Local Structure and Elasticity of Soft Gelatin Gels Studied with Atomic Force Microscopy V. I. Uricanu,* ,† M. H. G. Duits, † R. M. F. Nelissen, † M. L. Bennink, ‡ and J. Mellema † Physics of Complex Fluids Group and Biophysical Techniques Group, Faculty of Science and Technology, Associated with the J. M. Burgerscentrum for Fluid Mechanics, and Institute of Mechanics, Processes and ControlsTwente (IMPACT), University of Twente, Postbus 217, 7500 AE Enschede, The Netherlands Received April 24, 2003. In Final Form: July 8, 2003 Atomic force microscopy (AFM) measurements were done on aqueous gelatin gels submerged in dodecane. Use of home-built AFM instrumentation coupled with dedicated analysis of the recorded force-displacement curves, allowed the a posteriori extraction of both surface topography and elastic properties of these soft samples. Hertz theory was used to obtain (apparent) relative Young moduli (E*) from the force-indentation curves. For indentations smaller than 100 nm, scattered values of E* were found. This is partially attributed to the structural inhomogeneity of the polymer network at these length scales. At larger indentations, the relative Young moduli were found to be “compression-rate”-independent but to decrease with the indentation depth (δ). This independence of the compression rate indicates quasi equilibrium elastic behavior (i.e., the absence of stress relaxation by the gelatin), confirmed also by additional experiments, in which a truncated sawtooth driving voltage was used. In these latter AFM recordings, the compression regime is combined with a stationary piezo state when the AFM tip is in contact with the gel. The measured cantilever deflection due to gel relaxation was always below 10% from the indentation depth. Additional features were observed in the 3D recordings and associated with stiff fibrils lying on top of the soft gelatin network. Depending on aging time and location along the sample surface, mobile single fibrils as well as tough, compact, immobile fibril bundles were observed. A comparison was made between the relative Young moduli measured with AFM and the elastic (storage) moduli as measured in a conventional rheometer. Taking as variable the sample’s “age”, the microscopic and the macroscopic moduli turned out to be in good agreement in the limit of high (polymer) concentrated gels. Gel syneresis, with water exudation from the 10% gelatin network, was found to drastically increase the E* values found with AFM (at δ ) 250 nm). The fact that this was not found with conventional rheometry might suggest a different syneresis behavior in dodecane. Introduction Gelatin is an abundant and relatively inexpensive protein derived from collagen, with the capacity to form network structures and gels. It is used in a variety of gel applications including photography, drug delivery, ho- lography, microencapsulation, and food preparation. Gelatin is also a promising structural biomaterial since it does not show antigenity and toxicity and can be completely resorbed in vivo. Its physicochemical properties can be suitably modulated and its mechanical properties can be improved and adjusted 2-5 by mixing gelatin with inorganic 1,6-8 materials or with other natural and synthetic polymers. 9-17 In native state, gelatin molecules are soluble in water at 40-45 °C, where the individual chains act like semiflexible coils. When the temperature drops below the gelation limit (which is around 30-35 °C, depending on the gelatin type and concentration), the initial solution undergoes a sol-gel transition. 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