Foldscope as a primary diagnostic tool for oral and urinary tract infections and its effectiveness in oral health education.

Due to its speed, accuracy and cost-effectiveness, microscopy has become an integral part of clinical examination for disease diagnosis. However, modern microscopes are very costly and require skilled personnel for their operation and maintenance, and specimen processing and analysis is labour-intensive. Further, lack of such expensive diagnostic tools in remote areas is a serious concern. Affordable point-of-care diagnostic tools are the most useful for timely disease diagnosis and management. The Foldscope is an affordable origami-based microscopy device composed of a series of paper clippings, which, upon assembly, can hold a specimen slide for observation, and this specimen can be viewed via a mobile phone camera attached to it. The present study evaluated the use of the Foldscope in the clinical diagnosis of oral and urinary tract infections and evaluated its efficacy as a motivational tool for improving oral health among school children in India. We qualitatively compared the Foldscope to a clinical microscope by examining five different types of clinical samples. Of the different types of clinical samples, the Foldscope was effective in detecting infection in dental plaque samples and urine samples. Thus, we further analysed 31 dental plaque samples of patients aged 3-13 years and 25 urine samples of patients aged 11-62 years. We also evaluated the use of the Foldscope as an educational tool for motivating oral hygiene among 80 school children aged 12 years and found that students in the Foldscope intervention group had better measures of oral hygiene than did students in the nonintervention group. In summary, our study indicated that the Foldscope is useful in detecting urinary tract infections and kidney stones in urine samples and is a useful motivational tool for oral health education among school-aged children. Furthermore, it may also be useful in oral health monitoring in resource poor settings. LAY DESCRIPTION: Poor and remote population often suffers due to lack of capacity to buy products or avail services which are expensive. In such a society people are not able to afford even the basic detection of diseases. Foldscope may come to rescue here! Foldscope is a paper-based, use-and-throw, affordable microscope. This study explores the use of Foldscope in clinics and diseases diagnosis using patient samples. Preliminary detection of disease associated symptoms in dental and urinary infections and digital record keeping via storage in mobile phone memory and hospital OPD records for monitoring patient's health are some of the advantages of Foldscope for clinical use in resource poor settings. It identifies that Foldscope not only can be used by students or teachers for educational purposes but it can also pave a path for promotion of dental hygiene among young children. The study also suggests that further improvement in design or resolution of Foldscope will broaden the scope of its application, in future.

Synthetic polypeptide adsorption to Cu-IDA containing lipid films: a model for protein-membrane interactions.

Adsorption of synthetic alanine-rich peptides to lipid monolayers was studied by X-ray and neutron reflectivity, grazing incidence X-ray diffraction (GIXD), and circular dichroic spectroscopy. The peptides contained histidine residues to drive adsorption to Langmuir monolayers of lipids with iminodiacetate headgroups loaded with Cu2+. Adsorption was found to be irreversible with respect to bulk peptide concentration. The peptides were partially helical in solution at room temperature, the temperature of the adsorption assays. Comparisons of the rate of binding and the structure of the adsorbed layer were made as a function of the number of histidines (from 0 to 2) and also as a function of the positioning of the histidines along the backbone. For peptides containing two histidines on the same side of the helical backbone, large differences were observed in the structure of the adsorbed layer as a function of the spacing of the histidines. With a spacing of 6 A, there was a substantial increase in helicity upon binding (from 17% to 31%), and the peptides adsorbed to a final density approaching that of a nearly completed monolayer of alpha-helices adsorbed side-on. The thickness of the adsorbed layer (17 +/- 2.5 A) was slightly greater than the diameter of alpha-helices, suggesting that the free, unstructured ends extended into solution. With a spacing of 30 A between histidines, a far weaker increase in helicity upon binding was observed (from 13% to 19%) and a much lower packing density resulted. The thickness of the adsorbed layer (10 +/- 4 A) was smaller, consistent with the ends being bound to the monolayer. Striking differences were observed in the interaction of the two types of peptide with the lipid membrane by GIXD, consistent with binding by two correlated sites only for the case of 6 A spacing. All these results are attributed to differences in spatial correlation between the histidines as a function of separation distance along the backbone for these partially helical peptides. Finally, control over orientation was demonstrated by placing a histidine on an end of the sequence, which resulted in adsorbed peptides oriented perpendicular to the membrane.

