Computer screen photoassisted off-null ellipsometry.

The ellipsometric measurement of thickness is demonstrated using a computer screen as a light source and a webcam as a detector, adding imaging off-null ellipsometry to the range of available computer screen photoassisted techniques. The results show good qualitative agreement with a simplified theoretical model and a thickness resolution in the nanometer range is achieved. The presented model can be used to optimize the setup for sensitivity. Since the computer screen serves as a homogeneous large area illumination source, which can be tuned to different intensities for different parts of the sample, a large sensitivity range can be obtained without sacrificing thickness resolution.

Effect of fingerprint conformation and spectral scaling on the performance of computer screen photo-assisted experiments.

The use of computer screens as controlled light sources and web cameras as image detectors (the so-called computer screen photo-assisted technique, CSPT) is an ubiquitous alternative for the evaluation of colorimetric quick tests at homes or in primary care units. The performance of CSPT for such evaluations depends on several factors, from which the most relevant are the composition of illuminating sequences and the conformation of CSPT substance signatures. In this work, with the aid of a CSPT model, the effect of the construction of the substance signatures on the classification performance of different representative substance sets is studied. The correlation of illuminating colors with such classification is investigated, allowing one to determine redundancy and limitations with respect to visible spectroscopy. The concept of spectral scaling is introduced and its properties compared with standard procedures.

Measurement strategy and instrumental performance of a computer screen photo-assisted technique for the evaluation of a multi-parameter colorimetric test strip.

A measuring strategy for the evaluation of a seven parameters colorimetric test using a computer screen photo-assisted technique (CSPT) is demonstrated. CSPT is a versatile approach aimed at point of care or home tests that uses regular computer sets and web cameras as the whole instrument. Issues such as the stability and the equivalency on different platforms of the determinations have been addressed in the present work. The method uses an embedded local reference simultaneously measured with the tests and solves the evaluation as a classification problem. The achieved performance tested along 580 classifications covering all the ranges of the assay, using synthetic samples, yielded 97.2% correct determinations compared with 89.7% for the case of colorimetric determinations. The errors were concentrated in only two parameters that show a significant correlation with a set of quality indices used to assess the performance of the classification.

ELISA test for anti-neutrophil cytoplasm antibodies detection evaluated by a computer screen photo-assisted technique.

The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation.

Alpha-helix-inducing dimerization of synthetic polypeptide scaffolds on gold.

Designed, synthetic polypeptides that assemble into four-helix bundles upon dimerization in solution were studied with respect to folding on planar gold surfaces. A model system with controllable dimerization properties was employed, consisting of negatively and positively charged peptides. Circular dichroism spectroscopy and surface plasmon resonance based measurements showed that at neutral pH, the peptides were able to form heterodimers in solution, but unfavorable electrostatic interactions prevented the formation of homodimers. The dimerization propensity was found to be both pH- and buffer-dependent. A series of infrared absorption-reflection spectroscopy experiments of the polypeptides attached to planar gold surfaces revealed that if the negatively charged peptide was immobilized from a loading solution where it was folded, its structure was retained on the surface provided it had a cysteine residue available for anchoring to gold. If it was immobilized as random coil, it remained unstructured on the surface but was able to fold through heterodimerization if subsequently exposed to a positively charged polypeptide. When the positively charged peptide was immobilized as random coil, heterodimerization could not be induced, probably because of high-affinity interactions between the charged primary amine groups and the gold surface. These observations are intended to pave the way for future engineering of functional surfaces based on polypeptide scaffolds where folding is known to be crucial for function.

Penetration and loading of human serum albumin in porous silicon layers with different pore sizes and thicknesses.

Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 microm and with different mean pore radius in the range 4.5-10 nm. The adsorbed amount of protein was quantified by I(125) radioactive labeling techniques and ellipsometry. The results show that albumin penetrated into the pores when the mean pore radius was larger than 5.5 nm, but could not totally occupy the available surface area when the layer thickness was larger than 1 microm. Loading of albumin both into porous layers and onto plane silicon as a function of albumin concentration was also investigated. These measurements show that loading of protein increased with protein concentration at least up to 10 mg/ml for porous silicon and up to 1 mg/ml for plane silicon. The maximum deposition into the type of porous layers used here was 28 microg/cm(2), compared to 0.36 microg/cm(2) for plane silicon.

Altered impedance during pigment aggregation in Xenopus laevis melanophores.

Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

A model for switch-like phenomena in biological systems.

