schmitt et al. (2002) Anal Chem 74:3865–3868]. Experimental data from sera of animals in the preclinical stages of scrapie revealed subtle but reproducible spectral variations that permitted the identification of a preclinical scrapie infection at 100 dpi and later, but not at 70 dpi. The IR spectral features that were discriminatory for the preclinical stages differed from those of the terminally ill individuals. In order to reliably identify scrapie-negative as well as preclinical (100 and 130 dpi) and terminal scrapie-positive animals, a hierarchical classification system of independent artificial neural networks (ANN) was established. A “toplevel” ANN was designed which discriminates between animals in the terminal stage of scrapie and preclinical scrapie-positive or control animals. Spectra identified by the “toplevel” ANN as preclinical or controls were then further analyzed by a second classifier, the “sublevel” ANN. Using independent external validation procedures, the toplevel classifier produced an overall classification accuracy of 98%, while the sublevel classifier yielded an accuracy of 93%, indicating that scrapie-specific serum markers were also present for preclinical disease stages. Possible spectral markers responsible for the discrimination capacity of the two different ANNs are discussed.

Keywords: Scrapie; Transmissible spongiform encephalopathy; Serum diagnostics; Infrared spectroscopy; Pattern recognition

Multivariate feature selection and hierarchical classification for infrared spectroscopy: serum-based detection of bovine spongiform encephalopathy by Bjoern H. Menze; Wolfgang Petrich; Fred A. Hamprecht (pp. 1801-1807).

A hierarchical scheme has been developed for detection of bovine spongiform encephalopathy (BSE) in serum on the basis of its infrared spectral signature. In the first stage, binary subsets between samples originating from diseased and non-diseased cattle are defined along known covariates within the data set. Random forests are then used to select spectral channels on each subset, on the basis of a multivariate measure of variable importance, the Gini importance. The selected features are then used to establish binary discriminations within each subset by means of ridge regression. In the second stage of the hierarchical procedure the predictions from all linear classifiers are used as input to another random forest that provides the final classification. When applied to an independent, blinded validation set of 160 further spectra (84 BSE-positives, 76 BSE-negatives), the hierarchical classifier achieves a sensitivity of 92% and a specificity of 95%. Compared with results from an earlier study based on the same data, the hierarchical scheme performs better than linear discriminant analysis with features selected by genetic optimization and robust linear discriminant analysis, and performs as well as a neural network and a support vector machine.

Keywords: Diagnostic pattern recognition; Random forest; Gini importance; Feature selection; Hierarchical classification

Quantification of serum apolipoprotein B by infrared spectroscopy by Kan-Zhi Liu; Angela Man; Thomas C. Dembinski; R. Anthony Shaw (pp. 1809-1814).

While the conventional approach to assessing both the risk of coronary artery disease and the adequacy of therapy is LDL cholesterol testing, there is compelling evidence to suggest that apolipoprotein B (apoB) is superior to LDL cholesterol for both of these purposes. However, the measurement of apoB requires techniques that can be expensive and difficult to standardize. The aim of this study was, therefore, to develop a new method, based on infrared (IR) spectroscopy, for the routine quantification of apoB in human serum. A total of 366 serum samples were obtained from patients with various disorders. Small volumes (2 μl) of serum specimens were dried to films, and duplicate IR absorption spectra measured. The reference apoB concentrations were determined separately using a standard method, and the proposed IR method was then calibrated using partial least squares (PLS) regression analysis to quantitatively correlate the IR spectra with the reference results. The apoB concentrations predicted from the IR spectra of serum were highly correlated and in excellent agreement with those determined by the reference method. The correlation coefficient (r) for apoB was 0.94, with the standard error between IR-predicted and reference values was 0.10 g/L. In combination with earlier work demonstrating the accurate determination of LDL-C, HDL-C, total cholesterol, and triglycerides from a single infrared spectroscopic measurement, the addition of accurate apoB determination from the same spectrum makes the method very attractive for laboratory use in the routine evaluation of coronary artery disease risk.

Keywords: Apolipoproteins; LDL; Lipid panel; Cardiovascular disease; Cholesterol; PLS; Clinical chemistry; Quantification; Serum; Infrared

Clinical chemistry without reagents? An infrared spectroscopic technique for determination of clinically relevant constituents of body fluids by Gamze Hoşafçı; Oliver Klein; Gerhard Oremek; Werner Mäntele (pp. 1815-1822).

