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We have previously reported on the IR spectra of styrene, acrylonitrile, and methyl methacrylate (known as the SAN co-polymer), as well as the glass transitions. Not too much work on the refractive indices has been performed, and the refractive indices for a variety of these mixtures and some selected TEM pictures will be presented in this note.
Optimization of the optical design for pulsed-laser crossed-beam thermal lens (PLCBTL) spectrometry has been investigated. Experiments have been carried out with large samples as well as for very small samples in a microchannel and using different lens combinations to focus the probe and excitation beams. The results have been interpreted in terms of the influence of the excitation beam size as well as the degree of mode-mismatching of the excitation and probe beams on the optimum sample position and on the amplitude and decay of the photothermal signal. A semi-empirical formula that describes the influence of the sample position with respect to the probe beam waist has been established. We have shown that the optimum signal is inversely proportional to the waist of the excitation beam and is independent of the sample size as long as the size of the excitation beam is smaller than the microchannel. Time-resolved experiments have also shown that when the excitation beam is smaller than the sample, the signal decay depends not only on the size of the excitation beam but also on the mode-mismatching factor. Otherwise, the temporal characteristics are closely related to the size of the microchannel.
Gradient ion-exchange chromatography (IEC) was interfaced with a low-angle laser light scattering photometer (LALLS) and ultraviolet (UV) and refractive index (RI) detectors connected in series for on-line determination of the differential refractive index (dn/dc) of proteins and eventually their molecular weights (Mw's). Interfacing of gradient HPLC with a RI detector was made possible by using two isorefractive buffers, which helped generate stable baselines for the LALLS and RI detectors. An optically modified, laser based RI detector was used for determination of dn/dc. On-line determinations of dn/dc required smaller amounts of sample compared to off-line determinations (3-4 mg vs 200 mg). Also, dn/dc could be measured by making one repetitive injection of the sample, if the molar absorptivity of the sample was known. dn/dc values determined on-line were in close agreement with those determined off-line. Additionally, percent recovery of the sample was calculated on-line, and this provided true concentration detected at the UV detector, which was then used for determination of Mw.
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An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.
A simple method for converting local reflectivity changes measured in surface plasmon resonance (SPR) microscopy to effective adlayer thicknesses and absolute surface coverages of adsorbed species is presented. For a range of high-contrast angles near the SPR resonance where the local metal surface's reflectivity changes linearly with angle, the change in reflectivity at fixed angle is proportional to the change in effective refractive index (eta(eff)) near the surface. This change in eta(eff) can be converted to absolute adsorbate coverage using methods developed for quantitative SPR spectroscopy. A measurement of the change in reflectivity due to changes in refractive index of bulk solutions, i.e., percent reflectivity change per refractive index unit (RIU), is the only calibration required. Application of this method is demonstrated for protein adsorption onto protein/DNA arrays on gold from aqueous solution using an SPR microscope operating at 633 nm. A detection limit of 0.072% change in absolute reflectivity is found for simultaneous measurements of all 200 microm x 200 microm areas within the 24-mm(2) light beam with 1-s time averaging. This corresponds to a change in effective refractive index of 1.8 x 10(-5) and a detection limit for protein adsorption of 1.2 ng/cm(2) (approximately 0.5 pg in a 200-microm spot). The linear dynamic range is Deltaeta(eff) = approximately 0.011 RIU or approximately 720 ng/cm(2) of adsorbed protein. Using a nearby spot as a reference channel, one can correct for instrumental drift and changes in refractive index of the solutions in the flow cell.
