DEVELOPMENT AND CHARACTERIZATION OF SMALL-MOLECULE-FUNCTIONALIZED CAPTURE SURFACES
Open Access
- Author:
- