A novel combination of DLS-optical microrheology and low frequency Raman spectroscopy to reveal underlying biopolymer self-assembly and gelation mechanisms

Amin, Samiul; Blake, Steven; Kenyon, Stacy M.; Kennel, Rachel; Lewis, E. Neil

doi:10.1063/1.4903785

Cited by 13 publications

(14 citation statements)

References 24 publications

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“…More detailed information could be found in prior work [34][35][36]. is a constant for the short delay-time baseline, is an instrument specific constant, is the self diffusion coefficient (when protein concentrations are low), is the magnitude of the scattering vector defined in Equation [4], n is the refractive index of solvent, is the laser wavelength (658.9 nm), and is the scattering angle, is the decay time, and is the second cumulant and is related to the sample polydispersity index.…”

Section: Far-uv Circular Dichroismmentioning

confidence: 98%

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Barnett

Wei

Amin

et al. 2015

Biophysical Chemistry

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Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).

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Section: Far-uv Circular Dichroismmentioning

confidence: 98%

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Barnett

Wei

Amin

et al. 2015

Biophysical Chemistry

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“…9 More recent measurements have combined Raman spectroscopy with dynamic light scattering-based microrheological measurements to characterize gelation processes. 10 Despite the benefit of combined Raman spectroscopy and rheological information, these prior systems lack the means to characterize the microstructure evolution of the sample or to control the location of the Raman beam with respect to the microstructure. Optical microscopy provides a direct measure of structure within many samples of interest and has been used successfully in the past in combination with rheology.…”

Section: Introductionmentioning

confidence: 99%

The rheo-Raman microscope: Simultaneous chemical, conformational, mechanical, and microstructural measures of soft materials

Kotula

Meyer

Vito

et al. 2016

Review of Scientific Instruments

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The design and performance of an instrument capable of simultaneous Raman spectroscopy, rheology, and optical microscopy are described. The instrument couples a Raman spectrometer and optical microscope to a rotational rheometer through an optically transparent base, and the resulting simultaneous measurements are particularly advantageous in situations where flow properties vary due to either chemical or conformational changes in molecular structure, such as in crystallization, melting, gelation, or curing processes. Instrument performance is demonstrated on two material systems that show thermal transitions. First, we perform steady state rotational tests, Raman spectroscopy, and polarized reflection microscopy during a melting transition in a cosmetic emulsion. Second, we perform small amplitude oscillatory shear measurements along with Raman spectroscopy and polarized reflection microscopy during crystallization of a high density polyethylene. The instrument can be applied to study structure-property relationships in a variety of soft materials including thermoset resins, liquid crystalline materials, colloidal suspensions undergoing sol-gel processes, and biomacromolecules. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States.

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“…The gelation can be attributed to aggregation of the protein at the higher temperatures at this pH. This observation is further validated when the elastic modulus is analyzed against the low-frequency Raman data that measures the enhanced hydrogen bonding in water and the degree of water confinement [ 39 , 40 ]. This portion of the Raman spectrum is seen to correlate well with the G ′ evolution ( Figure 7 B).…”

Section: Resultsmentioning

confidence: 78%

“…In order to further investigate this gelation effect, DLS-microrheology was carried out. In general, the elastic modulus ( G ′) acquired by optical microrheology is a strong indicator of microstructural evolution and network formation in self-assembling systems, such as agarose [ 39 ] and surfactant-based wormlike micelles [ 40 ]. Here, the evolution toward gelation is illustrated by the rapid increase in G ′ as a function of temperature, especially at pH 5.5 ( Figure 7 A).…”

Section: Resultsmentioning

confidence: 99%

Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly

Blake¹,

Amin²,

Wei³

et al. 2015

IJMS

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A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS), optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC). The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein’s secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5), oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance.

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A novel combination of DLS-optical microrheology and low frequency Raman spectroscopy to reveal underlying biopolymer self-assembly and gelation mechanisms

Cited by 13 publications

References 24 publications

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations

The rheo-Raman microscope: Simultaneous chemical, conformational, mechanical, and microstructural measures of soft materials

Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly

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