Abstract
We combined optical tweezers and microrheological technique to measure the viscosity and viscoelasticity of collagen solutions in the denature process. Using optical tweezers to measure the collagen viscosity and viscoelasticity enabled us to extend the range of frequency and reduced the amount of samples used compared to traditional viscometers and rheometers. This study could be used in biomaterials and biotechnology research in the future.Collagen is a natural biomaterial and is one of the most abundant proteins in the human body. It has often been applied in the fields of tissue engineering and biomaterials. Collagen denatures at about 40°C and causes the collagen’s triple helix structure becoming random coils.In this study, we utilized optical tweezers system to measure the effects of thermal denaturation with varying pH and additives, (i.e. glycerol, urea, glucose, and NaCl) on viscosity of collagen Type I. Rat tail collagen Type I was diluted with PBS buffer and acetic acid to varying pH, with different additives. Collagen samples are then heated to target temperature and cooled down to room temperature (25°C) for measurement. Viscosity of collagen samples were determined by measuring the thermal motion of polystyrene microspheres (d=2.07μm) by optical tweezers. We used distilled water to calibrate the voltage-displacement coefficient and trap stiffness. After obtaining the system parameters, we used Kramers-Kornig relation to calculate water’s elastic modulus G' and viscous modulus G''. And then, we measured the collagen’s viscosity with varying pH and additives under thermal denaturation conditions. Our results indicate that as the pH of collagen approaches neutral, the aggregation of collagen fibrils causes the viscosity to increase significantly. The addition of glucose would inhibit collagen aggregation, causes the viscosity to decrease, and the denaturation temperature was measured to be 1°C higher when compared to pH 6.8. The addition of glycerol would protect collagen fibrils, and increase the denaturation temperature about 2°C. The addition of urea would break the collagen’s intermolecular hydrogen bonds, causes the viscosity to decrease, and lower the denaturation temperature about 4°C. The addition of NaCl would degrade collagen, causes the viscosity to decrease, and lower the denaturation temperature about 4°C.Different additives and pH would alter the collagen’s degree of cross-linking, intermolecular interactions, intramolecular interactions, hydrogen bonds, hydrophobic interactions, and electrostatic interactions. This would influence the collagen’s denature temperature, folding equilibrium, molecular structure, thermal stability, viscosity, and viscoelasticity.