Browsing by Subject "Immobilization"

Background

Methods

Results

Conclusions

The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.

In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.

In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.

In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.