A quartz crystal microbalance method to quantify the size of hyaluronan and other glycosaminoglycans on surfaces

Abstract

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of soluble HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift (ΔD) and the negative frequency shift (-Δf) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from a tetrasaccharide to a 1 MDa polysaccharide, we establish a standard curve of the ΔD/-Δf ratio as a function of HA size. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, and with a resolution better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduced the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient

“…The obtained biotinylated heparin was characterized by biotin-streptavidin binding assays using QCM-D. Lowsulfated HS was derived from porcine intestinal mucosa (Celsus Laboratories, Cincinnati, OH, USA), and high-sulfated HS was purified in the laboratory of Hughes Lortat-Jacob (Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France) and treated in the same manner. The average mass of all the heparin and HS isolates, when anchored to the surface, was estimated at 9 kDa (~18 disaccharide units) by QCM-D analysis 82 .…”

Practical Use of Quartz Crystal Microbalance Monitoring in Cartilage Tissue Engineering

“…It is particularly useful when protein (e.g., ECM components) adsorption to a base biomaterial is intended [ 87 ]. This is a common strategy in designing the scaffold for CTE [ 81 , 103 ] since it is well-known that the protein adsorption and morphology of the protein layer on the biomaterial surface govern cellular adhesion, cell function, and activity, such as proliferation, survival, and gene expression [ 35 , 104 , 105 ]. QCM is also most suitable on the nanoscale, as it becomes very difficult to determine the physical properties of the load on the microscale and impossible on the macroscale.…”