Preparation of End-Grafted Polyelectrolyte Brushes on Nanoscale Probe Tips Using an Electric Field

Nanomechanics of opposing glycosaminoglycan macromolecules

“…electrically functionalized probe tip was estimated to be $6 nm using a protocol described previously (Seog et al, 2004). Taken together, these methods enabled direct experimental measurement and interpretation of forces between an array of $40 GAGs on the substrate and $40 GAGs on the probe tip, thereby simulating the interaction between GAGs of adjacent aggrecan molecules in cartilage extracellular matrix (ECM), consistent with the objective of probing molecular level biomechanical interactions relevant to tissue ECM function.…”

“…The average distance between neighboring GAGs on the substrate (s) was found to be $11 nm for the 2 h incubation time and $6.5 nm for the 72 h incubation time. Carboxyl-functionalized probe tips were prepared by immersing Au-coated Si 3 N 4 cantilever probe tips (Thermomicroscopes, Inc. V-shaped cantilever unsharpened Microlevers, spring constant ¼ 0:01 N/m, probe tip end-radius R TIP $100 nm measured by scanning electron microscopy (SEM)) in 5 mM HS-(CH 2 ) 15 -COOH (Aldrich) in ethanol for 24 h. GAGs were end-grafted to similar Au-coated Si 3 N 4 cantilever probe tips (R TIP $50 nm) by applying an electric field between the tip and a platinum electrode, spaced $100 mm from each other, while both were immersed in 1 mg/ml solution of GAG in PBS within the closed liquid cell of an AFM (Multimode IIIa, Digital Instruments, Santa Barbara, CA) for 9 h as described previously (Seog et al, 2004). The same backfilling procedure was employed for the GAG-functionalized probe tips as the GAG-functionalized planar substrates.…”

“…The same backfilling procedure was employed for the GAG-functionalized probe tips as the GAG-functionalized planar substrates. The molecular spacing between GAGs, s, on the probe tip was found previously to be $6 nm from highresolution force spectroscopy measurements (Seog et al, 2004). In this procedure, the force between the GAGfunctionalized probe tip and an OH-terminated alkanethiol self-assembled monolayer (SAM) planar substrate functionalized with HS-(CH 2 ) 11 -OH (Seog et al, 2002) was compared to a Poisson-Boltzmann-based charged rod model (Dean et al, 2003) (data not shown).…”

“…Based on biochemical and biophysical measurements on rat chondrosarcoma GAGs, the average GAG chain was assumed to be 25 disaccharides long (Dean et al, 2003). Therefore, the following fixed parameters values were used in the model: total GAG (cylinder) charge, Q rod ¼ 8 Â 10 À18 C (assuming one ionized COO À and SO 3 À group per disaccharide); probe tip end-radius R TIP ¼ 50 nm (Seog et al, 2004); h ¼ 45 nm (measured by VASE at 0.1 M NaCl), effective rod radius, w ¼ 2 nm, and inter-GAG spacing, s ¼ 627 nm (measured by scintillation counting).…”

“…In this study, we have used recently reported methods to prepare end-grafted chondroitin sulfate (CS) GAGfunctionalized planar substrates (Seog et al, 2002) and nanoscale probe tips (Seog et al, 2004) with a range of grafting densities close to that found in native cartilage. The interaction force (F ) between a GAG-functionalized probe tip and a GAG-functionalized planar substrate was measured on approach (compression), at a constant rate, as a function of probe tip-substrate separation distance (D).…”