We present a model for the activity of protein clusters based on a simultaneous desorption of an activator (agonist, substrate molecule, etc.) and an inactivator (antagonist, inhibitor, etc.) caused by the collision or interaction between two effector molecules (e.g. receptors, enzymes). This model gives rise to switch-like dose-response curves, which are difficult to explain by ordinary co-operativity. It fits with recent experimental results obtained on single cells. Some other interesting aspects of the model are also pointed out. The model is similar to the model used to explain steep 'dose-response curves' in heterogeneous catalysis, caused by the reaction between two different molecules or atoms on the surface of the catalyst.

Characterization of [3H]flunitrazepam binding to melanin.

In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 +/- 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.

Electrochemical evaluation of the interfacial capacitance upon phosphorylation of amino acid analogue molecular films.

An approach based on electrochemistry to differentiate between phosphorylated and nonphosphorylated amino acid analogues adsorbed on gold is presented. Analogues of serine, threonine, and tyrosine, containing thiohexadecyl headgroups, were synthesized and assembled on gold, and the surface capacitance was evaluated using electrochemical impedance spectroscopy. A procedure for deprotection of tert-butyl phosphate protecting groups, on the monolayer, is also described. Characterizations of the assembled analogues by cyclic voltammetry, infrared spectroscopy, and ellipsometry are used to confirm the insulating properties of the monolayers and the outcome of surface modifications. The results from cyclic voltammetry show good insulating properties for the monolayers even after phosphate deprotection. The infrared measurements reveal well-ordered monolayers, and the thickness from ellipsometry is in good agreement with expectations from molecular modeling. The impedance experiments show a capacitance increase up to 0.6 microF/cm2 as phosphate groups are introduced. The results in this study indicate the possibility of using a surface chemical and impedance spectroscopy approach to detect the kinase/phosphatase activity and kinetics involved in phosphorylation reactions.

Subtle differences in dissociation rates of interactions between destabilized human carbonic anhydrase II mutants and immobilized benzenesulfonamide inhibitors probed by a surface plasmon resonance biosensor.

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s(-1). For the mutants, dissociation rates were faster (0.022-0.025 s(-1)), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Iron and vitamin deficiencies, endocrine and immune status in patients with primary Sjögren's syndrome.

OBJECTIVES: To study the prevalence of iron and vitamin deficiencies, endocrine disorders and immunological parameters in patients with primary Sjogren's syndrome (1 degree SS). DESIGN AND SUBJECTS: At the time of the establishment of the diagnosis of 1 degree SS in 43 consecutive patients, a clinical examination including haematological analyses was performed. The patients' medical records were also reviewed. SETTING: Patients referred for diagnosis to The University Hospital, Linköping, a secondary or tertiary referral hospital serving the middle part of southern Sweden. RESULTS: In total, current or previously treated iron and vitamin deficiencies were registered for 63% of the 1 degree SS patients (iron 51%, vitamin B12 25%, folate 9%). Current low ferritin was noted in 24%, low iron saturation in 37%, decreased vitamin B12 in 13% and folate in 9%. Thyroid disease was found in a total of 33% and 30% had had autoimmune thyroiditis. Three patients (7%) had verified diabetes mellitus. Erythrocyte sedimentation rate (ESR) was raised in 65% of the patients and 84% had a polyclonal increase of Ig. Rheumatoid factor (RF) was detected in 85%, antinuclear antibody (ANA) in 74%, anti-SS-A in 88% and anti-SS-B in 73% of the patients. CONCLUSION: Iron and vitamin deficiencies and thyroid diseases are common in patients with 1 degree SS. Since these disorders often are treatable and may affect the patients' distress as well as their immune and exocrine function, an active, recurrent search for deficiencies, endocrine diseases and other frequently recorded disorders is recommended.

Social inclusion and career development--transition from upper secondary school to work or post-secondary education among hard of hearing students.

The aim of this study was to investigate the decision process and to analyse the mechanisms involved in the transition from upper secondary education to post-secondary education or the labour market. Sixteen students with sensorioneural hearing loss were selected. Among these eight of the students continued to university and eight did not. Twenty-five per cent of the students were women and the average age was 28 years. The investigation was conducted about 5 years after graduation from the upper secondary school. Both quantitative and qualitative methods were used. The results showed that none of the students came from a family where any or both of the parents had a university or comparable education. The differences in choice between the two groups cannot be explained in terms of social inheritance. Our study indicates that given normal intellectual capacity the level of the hearing loss seems to have no predictive value regarding future educational performance and academic career. The conclusion is that it is of great importance that a hearing impaired pupil with normal intellectual capacity is encouraged and guided to choose an upper secondary educational programme which is orientated towards post-secondary education (instead of a narrow vocational programme). Additional to their hearing impairment and related educational problems, hard of hearing students have much more difficulty than normal hearing peers in coping with changes in intentions and goals regarding their educational career during their upper secondary education.

