Information-weighted constrained regularization for particle size distribution recovery in multiangle dynamic light scattering.

In particle size measurement with dynamic light scattering (DLS), it is difficult to get an accurate recovery of a bimodal particle size distribution (PSD) with a peak position ratio less than ~2:1, especially when large particles (>350nm) are present. This is due to the inherent noise in the autocorrelation function (ACF) data and the scarce utilization of PSD information during the inversion process. In this paper, the PSD information distribution in the ACF data is investigated. It was found that the initial decay section of the ACF contains more information, especially for a bimodal PSD. Based on this, an information-weighted constrained regularization (IWCR) method is proposed in this paper and applied in multiangle DLS analysis for bimodal PSD recovery. By using larger (or smaller) coefficients for weighting the ACF data, more (or less) weight can then be given to the initial part of the ACF. In this way, the IWCR method can enhance utilization of the PSD information in the ACF data, and effectively weaken the effect of noise at large delay time on PSD recovery. Using this method, bimodal PSDs (with nominal diameters of 400:608 nm, 448:608 nm, 500:600 nm) were recovered successfully from simulated data and it appears that the IWCR method can improve the recovery resolution for closely spaced bimodal particles. Results of the PSD recovery from experimental DLS data confirm the performance of this method.

Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes.

OBJECTIVE: To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex-(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum. METHODS: The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. RESULTS: The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m(2) were put in contact with undiluted sera in buffer borate pH 8-20 mm containing glycine (0.1 m) and polyethyleneglycol 8000 (3% w/v). Finally, the latex-protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. CONCLUSION: The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI.

Molar mass distributions of linear homopolymers by size exclusion chromatography with light scattering detection: A method for automatic band broadening correction.

Size exclusion chromatography (SEC) equipped with a differential refractometer (DR) and a light scattering (LS) detector is a well-known technique for determining the molar mass distribution (MMD) of many polymers. In the case of narrow polymers, correction of the band broadening (BB) effect is necessary; but unfortunately, the available BB correction methods are rather impractical for most SEC users. This work proposes an automatic BB correction method for determining the MMD of narrow linear homopolymers (or multimodal homopolymers that include narrow modes) on the basis of SEC/(DR + LS) measurements. The required data are: the baseline-corrected DR and LS chromatograms, the inter-detector volume (IDV), and a molar mass calibration independently determined from narrow standards. In comparison to other available alternatives for BB correction, the here-proposed method has the following key advantages: a) no previous knowledge on the BB function is required; b) the detectors gain constants are unnecessary; and c) no numerical regularization method is required. Moreover, if the IDV is unknown, then the proposed method could also be used for estimating the IDV from the knowledge of the dispersity index of a narrow homopolymer. The proposal was experimentally assessed by analyzing narrow standards of poly(styrene) and poly(methyl-methacrylate). The proposed method estimated the dispersity indexes of the standards with errors lower than 0.9% with respect to values reported by manufacturers (between 1.015 and 1.044); while the classical approaches based on SEC/DR and SEC/(DR + LS), produced errors of up to -11% and 3%, respectively.

Arthroscopic fixation of displaced tibial eminence fractures: a new growth plate-sparing method.

PURPOSE: Fractures of the tibial eminence can be treated arthroscopically. Numerous ways to attach an anterior cruciate ligament avulsion from the tibial eminence have been designed. This report describes a new physis-sparing reduction and fixation technique using an anchor passing nonabsorbable braided sutures through the substance of the anterior cruciate ligament, holding the avulsed bone fragment by tying a locking knot. This study was performed to evaluate a consecutive group of patients who underwent reduction and fixation of tibial avulsion fractures fixed with an anchor with sutures. METHODS: The evaluation was performed by use of objective and subjective International Knee Documentation Committee (IKDC) scores, KT-1000 measurement (MEDmetric, San Diego, CA), Lachman and pivot-shift tests, and Lysholm score. RESULTS: The global IKDC objective score was normal (A) in 4 knees and nearly normal (B) in 3, without extension or flexion limitations. The mean IKDC subjective score was 92 out of 100 (range, 86 to 98). The results of the anterior drawer, Lachman, and pivot-shift tests were negative. The mean Lysholm score improved from 29 to 94. The mean side-to-side difference in anterior tibial translation was 2 mm (range, 1 to 3 mm). CONCLUSIONS: Arthroscopic stabilization by use of an anchor with sutures was possible in all cases of tibial spine fracture. We were able to obtain excellent results in this series using this fixation method. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Physicochemical characterization of water-soluble chitosan derivatives with singlet oxygen quenching and antibacterial capabilities.