Oligomerization of membrane-bound diphtheria toxin (CRM197) facilitates a transition to the open form and deep insertion.

Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane.

In-situ X-ray reflectivity study of alkane films grown from the vapor phase.

We carried out in-situ X-ray reflectivity study of nine n-alkane chains (CnH2n+2) on Si substrate, n in the range of 17-30. These films formed under vacuum at equilibrium vapor pressure of the respective alkane molecule. For all the alkanes studied we found a bilayer structure on the substrate, a higher density vertical layer at the air-film interface with the layer thickness equal to the all-trans length of the respective molecule, and a lower density layer below it with the molecules lying horizontal on the substrate. This model was earlier proposed for C32 films on Si by Volkmann et al.11 We observe that this model can fit the entire range of data from C17 to C30 in our experiments.

Comparison of critical adsorption scaling functions obtained from neutron reflectometry and ellipsometry.

Carpenter et al. [Phys. Rev. E 59, 5655 (1999); 61, 532 (2000)] managed to explain ellipsometric critical adsorption data collected from the liquid-vapor interface of four different critical binary liquid mixtures near their demixing critical temperature using a single model. This was the first time a single universal function had been found which could quantitatively describe the surface critical behavior of many different mixtures. There have also been various attempts to investigate this surface critical behavior using neutron and x-ray reflectometries. Results have been mixed and have often been at variance with Carpenter et al. In this paper, the authors show that neutron reflectometry data collected from a crystalline quartz-critical mixture interface, specifically deuterated water plus 3-methylpyridine, can be quantitatively explained using the model of Carpenter et al. derived from ellipsometric data.

Rearrangement of lipid ordered phases upon protein adsorption due to multiple site binding.

This study involves the interactions of proteins with Langmuir monolayers of a metal-chelating lipid, where adsorption is driven by a strong specific interaction between histidines on the proteins and divalent metal ions loaded into the lipid headgroups. A comparison of the structural rearrangement of the lipid film upon adsorption of myoglobin and a synthetic peptide, each of which have multiple histidines, with that upon the adsorption of lysozyme, which has only one histidine, suggests that the lipid rearrangement in the former case is due to the multiplicity of binding sites. The kinetics and manner of rearrangement change with the binding energy and film pressure.

Supercritical fluid processing of polymer thin films: an X-ray study of molecular-level porosity.

This paper reviews our recent experimental results that address the effects of solvent density inhomogeneities in supercritical carbon dioxide (scCO(2)) on polymer thin film processing. The key phenomenon is excess sorption of CO(2) molecules into polymer thin films even when the bulk miscibility with CO(2) is very poor. We have found that the amount of the excess sorption is attributed to the large density fluctuations in scCO(2) near the critical point. Further, taking advantage of the vitrification process of polymer chains through quick evaporation of CO(2), we can preserve the "expanded" structures as they are. The resultant films have large degree of molecular-level porosity that is very useful in producing coatings with low dielectric constants, enhanced adhesion, and metallization properties. These characteristics can be achieved in an environmentally "green" manner, without organic solvents, and are not specific to any class of polymers.

Evidence for vertical phase separation in densely grafted, high-molecular-weight poly(-isopropylacrylamide) brushes in water.

The detailed conformational change of poly(N-isopropylacrylamide) (PNIPAM) brushes at high grafting density in D2O was investigated as a function of temperature using neutron reflection. PNIPAM chains were grafted at high surface density from gold and silicon oxide surfaces by atom transfer radical polymerization. Whereas single layer profiles were observed for temperatures below and above the transition region, bilayer profiles were observed for a narrow range of temperatures near the transition. This nonmonotonic change in the concentration profile with temperature is discussed in the context of theoretical models of vertical phase separation within a brush.

