In-Situ AFM Studies of the Phase-Transition Behavior of Single Thermoresponsive Hydrogel Particles ² Justyna Wiedemair, ‡ Michael J. Serpe, ‡,§ Jongseong Kim, ‡,§ Jean-Francois Masson, ‡ L. Andrew Lyon,* ,‡,§ Boris Mizaikoff, ‡ and Christine Kranz* ,‡ School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed May 8, 2006. In Final Form: July 5, 2006 The volume phase transition (VPT) behavior of individual thermally responsive poly(N-isopropylacrylamide-co- acrylic acid) (pNIPAm-co-AAc) hydrogel microparticles was studied by in-situ dynamic mode atomic force microscopy (AFM) and force spectroscopy during heating and cooling cycles. Hydrogel samples were prepared by electrostatic immobilization of microparticles to amine-modified gold surfaces. The AFM studies of particle deswelling were performed by varying the force applied on the particles during imaging as a function of the geometry and material of the AFM probe. Aluminum-coated silicon cantilevers were found to influence substantially the behavior of the particles during the VPT, leading to a significant shape change. Low force impact magnetic excitation of the AFM probe (MAC mode) during dynamic mode measurements resulted in an undisturbed deswelling behavior enabling observation of the expected volume changes of the particles without significant tip-sample interaction. Hence, MAC- mode AFM was determined to be the most suitable technique for in-situ AFM studies on volume and shape changes at single hydrogel particles during VPT. Elasticity measurements performed at single particles at temperatures below and above the VPT revealed a 15-fold increase in the Young’s modulus after passing the VPT, indicating the transition from a soft, swollen network to a stiffer, deswollen state. Introduction Thermoresponsive hydrogels are composed of amphiphilic cross-linked polymer chains and are characterized by an abrupt change in the physical properties of the polymer network at a specific transition temperature. 1 In the solvent swollen state, polymer-solvent interactions are thermodynamically favorable. However, increasing the temperature above the intrinsic lower critical solution temperature (LCST) of the polymer leads to a change in the solvation behavior of the polymer chains. As polymer-polymer interactions become increasingly preferable, an abrupt and reversible volume change of the network is observed, which is called the volume phase transition (VPT). Poly(N-isopropylacrylamide) (pNIPAm)-based hydrogels are among the most common types of temperature-responsive materials. 1-5 Polymers can be synthesized as hydrogel films 6-8 or as small particles ranging from micrometer to nanometer dimensions (e.g., microgels or nanogels). 1,9,10 Copolymerization of acidic groups (e.g., acrylic acid) leads to additional pH- responsive behavior, which manifests itself as a shift in the volume phase transition temperature (VPTT) as a function of pH. 11 These polymers are characterized by a highly complex phase transition behavior, which in addition is dependent on the ionic strength of the solution and the size of the ions. 12 The responsive properties render such hydrogels suitable as matrixes for a variety of applications ranging from drug delivery 2,3 to biosensor design. 13 The shrinking behavior during their volume phase transition leads to the discharge of the solvent including potentially loaded molecules (e.g., drugs). 2,14-18 Furthermore, most hydrogels are biocompatible with interfaces resistant to biofouling. 19,20 To explore these applications, fundamental knowledge on the VPT behavior, ideally at individual immobilized particles, is a prerequisite. Dynamic light scattering techniques (DLS) are frequently applied to study particle size and size distribution in microgels. 1,21 However, these techniques are ensemble-averaged measurements, and single hydrogel particle analysis would certainly provide more detailed information. Optical microscopy has been applied in the study of immobilized hydrogel microspheres. 22 To date, the volume phase transition of single particles has been investigated using particles with diameters >1 μm. Hydrogel films have been extensively investigated with surface plasmon resonance (SPR) techniques. 23,24 The combination of this surface- ² Part of the Stimuli-Responsive Materials: Polymers, Colloids, and Multicomponent Systems special issue. ‡ School of Chemistry and Biochemistry. § Petit Institute for Bioengineering and Bioscience. (1) Pelton, R. AdV. Colloid Interface Sci. 2000, 85,1-33. (2) Soppimath, K. S.; Aminabhavi, T. M.; Dave, A. M.; Kumbar, S. G.; Rudzinski, W. E. Drug DeV. Ind. 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