The physicochemical properties of nanosized colloidal drug carrier systems are of great influence on drug efficacy. Consequently, a broad spectrum of analytical techniques is applied for comprehensive drug carrier characterization. It is the primary objective of this paper to present asymmetrical flow field-flow fractionation (AF4), coupled online with multiangle light scattering detection, for the characterization of gelatin nanoparticles. Size and size distribution of drug-loaded and unloaded nanoparticles were determined, and data were correlated with results of state-of-the-art methods, such as scanning electron microscopy and photon correlation spectroscopy. Moreover, the AF4 fractionation of gelatin nanoparticulate carriers from a protein model drug is demonstrated for the first time, proposing a feasible way to assess the amount of loaded drug in situ without sample preparation. This hypothesis was set into practice by monitoring the drug loading of nanoparticles with oligonucleotide payloads. In this realm, various fractions of gelatin bulk material were analyzed via AF4 and size-exclusion high-pressure liquid chromatography. Mass distributions and high-molecular-weight fraction ratios of the gelatin samples varied, depending on the separation method applied. In general, the AF4 method demonstrated the ability to comprehensively characterize polymeric gelatin bulk material as well as drug-loaded and unloaded nanoparticles in terms of size, size distribution, molecular weight, and loading efficiency.
An integrated optical metal clad leaky waveguide (MCLW) sensor device has been developed for the detection of bacteria. This is more sensitive than waveguide sensors currently in use. The MCLW device has been fabricated to extend the evanescent field to provide significant light intensity over the entire volume of the bacteria bound on the chip surface within this field. This in turn increases the interaction of the light with the entire volume of the bacteria. MCLW devices have been used for detecting refractive index changes, scattering, and fluorescence from bacterial spores captured on an immobilized antibody. The detection limit of Bacillus subtilis var. niger bacterial spores using refractive index detection was 8 x10(4) spores/mL. The scattering intensity of the BG spores was found to be three times greater than the scattering intensity generated using surface plasmon resonance. The extended light propagation along the direction of flow for a few millimeters provides an effective interrogation approach to increase the area of detection to detect low concentrations down to 1 x 10(4) spores/mL. The sensor was then optimized by studying the key factors affecting sensor performance including changing the pH of the medium, type of antibody immobilization matrix, sensor surface regeneration approaches, and longevity of the sensor.
The unique optical properties of noble metal nanoparticles have been used to design a label-free biosensor in a chip format. In this paper, we demonstrate that the size of gold nanoparticles significantly affects the sensitivity of the biosensor. Gold nanoparticles with diameters in the range of 12-48 nm were synthesized in solution and sensor chips were fabricated by chemisorption of these nanoparticles on amine-functionalized glass. Sensors fabricated from 39-nm-diameter gold nanoparticles exhibited maximum sensitivity to the change of the bulk refractive index and the largest "analytical volume", defined as the region around the nanoparticle within which a change in refractive index causes a change in the optical properties of the immobilized nanoparticles. The detection limit for streptavidin-biotin binding of a sensor fabricated from 39-nm-diameter nanoparticles was 20-fold better than a previously reported sensor fabricated from 13-nm-diameter gold nanoparticles. We also discuss several other factors that could improve the performance of the next generation of these immobilized metal nanoparticle sensors.
An absolute calibration of the light scattering region in an aerosol time-of-flight mass spectrometer (ATOFMS) has been performed enabling a direct comparison of the average experimentally measured light scattering intensity to theory. A fitting procedure allows for the determination of both refractive index and density for spherical homogeneous particles. The scattering information has been correlated with the other single-particle information measured by the ATOFMS. Size, chemical composition, and scattering intensity can all be linked to establish a better understanding of the relationships between the chemical and physical properties of aerosol particles. Currently, inputs into climate models are derived from data acquired from bulk aerosol composition measurements, and therefore, assumptions must be made regarding the chemical associations within individual particles (mixing state) to enable modelers to calculate the relevant aerosol optical properties. These new measurements aim for the goal of directly testing the model assumptions by utilizing single-particle chemical information to derive the optical properties of the different particle classes.
Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.