A spectroscopic method based on attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has been developed for reagent-free analysis of blood and urine constituents in the clinical laboratory and for point-of-care-applications. Blood plasma, whole blood, and urine were analyzed without any sample preparation, such as drying, concentration, or enrichment. Sample volumes as small as 5 μL (a single drop of blood) can be used. Mathematical models, including partial least-squares regression, were used to construct a prediction model which can calculate the concentration of albumin, cholesterol, glucose, total protein, urea, and triglycerides in whole blood or blood plasma samples and the concentration of urea, uric acid, phosphate and creatinine in urine samples. The absolute precision and reproducibility of the prediction reached is sufficient for routine clinical analysis and is only limited by the precision of the reference analysis used for calibration. This was achieved by use of a large number of calibration samples (approx. 400 for blood samples and approx. 100 for urine samples) carefully selected for physiological and pathological range and for specific disease profiles.

Keywords: Blood analysis; Urine analysis; Body fluids; Attenuated total reflection (ATR); Infrared spectroscopy

Analysis of biosensors by chemically specific optical techniques. Chemiluminescence-imaging and infrared spectroscopic mapping ellipsometry by K. Hinrichs; M. Gensch; N. Esser; U. Schade; J. Rappich; S. Kröning; M. Portwich; R. Volkmer (pp. 1823-1829).

The standard methods currently used to read out microarrays are fluorescent and chemiluminesent imaging techniques. These methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected. A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.

Keywords: IR spectroscopy/Raman spectroscopy; Bioanalytical methods; Interface/Surface analysis; Spectroscopy/Instrumentation; Biosensors

Recent advances in surface plasmon resonance based techniques for bioanalysis by K. Scott Phillips; Quan Cheng (pp. 1831-1840).

Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright.Surface plasmon resonance (SPR) is a powerful and versatile spectroscopic method for biomolecular interaction analysis (BIA) and has been well reviewed in previous years. This updated 2006 review of SPR, SPR spectroscopy, and SPR imaging explores cutting-edge technology with a focus on material, method, and instrument development. A number of recent SPR developments and interesting applications for bioanalysis are provided. Three focus topics are discussed in more detail to exemplify recent progress. They include surface plasmon fluorescence spectroscopy, nanoscale glassification of SPR substrates, and enzymatic amplification in SPR imaging. Through these examples it is clear to us that the development of SPR-based methods continues to grow, while the applications continue to diversify. Major trends appear to be present in the development of combined techniques, use of new materials, and development of new methodologies. Together, these works constitute a major thrust that could eventually make SPR a common tool for surface interaction analysis and biosensing. The future outlook for SPR and SPR-associated BIA studies, in our opinion, is very bright.

Keywords: Surface plasmon resonance; Surface plasmon resonance fluorescence; Surface plasmon resonance imaging; Biosensors; Biomolecular interaction analysis

Effects of sample pretreatment and storage conditions in the determination of pyrethroids in water samples by solid-phase microextraction and gas chromatography–mass spectrometry by Vanessa Casas; María Llompart; Carmen Garcia-Jares; Rafael Cela; Thierry Dagnac (pp. 1841-1849).

The aqueous instability of pyrethroids and other compounds usually found in commercial pesticide formulations has been demonstrated in this work. Several types of sample treatment have been studied to avoid analyte losses during sample manipulation and storage. Analysis was performed by SPME–GC–MS. Addition of sodium thiosulfate to tap water prevented pyrethroid degradation as a result of oxidation by free chlorine. The amount added was optimized to minimize the effect of the salt on the analytical results. Analysis of samples that had been stored at 4 °C for several days revealed loss of some of the pyrethroids in the first period of storage. The effect of freezing the samples was studied and it was confirmed that samples could be stabilized for at least one week by freezing. Finally, addition of a miscible organic solvent, for example acetone, led to improvement of the analytical precision. The quality of the SPME–GC–MS method was studied. Linearity (R > 0.993), repeatability (RSD < 15%), and sensitivity (detection limits between 0.9 and 35 pg mL⁻¹) were good. When the procedure was applied to real samples including run off and waste water some of the target compounds were identified and quantified.