New water-soluble chitosan derivatives (WSCh) were obtained by Maillard reaction (MR) between glucosamine (GA) with both low and medium molecular weight chitosans (Ch). The WSCh showed larger solubility than the respective Ch, while their deacetylation degree (DD) decreased by approximately 12%. Infrared spectroscopy experiments of WSCh confirmed the formation of imine bonds after MR with intensified pyranose structure, and sugar molecules as polymer branches. However, a 6-times reduction of the molecular weight of WSCh was measured, indicating the breakdown of the polysaccharide chain during the MR. The polysaccharides quenched singlet molecular oxygen (1O2), with rate quenching constants correlating with the DD value of the samples, suggesting the important role of amino groups (-NH2) in the deactivation of 1O2. Additionally, all polysaccharides presented antimicrobial activity against pathogenic bacteria, e.g. Staphylococcus aureus, Escherichia coli, Salmonella sp., Enterococcus faecalis and Listeria ivanovii, as tested by their minimum inhibitory concentration (MIC). This way we obtained new water-soluble polysaccharides, with similar functional properties to those presented by native Ch, enhancing its potential application as carrier material for bioactive compounds.

Estimation of band broadening in size-exclusion chromatography. I. A method based on analyzing narrow standards with a molar mass-sensitive detector.

A method is proposed for estimating the (asymmetrical and non-uniform) band broadening function (BBF) in size-exclusion chromatography (SEC). The following data are required: the molar mass calibration and the concentration- and molar mass chromatograms of a set of narrow standards. In the narrow range of each standard, the BBF is uniform but skewed. Each uniform BBF is estimated through a nonlinear optimization procedure that compares one (of the two) measured chromatograms with its theoretical prediction based on the other chromatogram. The method is validated with numerical examples that simulate the analyses of narrow standards exhibiting log-normal and Poisson weight chain length distributions. The BBF can be assumed of arbitrary shape, or represented by an exponentially-modified Gaussian (EMG). From the uniform BBF estimate, the true polydispersity of the standard can be determined. The global non-uniform BBF is obtained by interpolation between a set of uniform BBFs covering a wide range of elution volumes.

Alternative approaches for the estimation of the band broadening parameters in single-detection size exclusion chromatography.

New approaches for the determination of the extent of symmetric and asymmetric band broadening (BB) in size exclusion chromatography (SEC) are presented. For this purpose raw data was simulated by starting with either a theoretical Poisson number chain length distribution (NCLD), or a log-normal weight chain length distribution (WCLD). Each distribution was first converted to a BB-free mass chromatogram, as typically obtained from a standard differential refractive index detector. Then, the broadened (or "measured") chromatograms were simulated by convoluting the BB-free chromatograms with a BB function, which was assumed to follow symmetrical (Gauss) as well as unsymmetrical (exponentially modified Gauss) function. A broad range of BB parameters (standard deviation, sigma(BB), and exponential decay, tau(BB)) was used for the simulations. The approaches are based on the determination of the points of inflection belonging to the peak of the broadened chromatogram, and closed as well as empirically derived equations connecting the peak width, its variance, and the parameters sigma(BB) and tau(BB). The developed methods are applicable for Poisson distributions well above a peak chain length of 100.

Contamination by larger particles of two almost-uniform latices: analysis by combined dynamic light scattering and turbidimetry.

Multiangle dynamic light scattering (MDLS) and turbidimetry (T) were applied (both individually and combined) for determining the contamination by larger particles of two almost-uniform polystyrene (PS) latices. Latex 1 was synthesized in our laboratories, and it contained a main population diameter of 340 nm together with a small fraction of larger particles. This latex was used as the base material for producing an immunoassay kit. Latex 2 was obtained by a simple blend of two uniform PS standards. The proposed data treatment calculates the diameter and number fraction of the large particles contamination assuming that the PSDs are bimodal. The calculation involves minimizing the errors between the measurements and their theoretical predictions. When analyzed by combined MDLS-T, the contamination of Latex 1 involved number fraction 0.6% and particle diameter 865 nm. The T average diameter is a function of the measurement wavelength, and the highest deviations of this average to an increasing contamination by large particles were always observed at the higher wavelengths. The DLS average diameter is a function of the measurement angle, but in this case it is impossible to determine a priori the angle of observation that provides the largest deviation of this average diameter to an increasing contamination.

Latex particle size distribution by dynamic light scattering: novel data processing for multiangle measurements.