Pooled human milk oligosaccharides were fractionated by anion-exchange chromatography on AG 1-X2 and by an improved gel filtration procedure that allowed the separation of large oligosaccharides on Toyopearl HW 40 (S) and Bio-Gel P-6 columns, respectively. The analysis of the resulting nonderivatizated fractions by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed several neutral and acidic high-molecular-weight oligosaccharides. So far unknown acidic oligosaccharides containing up to 20 monomers were detected in a molecular mass range of 2094-3626 Da. Furthermore, neutral structures containing up to 35 monosaccharides were identified after fractionation on Toyopearl HW 40 (S) and subsequent P-6 fractionation, demonstrating the suitability of the applied method for the preparation of oligosaccharides in this high-molecular-mass range. The composition of the detected oligosaccharides was found to be the same as those previously identified in oligosaccharides of lower masses. However, an enormous structural heterogeneity was observed when acidic and neutral fractions were characterized by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). From our analysis we may conclude that each molecular mass identified by MALDI-MS corresponds to a variety of isomeric structures. The total number of oligosaccharides occurring in human milk may consequently be much higher than estimated before.
Resonance Raman spectroscopy was performed on peripheral light-harvesting proteins from Rhodobacter sphaeroides in which the residue betaArg-10 has been modified by site-selected mutagenesis. We show that this residue is indeed involved (as proposed by X-ray crystallographic studies on the LH2 complex from Rhodopseudomonas acidophila), in an H-bond with the acetyl carbonyl of the 800 nm-absorbing BChl in these proteins (B800), and that the presence of such an H-bond induces a ca. 10 nm red shift of the lowest energy transition (Qy) of this molecule. Moreover, other parameters involved in the fine tuning of the absorption of the B800 molecules may be determined from our experiments, and we propose that the local electromagnetic properties of the B800 binding site may induce an additional 10 nm red shift of this transition. These results constitute the first experimental evidence for the parameters able to modify in vivo the absorption of "monomeric" BChl molecules, i.e. BChl not involved in strong excitonic interactions, and will be of great help for understanding the absorption properties of such pigments in other light-harvesting systems.
The interactions between polystyrenesulfonate (PSS)/polyallylamine (PAH) multilayers with human serum albumin (HSA) were investigated by means of scanning angle reflectometry (SAR). We find that albumin adsorbs both on multilayers terminating with PSS (negatively charged) or PAH (positively charged) polyelectrolytes. On films terminating with PSS only, an albumin equivalent monolayer is found whereas when PAH constitutes the outer layer, albumin interacts with the multilayer in such a way as to form a protein film that extends over thicknesses that can be as high as four times the largest dimension of the native albumin molecule. Once the protein film is formed, it is found that when the albumin solution is replaced by a pure buffer solution of same ionic strength as the adsorption solution almost no desorption takes place. On the other hand, when a buffer solution of higher ionic strength is brought in contact with the albumin film, a significant amount of adsorbed proteins is released. One also observes that, for albumin solutions of a given protein concentration, the adsorbed protein amount depends on the ionic strength of the adsorption solution. On surfaces terminating with PAH, the adsorbed protein amount first increases rapidly but passes through a maximum and decreases with the ionic strength. The ionic strength corresponding to the maximum of the adsorbed albumin amount itself depends on the albumin concentration. On the other hand, on films terminating with PSS the adsorbed amount increases with the salt concentration before leveling-off. These results show that the underlying complexity of concentration and pH dependent adsorption/desorption equilibria often simply termed "protein adsorption" is the result of antagonist competing interactions that are mainly of electrostatic origin. We also propose two microscopic models, that are compatible with our experimental observations.
Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.
