Multivariate Calibration Models for Lysozyme from Near-Infrared Transmission Spectra in Scattering Solutions of Monodisperse Microspheres

Abstract

The ability to quantify lysozyme is demonstrated for a series of aqueous samples with different degrees of scattering. Near-infrared spectra are collected for two sets of lysozyme/scattering solutions. In both sets of samples, the solutions are composed of lysozyme dissolved in acetate buffer with suspended monodisperse latex microspheres of polystyrene. The diameter of the microspheres is 6.4 microm for the first set and 0.6 microm for the second. For each set, the amount of microspheres range from 0.005 to 0.998 wt %, the lysozyme concentrations range from 0.834 to 28.6 mg/mL, and solution compositions are designed to minimize correlations between the concentration of lysozyme and percentage of microspheres. Near-infrared spectra are collected individually for each set of solutions. Single-beam spectra are collected over the combination spectral range (5000-4000 cm(-1), 2.0-2.5 microm) by transmitting the incident radiation through a 1.5-mm-thick sample that is maintained at 21 degrees C. Partial least-squares calibration models are evaluated individually for each data set both with and without wavelength optimization. Results indicate that models from raw, nonmodified, single-beam spectra are incapable of extracting lysozyme concentration from these highly scattering solutions. Accurate concentration measurements are possible, however, by implementing either a multiplicative scatter correction to the single-beam spectra or by taking the ratio of these single-beam spectra to an appropriate reference spectrum. In addition, digital Fourier filtering of these spectra enhances model performance. The best calibration model in the presence of 6.4-microm microspheres is obtained from multiplicative scatter corrected single-beam spectra over the 4550-4190-cm(-1) spectral range. The mean percent error of prediction (MPEP) and standard error of prediction (SEP) for this model are 2.2% and 0.28 mg/mL, respectively. Likewise, the multiplicative scatter corrected spectra with wavelength optimization provided the best calibration model for the 0.6-microm data set. In this case, the MPEP and SEP are 2.3% and 0.44 mg/mL, respectively. In addition, the ability to predict lysozyme concentrations is evaluated for the situation where the degree of scattering is greater in the predication samples compared to the calibration samples. Differences in the prediction ability are noted between the 6.4- and 0.6-microm data sets.

Surface Structural Characterization of Protein- and Polymer-Modified Polystyrene Microspheres by Infrared−Visible Sum Frequency Generation Vibrational Spectroscopy and Scanning Force Microscopy

“…Polymer microspheres (beads) of various diameters have been popularly used as solid substrates for biomolecular immobilization. These beads are primarily made of polystyrene and another polymer containing either carboxyl, amine, or hydroxyl groups as a minor component in the bead formulation. − These functional groups are used to selectively and specifically adsorb various biomolecules, such as proteins and DNA, via direct chemical coupling. , Surface-modified beads prepared in this manner are routinely used in diagnostic immunoassays to quantitate, for example, the concentrations of drugs of abuse or therapeutic drugs in bodily fluids. , The accuracy and limit of detection for systems involving modified beads depend primarily on the structure and orientation of biomolecules adsorbed at the surface.…”

“…Various techniques, including light scattering, spectroscopy, microscopy, and flow-adhesion assays have been used to structurally characterize bead adsorbates. ,− Although these techniques have produced valuable information regarding the chemical structure and concentration of adsorbates, more detailed structural information at the molecular level is anticipated. Surface-sensitive infrared−visible sum frequency generation (SFG) vibrational spectroscopy and scanning force microscopy (SFM) have emerged as powerful surface analytical tools to interrogate biological interfaces without background interference in solution.…”