Effect of density fluctuating supercritical carbon dioxide on polymer interfaces.

We investigated an effect of CO2 sorption on the compatibility of immiscible polystyrene (PS) and polybutadiene (PB) bilayers by using in situ neutron reflectivity. By labeling either polymer with deuterium, we found that the excess CO2 molecules were adsorbed to both top PS and bottom PB layers when the bilayers were exposed to CO2 at the narrow T and P regime near the critical point of pure CO2. Furthermore, we clarified that this excess sorption of CO2 molecules increased the interfacial width between the layers up to 100 angstroms even near room temperature, while the interfacial width without CO2 exposure has been reported to be at most 40 A even at the highest temperature (T congruent with 175 degrees C).

Influence of the electric field on a bio-mimetic film supported on a gold electrode.

A model biological membrane was formed by fusion of mixed cholesterol and DMPC (dimyristoylphosphatidylcholine) phospholipid vesicles onto a gold-coated quartz support. The gold surface was charged and the influence of the charge at the solid support on the structure and integrity of the phospholipid bilayer was investigated using the specular reflection of neutrons and electrochemical measurements. When the surface charge density is close to zero, the lipid vesicles fuse directly on the surface to form a bilayer with a small number of defects and hence low water content. When the support's surface is negatively charged the film swells and incorporates water due to the field driven poration of the membrane. When the charge density is more negative then -8 microC cm(-2) the bilayer is detached from the metal surface. However, it remains in close proximity to the metal electrode, suspended on a thin cushion of water. The film thicknesses, calculated from neutron reflectivity, have allowed us to determine the tilt angle of the lipid molecules as a function of the support's charge density. The lipid molecules are tilted 55 degrees from the surface normal at zero charge density but become significantly more perpendicular (30 degrees tilt angle) at charge densities more negative than -8 microC cm(-2). The tilt angle measurements are in very good agreement with previous IR studies. This paper describes the highlights of a more in-depth study which is fully described in [1].

Neutron and X-ray scattering studies of cholera toxin interactions with lipid monolayers at the air-liquid interface.

Using neutron/X-ray reflectivity and X-ray grazing incidence diffraction (GID), we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. Both CTAB(5) and CTB(5) were measured to have approximately 50% coverage when bound to the lipid monolayer. X-ray GID experiments show that both the lipid monolayer and the cholera toxin layer are crystalline. The effects of X-ray beam damage have been assessed and the monolayer/toxin structure does not change with time after protein binding has saturated.

Cholera toxin assault on lipid monolayers containing ganglioside GM1.

Many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components. Using neutron reflectivity, we have characterized the structure of mixed DPPE:GM(1) lipid monolayers before and during the binding of cholera toxin (CTAB(5)) or its B-subunit (CTB(5)). Structural parameters such as the density and thickness of the lipid layer, extension of the GM(1) oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. The density of the lipid layer was found to decrease slightly upon protein binding. However, the A-subunit of the whole toxin is clearly located below the B-pentameric ring, away from the monolayer, and does not penetrate into the lipid layer before enzymatic cleavage. Using Monte Carlo simulations, the observed monolayer expansion was found to be consistent with geometrical constraints imposed on DPPE by multivalent binding of GM(1) by the toxin. Our findings suggest that the mechanism of membrane translocation by the protein may be aided by alterations in lipid packing.

Electric field-driven transformations of a supported model biological membrane--an electrochemical and neutron reflectivity study.