Keywords: Water analysis; Solid-phase microextraction; Gas chromatography–mass spectrometry; Pyrethroids; Sample stability

ICP-MS as a novel detection system for quantitative element-tagged immunoassay of hidden peanut allergens in foods by Maria Careri; Lisa Elviri; Alessandro Mangia; Claudio Mucchino (pp. 1851-1854).

A novel ICP-MS-based ELISA immunoassay via element-tagged determination was devised for quantitative analysis of hidden allergens in food. The method was able to detect low amounts of peanuts (down to approximately 2 mg peanuts kg⁻¹ cereal-based matrix) by using a europium-tagged antibody. Selectivity was proved by the lack of detectable cross-reaction with a number of protein-rich raw materials.

Keywords: ICP; MS detection; ELISA; Food allergens

Separation of molecular tracers sorbed onto atmospheric particulate matter by flash chromatography by Audrey Lottmann; Emilie Cadé; Majdi Lahd Geagea; Olivier Delhomme; Catherine Grand; Claire Veilleraud; Anne-Laure Rizet; Philippe Mirabel; Maurice Millet (pp. 1855-1861).

In to order increase sensitivity and to reduce the background induced by matrix effects, a method was developed that uses flash chromatography to separate various compounds present in atmospheric aerosol samples prior to their analysis with different analytical techniques (GC–MS, GC–FID, HPLC). For this purpose, flash chromatography using a 4 g silica gel column crossed by eluent at a flow rate of 20 mL min⁻¹ was used. An eluent with enhanced polarity is needed to separate nonpolar (linear and branched alkanes), semipolar (PAH, nitro-PAH and cholesterol) and polar (methoxyphenols, alkanoic acids, and levoglucosan) compounds. Three combinations of solvents were used: hexane for the nonpolar fraction (F1), toluene/hexane for the semipolar fraction (F2) and dimethylformamide for the polar fraction (F3). The use of different eluents for each fraction allows separation of the sample to be accomplished with good repeatability and satisfying yields [85 ± 5% for F1, 81 ± 8% (PAHs), 89 ± 6% (nitro-PAHs) and 74 ± 7% (cholesterol) for F2 and 79 ± 7% (n-alkanoic acids), 40 ± 11% (methoxyphenols) and 77 ± 6% (levoglucosan) for F3]. The methoxyphenol yields were low due to losses during the concentration/evaporation step. This method was then applied to analyse the organic composition of particles collected at an urban site in Strasbourg (France).

Keywords: Flash chromatography; Silica gel column; Separation; Organic aerosols

Multivariate curve resolution of rapid-scan FTIR difference spectra of quinone photoreduction in bacterial photosynthetic membranes by L. Blanchet; A. Mezzetti; C. Ruckebusch; J.-P. Huvenne; A. de Juan (pp. 1863-1873).

Photosynthetic reaction centres and membranes are systems of particular interest and are often taken as models to investigate the molecular mechanisms of selected bioenergetic reactions. In this work, a multivariate curve resolution by alternating least squares procedure is detailed for resolution of time-resolved difference FTIR spectra probing the evolution of quinone reduction in photosynthetic membranes from Rhodobacter sphaeroides under photoexcitation. For this purpose, different data sets were acquired in the same time range and spectroscopic domain under slightly different experimental conditions. To enable resolution and provide meaningful results the different data sets were arranged in an augmented matrix. This strategy enabled recovery of three different species despite rank-deficiency conditions. It also results in better definition (identity and evolution) of the contributions. From the resolved spectra, the species have been attributed to: 1. the formation of ubiquinol, more precisely the disappearance of Q/appearance of QH₂; 2. conformational change of the protein in the surrounding biological medium; 3. oxidation of diaminodurene, a redox mediator. Because, moreover, results obtained from augmented data sets strategies enable quantitative and qualitative interpretation of concentration profiles, other effects, for example the consequence of repeated light excitation of the same sample, choice of illumination power, or the number of spectra accumulated could be compared and discussed.

Keywords: Factor analysis; MCR ALS; Rapid-scan FTIR; Difference spectra; Purple bacteria

Surface plasmon resonance biosensor based on Hg/Ag–Au film by Ying Sun; Daqian Song; Zhongqiu Li; Yu Bai; Hanqi Zhang (pp. 1875-1882).