Well-defined amphiphilic cubic silsesquioxane-poly(ethylene oxide) (CSSQ-PEO) was prepared from octakis (dimethylsiloxy)octasilsesquioxane (Q8M8(H)) and allyl-PEO through a hydrosilylation reaction. The structure of CSSQ-PEO was characterized by nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and gel permeation chromatography (GPC). The amphiphilic properties and aggregation process of CSSQ-PEO in aqueous solution were investigated by fluorescence, dynamic and static light scattering (DLS and SLS), and transmission electron microscopy (TEM). The critical aggregation concentration (CAC) determined by fluorescence measurements was found to be 0.28 mg/mL. Combinations of DLS, SLS, and TEM studies showed the existence of core-corona micelle with hydrophobic CSSQ as the core and hydrophilic PEO as the corona in aqueous solution. The observation of two size distribution peaks from DLS measurements revealed the coexistence of small amounts of unassociated unimolecular micelles (approximately 10% of the scattered intensity) together with micellar aggregates when the CSSQ-PEO concentration was < or = 2 mg/mL. The hydrodynamic radii (R(h)) of unassociated unimolecular micelle and micellar aggregates were found to be 26 and 79 nm, respectively. A large R(g)/R(h) ratio (1.46) and the extremely small value of average chain density (4 x 10(-4) g/cm3) indicate the small hydrophobic CSSQ core was surrounded by the extended PEO coronae. The aggregation number (N(agg)) of CSSQ-PEO in aqueous solution was found to be 38 +/- 2 from SLS and 31-40 from TEM, respectively. The long PEO segments act as a spacer between the spherical aggregates, which facilitate the formation of a network-like structure at high concentration.
The tin oxide and silicon oxide films have been deposited on polycarbonate substrates as gas barrier films, using a thermal evaporation and ion beam assisted deposition process. The oxide films deposited by ion beam assisted deposition show a much lower water vapor transmission rate than those by thermal evaporation. The tin oxide films show a similar water vapor transmission rate to the silicon oxide films in thermal evaporation but a lower water vapor transmission rate in IBAD. These results are related to the fact that the permeation of water vapor with a large dipole moment is affected by the chemistry of oxides and the packing density of the oxide films. The permeation mechanism of water vapor through the oxide films is discussed in terms of the chemical interaction with water vapor and the microstructure of the oxide films. The chemical interaction of water vapor with oxide films has been investigated by the refractive index from ellipsometry and the OH group peak from X-ray photoelectron spectroscopy, and the microstructure of the composite oxide films was characterized using atomic force microscopy and a transmission electron microscope. The activation energy for water vapor permeation through the oxide films has also been measured in relation to the permeation mechanism of water vapor. The diffusivity of water vapor for the tin oxide films has been calculated from the time lag plot, and its implications are discussed.
Several recent conceptual advances, which take advantage of the polymer conformation in the near critical point of coexisting liquid phases and practical techniques of some unique molecular interactions between polymer chain and the solvent molecules, have been made to allow the investigation of the effect of the well-defined polymer in phase separation of binary mixtures. The behavior of a flexible linear or branched chain polymer (polyethylene oxide, PEO, MW = 9 x 10(5), as an impurity) in the critical binary mixture of isobutyric acid (I) + water (W) was studied by the refractive index (n) measurements using a very accurate and sensitive refractometer. The refractive index in each phase of IW as well as three different PEO concentrations (C = 0.395, 0.796, and 1.605 mg/cm(3)) in the near critical composition of IW have been measured at temperatures below the system's upper critical point. We observed that the polymer was significantly affected in the critical region of IW and these various concentrations of PEO show an important behavior on the critical exponents (beta), the critical temperatures (T(c)), and critical composition (phi(c)), which are depicting the shape of the coexistence curve. The phase-transition region of coexisting phases of IW shifts down with the addition of PEO and T(c) decreases linearly with increasing PEO concentrations. This may indicate that the polymer chain entangles with each phase, thereby the polymer monomers strongly interact with neighbor solvent particles and also intrachain interaction between the polymer segments. At such conditions, the collapse of polymer chain is possible in the vicinity of the critical point. At temperatures T close enough to T(c), the critical exponent beta (defined by the relation (n(1) - n(2)) proportional, variant (T(c) - T)(beta), with n(1) and n(2) being the refractive indices of the coexisting phases) was found to decrease from 0.382 to 0.360 when the PEO concentration changes from 0.395 to 1.605 mg/cm(3). These values are higher than that of 0.326 +/- 0.005 of pure IW, which is compatible with the three-dimensional Ising value beta = 0.325. The observed critical exponents for the PEO in IW are fully renormalized Ising critical exponents. Besides, the phi(c) values decrease with increasing the C values in the mixture of IW. It appears that the shape of the PEO in IW coexistence curves is similar from that of pure IW.