A mixed bilayer of cholesterol and dimyristoylphosphatidylcholine has been formed on a gold-coated block of quartz by fusion of small unilamellar vesicles. The formation of this bilayer lipid membrane on a conductive surface allowed us to study the influence of the support's surface charge on the structure and hydration of the bilayer lipid membrane. We have employed electrochemical measurements and the specular reflection of neutrons to measure the thickness and water content in the bilayer lipid membrane as a function of the charge on the support's surface. When the surface charge density is close to zero, the lipid vesicles fuse directly on the surface to form a bilayer with a small number of defects and hence small water content. When the support's surface is negatively charged the film swells and incorporates water. When the charge density is more negative than -8 micro C cm(-2), the bilayer starts to detach from the metal surface. However, it remains in a close proximity to the metal electrode, being suspended on a thin cushion of the electrolyte. The field-driven transformations of the bilayer lead to significant changes in the film thicknesses. At charge densities more negative than -20 micro C cm(-2), the bilayer is approximately 37 A thick and this number is comparable to the thickness determined for hydrated multilayers of dimyristoylphosphatidylcholine from x-ray diffraction experiments. The thickness of the bilayer decreases at smaller charge densities to become equal to approximately 26 A at zero charge. This result indicates that the tilt of the acyl chains with respect to the bilayer normal changes from approximately 35 degrees to 59 degrees by moving from high negative charges (and potentials) to zero charge on the metal.

Analysis of myoglobin adsorption to Cu(II)-IDA and Ni(II)-IDA functionalized Langmuir monolayers by grazing incidence neutron and X-ray techniques.

The adsorption of myoglobin to Langmuir monolayers of a metal-chelating lipid in crystalline phase was studied using neutron and X-ray reflectivity (NR and XR) and grazing incidence X-ray diffraction (GIXD). In this system, adsorption is due to the interaction between chelated divalent copper or nickel ions and the histidine moieties at the outer surface of the protein. The binding interaction of histidine with the Ni-IDA complex is known to be much weaker than that with Cu-IDA. Adsorption was examined under conditions of constant surface area with an initial pressure of 40 mN/m. After approximately 12 h little further change in reflectivity was detected, although the surface pressure continued to slowly increase. For chelated Cu2+ ions, the adsorbed layer structure in the final state was examined for bulk myoglobin concentrations of 0.10 and 10 microM. For the case of 10 microM, the final layer thickness was approximately 43 A. This corresponds well to the two thicker dimensions of myoglobin in the native state (44 A x 44 A x 25 A) and so is consistent with an end-on orientation for this disk-shaped protein at high packing density. However, the final average volume fraction of amino acid segments in the layer was 0.55, which is substantially greater than the value of 0.44 calculated for a completed monolayer from the crystal structure. This suggests an alternative interpretation based on denaturation. GIXD was used to follow the effect of protein binding on the crystalline packing of the lipids and to check for crystallinity within the layer of adsorbed myoglobin. Despite the strong adsorption of myoglobin, very little change was observed in the structure of the DSIDA film. There was no direct evidence in the XR or GIXD for peptide insertion into the lipid tail region. Also, no evidence for in-plane crystallinity within the adsorbed layer of myoglobin was observed. For 0.1 microM bulk myoglobin concentration, the average segment volume fraction was only 0.13 and the layer thickness was < or = 25 A. Adsorption of myoglobin to DSIDA-loaded with Ni2+ was examined at bulk concentrations of 10 and 50 microM. At 10 microM myoglobin, the adsorbed amount was comparable to that obtained for adsorption to Cu2+-loaded DSIDA monolayers at 0.1 M. But interestingly, the adsorbed layer thickness was 38 A, substantially greater than that obtained at low coverage with Cu-IDA. This indicates that either there are different preferred orientations for isolated myoglobin molecules adsorbed to Cu-IDA and Ni-IDA monolayer films or else myoglobin denatures to a different extent in the two cases. Either interpretation can be explained by the very different binding energies for individual interactions in the two cases. At 50 microM myoglobin, the thickness and segement volume fraction in the adsorbed layer for Ni-IDA were comparable to the values obtained with Cu-IDA at 10 microM myoglobin.

Early-stage compositional segregation in polymer-blend films.