Multiangle dynamic light scattering (DLS) provides a better estimate of particle size distributions (PSD) than single-angle DLS. However, multiangle data treatment requires appropriate weighting of each autocorrelation measurement prior to calculation of the PSD. The weighting coefficients may be directly obtained from (i). the autocorrelation baselines or (ii). independent measurement of the average light intensity by elastic light scattering. However, the propagation of errors associated with such procedures may intolerably corrupt the PSD estimate. In this work, an alternative recursive least-squares calculation is proposed that estimates the weighting coefficients on the basis of the complete autocorrelation measurement. The method was validated through a numerical example that simulates the analysis of a polystyrene latex with a bimodal PSD and with "measurements" taken at 10 detection angles. The ill-conditioned nature of the problem determines that the "true" PSD cannot be recovered, even in the absence of errors. A sensitivity analysis was carried out to determine the effect of errors in the weighting coefficients on the PSD recoveries.

Scleroglucan compatibility with thickeners, alcohols and polyalcohols and downstream processing implications.

Thickening capacity and compatibility of scleroglucan with commercial thickeners (corn starch, gum arabic, carboxymethylcellulose, gelatin, xanthan and pectin), glycols (ethylene glycol and polyethylene glycol), alcohols (methanol, ethanol, 1-propanol and isopropanol) and polyalcohols (sorbitol, xylitol and mannitol) was explored. Exopolysaccharides (EPSs) from Sclerotium rolfsii ATCC 201126 and a commercial scleroglucan were compared. Compatibility and synergism were evaluated taking into account rheology, pH and sensory properties of different thickener/scleroglucan mixtures in comparison with pure solutions. S. rolfsii ATCC 201126 EPSs induced or increased pseudoplastic behaviour with a better performance than commercial scleroglucan, showing compatibility and synergy particularly with corn starch, xanthan, pectin and carboxymethylcellulose. Compatibility and a slight synergistic behaviour were also observed with 30% (w/v) ethylene glycol whereas mixtures with polyethylene glycol (PEG) precipitated. Scleroglucan was compatible with polyalcohols, whilst lower alcohols led to scleroglucan precipitation at 20% (v/v) and above. PEG-based scleroglucan downstream processing was compared to the usual alcohol precipitation. Downstream processed EPSi (with isopropanol) and EPS-p (with PEG) were evaluated on their yield, purity, rheological properties and visual aspect pointing to alcohol downstream processing as the best methodology, whilst PEG recovery would be unsuitable. The highest purified EPSi attained a recovery yield of ~23%, similar to ethanol purification, with a high degree of purity (88%, w/w vs. EPS-p, 8%, w/w) and exhibited optimal rheological properties, water solubility and appearance. With a narrower molecular weight distribution (M(w), 2.66×10(6) g/mol) and a radius of gyration (R(w), 245 nm) slightly lower than ethanol-purified EPSs, isopropanol downstream processing showed to be a proper methodology for obtaining a refined-grade scleroglucan.

Synthesis of latex-antigen complexes from single and multiepitope recombinant proteins. Application in immunoagglutination assays for the diagnosis of Trypanosoma cruzi infection.

The physical adsorption and the chemical coupling of recombinant proteins of Trypanosoma cruzi onto polystyrene and core-shell carboxylated particles were respectively investigated with the ultimate aim of producing latex-protein complexes to be used in an immunoagglutination assay able to detect the Chagas disease. To this effect, two single proteins (RP1 and RP5) and a multiepitope protein derived from three antigenic peptides (CP2) were evaluated, and sensitizations were carried out at different pHs. The maximum physical adsorption was produced at pHs close to the protein isoelectric point (i.e., pH 6 for RP5 and pH 5 for RP1 and CP2). High fractions of antigens were chemically bound to the carboxyl groups, and the highest surface density of linked protein was also observed at pHs close to the protein isoelectric point. The three latex-protein complexes obtained by covalent coupling at such pHs were tested with sera from a panel of 16 infected and 16 non-infected patients. In the immunoagglutination assays, the latex-CP2 complex produced the best discrimination between positive and negative sera.

Immunodiagnosis of Chagas disease: Synthesis of three latex-protein complexes containing different antigens of Trypanosoma cruzi.

This article describes the physical adsorption and the chemical coupling of 3 antigenic proteins of Trypanosoma cruzi onto polystyrene (PS) based latexes to be used as novel immunodiagnosis reagents for detecting the Chagas disease. The coupled proteins were a homogenate of T. cruzi, or a recombinant protein (either Ag36 or CP1). With the homogenate, between 30 and 60% of the total-linked protein was chemically coupled, showing a small dependence with the pH. For Ag36 and CP1, around 90% of the total-linked protein was chemically coupled, with a maximum coupling at pH 5 (i.e., close to the isoelectric point). The chemical coupling of CP1 was less affected by the pH than the coupling of Ag36.