Assessing the Thermal Sensitive Rheological Behavior in BSA Solution Using the BeNano 180 Zeta

Dynamic light scattering (DLS) microrheology involves using dynamic light scattering to obtain measurements of the mean square displacements (MSD) of added inert tracer particles in a solution environment and to acquire the rheological information of solutions.

Schematic diagram of the denaturalization of BSA during heating

Figure 1. Schematic diagram of the denaturalization of BSA during heating. Image Credit: Bettersize Instruments Ltd

DLS microrheology may characterize weakly structured polymer and protein solutions and gel systems, in which tracer particles can diffuse over large distances.

DLS microrheology enables quick and simple measurements and data treatment to be carried out compared to mechanical rheological methods. It can also facilitate the acquisition of high-frequency information in just one measurement, with the process described below:

  • Tracer particles are added to the system under investigation, e.g. a dilute polymer or protein solution.
  • Suspended in a solution system, particles move randomly via thermal energy exchange κΒΤ.
  • The surrounding environment's rheological properties determine these tracer particles' motion.
  • The detection of the tracer particles’ motion offers rheological information.

BSA solution is relatively stable under low-temperature conditions, but it becomes thermally denatured during heating, and protein agglomerates form. This results in the properties of the solution being altered significantly.

DLS microrheology may be employed to characterize the viscoelastic information of BSA solutions and various temperatures.

Theory

DLS microrheology involves the addition of colloidal particles, sized between 0.3 μm and 2.0 μm with known size information, as tracer particles. The rheological properties of the environment influence the motion of the tracer particles.

For a purely viscous fluid sample (Newtonian fluid), the tracer particles diffuse freely through the whole sample environment, and the MSD ⟨Δr2(τ)⟩ of the particle increases linearly with time:

⟨Δr2(τ)⟩ = 6Dτ

where D denotes the diffusion coefficient of the particle.

The standard Stokes-Einstein equation is used as follows:

where R denotes the radius of the tracer particle. From this equation, it can be seen that the relation between viscosity and MSD is as follows:

The viscosity of the Newtonian fluid, denoted by η, may be obtained by fitting the curve of ⟨Δr2(τ)⟩ with time. However, the generalized Stokes-Einstein equation can be utilized for non-Newtonian fluids that contain elastic components. This is provided below:

Here, the elastic (storage) modulus and the viscous (loss) modulus are derived by as a function of frequency. The complex viscosity η*(ω) and creep compliance J(t) may be obtained respectively, as follows:

Instrumentation

To carry out this study, Bettersize Instruments’ BeNano 180 Zeta nanoparticle size and zeta potential analyzer was employed.

This analyzer was equipped with a laser beam with a wavelength of 671 nm and a power of 50 mW as a light source. The instrument was also equipped with avalanche photodiode detectors (APD) to collect scattered light signals at 173°.

Sample Preparation and Measurement Condition

The BeNano’s integrated temperature control system was used to control measurement temperatures within the range of 25 ℃ to 70 ℃, with at least 300 seconds of temperature equilibration for each measurement.

A total of 10 μL of negatively charged 400 nm polystyrene spheres was added into 1 mL of the 10 mg/mL BSA solution to provide the tracer particles.

Results

Initially, the BSA solutions' zeta potentials and tracer particle suspension were measured in the same environment. The zeta potential of the BSA solution was determined to be -14.35 mV, while the zeta potential of the 400 nm tracer particle suspension was determined to be -51 mV.

The BSA and tracer particles were both negatively charged, inhibiting agglomeration due to the interaction of negative and positive charges. Calculating the fluctuation of the scattered signals provided the samples' correlation functions, as shown in Figures 1-6.

Correlation functions of BSA solutions at different temperatures

Figure 2. Correlation functions of BSA solutions at different temperatures. Image Credit: Bettersize Instruments Ltd

MSD curves of BSA solutions at different temperatures

Figure 3. MSD curves of BSA solutions at different temperatures. Image Credit: Bettersize Instruments Ltd

Viscoelastic modulus curves of BSA solutions at different temperatures

Figure 4. Viscoelastic modulus curves of BSA solutions at different temperatures. Image Credit: Bettersize Instruments Ltd

Complex viscosity curves of BSA solutions at different temperatures

Figure 5. Complex viscosity curves of BSA solutions at different temperatures. Image Credit: Bettersize Instruments Ltd

Creep compliance curves of BSA solutions at different temperatures

Figure 6. Creep compliance curves of BSA solutions at different temperatures. Image Credit: Bettersize Instruments Ltd

Figures 1-6 demonstrate that the correlation functions decayed faster as the temperature increased from 25 °C to 60 °C.

This indicates that the velocity of tracer particle motion increases as temperature increases because of the decrease in solution viscosity, while the solutions’ viscoelastic moduli decrease throughout this temperature range.

For this temperature range, the MSD curves show that higher temperatures correlate with higher MSD values and quicker tracer particle velocities. However, the correlation functions decayed more slowly with increasing temperature from 60 ℃ to 70 ℃.

This suggests that the tracer particles’ motion decreases with increasing temperature, resulting from the creation of aggregates caused by the thermal denaturation of BSA in this temperature range. These aggregates substantially increase the viscoelasticity of the solution.

The complex viscosity acquired using DLS microrheology quickly increased at approximately 65 ℃, as displayed in Figure 7. This results in the temperature dependence of particle size and complex viscosity and particle size being cross-verified.

Curves of BSA particle size and intensity versus temperature (left) and complex viscosity versus temperature @ 2096 rad/s (right).

Figure 7. Curves of BSA particle size and intensity versus temperature (left) and complex viscosity versus temperature @ 2096 rad/s (right). Image Credit: Bettersize Instruments Ltd

Conclusion

The measurement results presented in this article demonstrate BeNano’s microrheology detection ability for a protein sample. The thermal-sensitive microrheological information can accurately and sensitively exhibit the denaturing process of a protein solution with temperature.

The rheological parameters of the sample, including complex viscosity, mean square displacement, creep compliance, and viscoelastic moduli, can be quickly obtained in the high-frequency range by the microrheology measurements.

This offers a powerful tool for characterizing the rheological properties of liquids.

Acknowledgments

Produced from materials originally authored by Alia Yan from the Application Research Lab at Bettersize Instruments Ltd.

This information has been sourced, reviewed and adapted from materials provided by Bettersize Instruments Ltd.

For more information on this source, please visit Bettersize Instruments Ltd.

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