Refractive development in children with Down's syndrome: a population based, longitudinal study.

AIMS: To study the refractive development in children with Down's syndrome longitudinally. METHODS: An unselected population of 60 children with Down's syndrome was followed with repeated retinoscopies in cycloplegia for 2 years or more (follow up 55 (SD 23) months). Accommodation was assessed with dynamic retinoscopy. RESULTS: From longitudinal spherical equivalent values of the right eye, three main categories of refraction were defined: stable hypermetropia (<1.5 D difference between the first and last visit) (n=34), increasing hypermetropia ("hypermetropic shift"; >/=1.5 D difference) (n=11), and decreasing hypermetropia/development of myopia ("myopic shift"; >/=1.5 D difference) (n=9). Patients with anisometropia (n=6) were evaluated separately. In the stable hypermetropia group three sublevels were chosen: low (+4.0 D). An accommodation weakness was found in 55% of the children. Accommodation weakness was significantly less frequent in the stable, low grade hypermetropia group (22%) than in all the other groups (p=0.008). The frequency of astigmatism >/=1.0 D at the last visit was 57%, the direction of axis being predominantly "with the rule." All the eyes with oblique astigmatism had a side specific direction of axis; the right eyes belonging to the 135 degrees axis group and the left eyes to the 45 degrees axis group. CONCLUSION: A stable, low grade hypermetropia was significantly correlated with a normal accommodation. Accommodation weakness may be of aetiological importance to the high frequency of refractive errors encountered in patients with Down's syndrome. A striking right-left specificity in the oblique astigmatic eyes suggests that mechanical factors on the cornea from the upward slanting palpebral fissures may be a major aetiological factor in the astigmatism.

The electric charge of pigment granules in pigment cells.

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be -1.5 x 10(-16) and -1.7 x 10(-16) C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.

Volume changes of individual melanosomes measured by scanning force microscopy.

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G-protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three-dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P < 0.05, n=200). The average ellipsoidal volume was 0.14+/-0.01 (aggregated) and 0.17+/-0.01 microm3 (dispersed), a difference of 18%. The average major diameter was 810+/-20 and 880+/-20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non-destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.

Ellipsometric in vitro studies on the activation of complement by human immunoglobulins M and G after adsorption to methylated silicon.

Human serum immunoglobulin M (IgM) or human immunoglobulin G (IgG) were adsorbed to dichlorodimethyl silane (DDS) treated silicon. Subsequently, the model surfaces were incubated in normal-, complement factor 1q (C1q)-complement factor B or complement factor 2 (C2)-depleted human sera at 37 degrees C for up to 1.5 h. The serum deposition and binding of selected polyclonal complement antibodies into this layer were then quantified by null ellipsometry. Both types of precoated surfaces bound large amounts of anti-complement factor 3c (anti-C3c), anti-properdin and anti-C3d, after incubation in normal serum. In contrast to IgG coated surfaces, IgM coated surfaces bound no anti-C1q after the serum incubations and no anti-C3c deposition lag time was observed after incubations in EGTA serum. Upon immersions of IgM coated surfaces in the different sera, a rapid complement activation via a C1q factor B, and Ca(2+)-independent, but C2 dependent pathway, was indicated. When IgM was instead immobilized to APTES/glutaraldehyde surfaces, anti-C3c deposition was lower after incubations in EGTA than normal serum. The results suggest that, under the present experimental conditions, human IgM and IgG activate the complement system differently.

Melatonin-induced organelle movement in melanophores is coupled to tyrosine phosphorylation of a high molecular weight protein.

Melanophores, brown to black pigment cells from, for example, Xenopus laevis, contain mobile melanin filled organelles, and are well suited for studies on organelle movement. The intracellular regulation of the movement seems to be controlled by serine and threonine phosphorylations and dephosphorylations. Melatonin induces aggregation of the melanosomes to the cell centre through a G(i/o)-protein-coupled receptor, Mel1c, which leads to an inhibition of PKA and a stimulation of PP2A. However, this study shows that the melatonin-induced aggregation of melanosomes is also accompanied by tyrosine phosphorylation of a protein with a molecular weight of approximately 280 kDa. Cells pre-incubated with genistein, an inhibitor of tyrosine phosphorylations, showed inhibited melanosome movement after melatonin stimulation, and a lower degree of tyrosine phosphorylation of the approximately 280 kDa protein. The adenylyl cyclase activator forskolin, and the G(i/o) protein inhibitor pertussis toxin, also inhibited tyrosine phosphorylation of the approximately 280 kDa protein. The results indicate that melatonin stimulation generates tyrosine phosphorylation of a high molecular weight protein, an event that seems to be essential for melanosome aggregation.