Mercury or silver was electrodeposited on an Au surface to form an Hg–Au or Ag–Au film. Wavelength-modulation SPR biosensors based on this Hg/Ag–Au film were then used to determine human IgG and rabbit IgG. When direct immunoreactions were performed on the Au sensing surface, the range of concentrations of human IgG and rabbit IgG that could be determined were 2.00–40.00 μg/ml and 2.50–40.00 μg/ml, respectively. When Hg was electrodeposited onto the Au film for 1200 s, the range of concentrations of human IgG and rabbit IgG that could be determined were 0.50–40.00 μg/ml and 0.63–40.00 μg/ml, respectively. When Ag was electrodeposited onto the Au film for 1500 s, the range of concentrations of human IgG and rabbit IgG that could be determined were 0.25–20.00 and 0.42–20.00 μg/ml, respectively. The biosensor based on Ag–Au film was therefore found to be the most sensitive of the three types of biosensor tested, giving limits of determination that were up to eight times lower than those obtained with a biosensor based on Au film alone. Figure The relationship between the concentration of human IgG and the shift in the resonant wavelength Δλ _eq for different Ag electrodeposition times

Keywords: Surface plasmon resonance (SPR); Hg–Au film; Ag–Au film; Human IgG; Rabbit IgG

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence by Erwin Fundador; James Rusling (pp. 1883-1890).

Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 °C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.

Keywords: Capillary electrophoresis laser-induced fluorescence (CE-LIF); Abasic sites (AP); Fluorescent aldehyde-reactive probe (FARP); Methylmethane sulfonate (MMS); Depurination; N-7 methyl guanine

Electrocatalytic determination of reduced glutathione in human erythrocytes by Rita de Cássia Silva Luz; Flavio Santos Damos; Paulo Guimarães Gandra; Denise Vaz de Macedo; Auro Atsushi Tanaka; Lauro Tatsuo Kubota (pp. 1891-1897).

The determination of reduced glutathione (GSH) in human erythrocytes using a simple, fast and sensitive method employing a glassy carbon electrode modified with cobalt tetrasulfonated phthalocyanine (CoTSPc) immobilized in poly(l-lysine) (PLL) film was investigated. This modified electrode showed very efficient electrocatalytic activity for anodic oxidation of GSH, decreasing substantially the anodic overpotentials for 0.2 V versus Ag/AgCl. The modified electrode presented better performance in 0.1 mol l⁻¹ piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 7.4. The other experimental parameters, such as the concentration of CoTSPc and PLL in the membrane preparation, pH, type of buffer solution and applied potential, were optimized. Under optimized operational conditions, a linear response from 50 to 2,160 nmol l⁻¹ was obtained with a high sensitivity of 1.5 nA l nmol⁻¹ cm⁻². The detection limit for GSH determination was 15 nmol l⁻¹. The proposed sensor presented good repeatability, evaluated in terms of the relative standard deviation (1.5%) for n = 10. The modified electrode was applied for determination of GSH in erythrocyte samples and the results were in agreement with those obtained by a comparative method described in the literature The average recovery for these fortified samples was 100 ± 1)%. Applying a paired Student’s-t test to compare these methods, we could observe that, at the 95% confidence level, there was no statistical difference between the reference and the proposed methods.

Keywords: Electrocatalytic determination; Reduced glutathione; Erythrocyte samples

Amperometric biosensor for the determination of creatine by A. Ramanavicius (pp. 1899-1906).

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an $$ {{left[ {Fe{left( {CN} ight)}_{6} } ight]}^{{3 - }} } mathord{left/ {vphantom {{{left[ {Fe{left( {CN} ight)}_{6} } ight]}^{{3 - }} } {{left[ {Fe{left( {CN} ight)}_{6} } ight]}}}} ight. kern- ulldelimiterspace} {{left[ {Fe{left( {CN} ight)}_{6} } ight]}}^{{4 - }} $$ redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine

Keywords: Sarcosine; Creatine; Creatinine; Amperometric; Biosensor

A microscale device for measuring emissions from materials for indoor use by T. Schripp; B. Nachtwey; J. Toelke; T. Salthammer; E. Uhde; M. Wensing; M. Bahadir (pp. 1907-1919).