The existence of a transient period during the surface enrichment of a binary polymer blend by one of its components has been suggested by previous theoretical and experimental studies as well as computer simulations. Taking advantage of the high depth resolution of neutron reflectivity and the slow dynamics of polymers near their glass transition, we investigate this early-stage surface compositional enrichment in a phase separating polymer blend for the first time. Two stages of surface enrichment layer growth are observed. A rapid local surface enrichment at the chain segmental level occurs first, followed by a slower growth of a diffuse layer having a scale on the order of the bulk correlation length and the radius of gyration of the surface enriching polymer chains.

Materials Research With Neutrons at NIST.

The NIST Materials Science and Engineering Laboratory works with industry, standards bodies, universities, and other government laboratories to improve the nation's measurements and standards infrastructure for materials. An increasingly important component of this effort is carried out at the NIST Center for Neutron Research (NCNR), at present the most productive center of its kind in the United States. This article gives a brief historical account of the growth and activities of the Center with examples of its work in major materials research areas and describes the key role the Center can expect to play in future developments.

Ligand accessibility as means to control cell response to bioactive bilayer membranes.

We report a new method to create a biofunctional surface in which the accessibility of a ligand is used as a means to influence the cell behavior. Supported bioactive bilayer membranes were created by Langmuir-Blodgett (LB) deposition of either a pure poly(ethylene glycol) (PEG) lipid, having PEG head groups of various lengths, or 50 mol % binary mixtures of a PEG lipid and a novel collagen-like peptide amphiphile on a hydrophobic surface. The peptide amphiphile contains a peptide synthetically lipidated by covalent linkage to hydrophobic dialkyl tails. The amphiphile head group lengths were determined using neutron reflectivity. Cell adhesion and spreading assays showed that the cell response to the membranes depends on the length difference between head groups of the membrane components. Cells adhere and spread on mixtures of the peptide amphiphile with the PEG lipids having PEG chains of 120 and 750 molecular weight (MW). In contrast, cells adhered but did not spread on the mixture containing the 2000 MW PEG. Cells did not adhere to any of the pure PEG lipid membranes or to the mixture containing the 5000 MW PEG. Selective masking of a ligand on a surface is one method of controlling the surface bioactivity.

Comparison of tethered star and linear poly(ethylene oxide) for control of biomaterials surface properties.

Four different poly(ethylene oxide) [PEO] molecules were compared as grafted polymer layers for biomaterials' substrates: two linear polymers and two star polymers. Conditions maximizing surface coverage for each molecule were employed with the aim of inhibiting protein adsorption and increasing the density of end groups. Neutron reflectivities of the grafted layers immersed in deuterium oxide (heavy water) were measured and used to calculate volume fraction profiles of the polymer as a function of distance from the surface. These density profiles were combined with protein adsorption data on the grafted layers to compare with recent theoretical and experimental studies of protein resistance by PEO at surfaces. We found that the grafting density is maximized by coupling the linear PEO from a K2SO4 salt buffer, which is a poor solvent for PEO. However, the grafting density of star PEO was maximized when no K2SO4 was used and the stars were dissolved near the overlap concentration. Concentration profiles obtained from the reflectivity data show that the hydrated polymers swell to approximately 10 times the dried layer thickness and exhibit a low density (maximum volume fractions < 0.4 PEO) throughout the layer. The PEO surfaces obtained with both the star and linear polymers resisted adsorption of cytochrome-c and albumin except for a small amount of cytochrome-c adsorption on the short, many-armed star polymer surface. A hypothesis of adsorption on the star polymer layer is presented and criteria for controlling receptor-mediated cell-substrate interactions by ligand-modified chain ends are discussed.

Neutron Reflectivity and Grazing Angle Diffraction.

Over the last 10 years, neutron reflectivity has emerged as a powerful technique for the investigation of surface and interfacial phenomena in many different fields. In this paper, a short review of some of the work on neutron reflectivity and grazing-angle diffraction as well as a description of the current and planned neutron rcflectometers at NIST is presented. Specific examples of the characterization of magnetic, superconducting, and polymeric surfaces and interfaces are included.