Emission test chambers or cells are used to determine organic vapour emissions from construction products under controlled conditions. Polymeric car trim component emissions are typically evaluated using direct thermal desorption/extraction. The Microchamber/Thermal Extractor (μ-CTE, Markes International) was developed to provide both a complementary tool for rapid screening of volatile organic compound (VOC) emissions—suitable for industrial quality control—and a means for thermal extraction of larger, more representative samples of car trim components. To determine the degree of correlation between conventional emission test methods and the microchamber, experiments were carried out under different conditions of temperature, air change rate and sample conditioning time. Good quantitative and qualitative correlation was obtained for measurements at ambient temperature. Moreover, it was shown that ambient-temperature emissions data collected using the μ-CTE as rapidly as possible—i.e. with minimal or no sample conditioning time—nevertheless provided a reliable guide as to how well that material would perform in subsequent 3-day chamber tests of VOC emissions. The parameters found to have the greatest influence on data correlation for experiments carried out at elevated temperatures were the sample mass (for bulk emissions testing) and the conditioning time. The results also showed that, within the constraints of inherent sample homogeneity, the μ-CTE gave reproducible emissions data, despite its small sample size/capacity relative to that of conventional chambers. Preliminary results of modelling the air flow within a microchamber using computational fluid dynamics showed a high degree of turbulent flow and two potential areas of still air which could cause sink effects. However, the experimental data reported here and in previous studies showed enhanced recovery of semivolatile components from the μ-CTE relative to a recovery from a 1 m³ conventional chamber. This indicates that if these areas of relatively still air are present within the microchamber, they do not appear to be significant in practice.

Keywords: Microchamber; Thermal desorption; Indoor air; Material emissions; Industrial quality control; Emissions screening

Rapid protein expression analysis with an interferometric biosensor for monitoring protein production by Christian Hoffmann; Katrin Schmitt; Albrecht Brandenburg; Steffen Hartmann (pp. 1921-1932).

The concentration of a recombinantly expressed protein has to be monitored to select optimal expression conditions throughout the protein production process. Today this is usually achieved semiquantitatively with sodium dodecyl sulfate polyacrylamide gel electrophoresis/western blotting or with ELISAs, which are time- and labor-intensive methods. In this paper the applicability of a label-free sensor system based on a Young interferometer is presented as an alternative for the monitoring of recombinant protein production. Once a protein is successfully produced, the interferometric biosensor allows any protein–protein interaction to be characterized in a label-free manner. This is demonstrated with an antibody/antigen pair, where the antibody is directed against a four-amino-acid tag used for protein expression analysis as well as purification during recombinant protein production. Label-free detection of the tagged protein is shown both in buffer and in bacterial cell lysate as a sample matrix. The system exhibiting a low limit of detection, low drift and reliable operation is compared with a commercial surface plasmon resonance sensor and a competitive ELISA. Figure 1 Waveguide sensor chip; grating (green) illuminated by a red light source. Image courtesy of Unaxis Optics

Keywords: Label-free detection; Interferometric biosensor; Surface plasmon resonance; Competitive ELISA

Cryogenic grinding pre-treatment improves extraction efficiency of fluoroquinolones for HPLC-MS/MS determination in animal tissue by Manuel Lolo; Sandra Pedreira; Beatriz I. Vázquez; Carlos M. Franco; Alberto Cepeda; Cristina A. Fente (pp. 1933-1937).

An efficiency extraction of fluoroquinolones in chicken muscle was achieved by pulverizing it in a freezer mill before treatment with NaOH (10mM)/MeCN (1:1). The improvement of cryogenic grinding in the extraction was demonstrated for the same piece (whole leg) of four chickens treated with enrofloxacin in equal doses. A confirmatory method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. The chromatographic separation was achieved in 5min with a Synergi Fusion-RP 80A (50 x 2mm, 4μm) column filled with a hybrid polymer. The HPLC was coupled with a detector based in a quadrupole–linear ion trap Q-TRAP that allows a confirmatory detection according to the European legislation. The specificity of the method was assessed by testing a number of representative blank muscle samples (n = 10) to verify the absence of potential interfering compounds. The limits of detection and quantitation were 2 and 5ng g¹ of quinolones in muscle samples, respectively. The chromatographic method was demonstrated to be linear for the range studied (5–500ng g¹) with the P value for lack-of-fit in the ANOVA table greater or equal to 0.10 (calibration coefficient 0.9998 and 0.9996 for ciprofloxacin and enrofloxacin, respectively). The mean intra-day relative standard deviation (RSD) (n = 6, c = 50ng g¹) was 6%; inter-day assay gave a RSD of 12%. The extraction and clean-up were carried out in one step with very satisfactory recovery data (between 65 and 101%).

Keywords: Cryogenic grinding; HPLC-MS/MS; Chicken; Food residues

A binary matrix for background suppression in MALDI-MS of small molecules by Zhong Guo; Lin He (pp. 1939-1944).

Application of matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) to small-molecule detection is often limited, because of high matrix background signals in the low-mass region. We report here an approach in which a mixture of two conventional MALDI matrices with different proton affinity was used to suppress the formation of matrix clusters and fragments. Specifically, when acidic α-cyano-4-hydroxycinnamic acid (CHCA) and basic 9-aminoacridine (9-AA) were used as the binary matrix, fewer background matrix peaks were observed in both positive and negative-mode detection of small molecules. In addition, the presence of CHCA substantially reduced the laser fluence needed for analyte desorption and ionization; thus better signal-to-background ratios were observed for negatively charged inositol phosphates in complex plant extracts. The mixing of MALDI matrices of different protonaffinities leads to suppression of matrix clusterformation and subsequently yields cleaner MS spectraof fewer background peaks in both positive andnegative detection of small molecules

Keywords: MALDI-MS; Binary matrix; Matrix background suppression; Small molecule detection

Room-temperature, phosphorimetric determination of the beta-blocking agent pindolol in pharmaceutical tablets, urine and blood serum by A. Salinas-Castillo; B. Cañabate-Díaz; A. Segura-Carretero; A. Fernández-Gutiérrez (pp. 1945-1948).

It is already recognised that heavy-atom-induced, room-temperature phosphorescence can be used to determine pindolol in pharmaceutical samples and biological fluids. We describe here a new, simple, rapid and selective development of this technique. The phosphorescence signals derive from the interaction of pindolol with a relatively high concentration of heavy-atom salts in the presence of sodium sulphite as oxygen scavenger. Phosphorescence was registered in the presence of 1.2 M potassium iodide, 15 mM sodium sulphite and 30% v/v methanol at 450 nm, exciting at 285 nm. The detection limit was 21.1 ng mL⁻¹. The method has been successfully applied to the determination of pindolol in commercial pharmaceutical tablets, urine and blood serum.

Keywords: Pindolol; Pharmaceutical analysis; Biological fluids; Room-temperature phosphorescence

Speciation analysis of antimony in extracts of size-classified volcanic ash by HPLC–ICP-MS by R. Miravet; J. F. López-Sánchez; R. Rubio; P. Smichowski; G. Polla (pp. 1949-1954).

Although there is concern about the presence of toxic elements and their species in environmental matrices, for example water, sediment, and soil, speciation analysis of volcanic ash has received little attention. Antimony, in particular, an emerging element of environmental concern, has been less studied than other potentially toxic trace elements. In this context, a study was undertaken to assess the presence of inorganic Sb species in ash emitted from the Copahue volcano (Argentina). Antimony species were extracted from size-classified volcanic ash (<36 μm, 35–45 μm, 45–150 μm, and 150–300 μm) by use of 1 mol L⁻¹ citrate buffer at pH 5. Antimony(III) and (V) in the extracts were separated and quantified by high-performance liquid chromatography combined on-line with inductively coupled plasma mass spectrometry (HPLC–ICP-MS). Antimony species concentrations (μg g⁻¹) in the four fractions varied from 0.14 to 0.67 for Sb(III) and from 0.02 to 0.03 for Sb(V). The results reveal, for the first time, the occurrence of both inorganic Sb species in the extractable portion of volcanic ash. Sb(III) was always the predominant species.

Keywords: Antimony speciation; Citrate extraction; Volcanic ash; HPLC–ICP-MS

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Analytical and Bioanalytical Chemistry (v.387, #5)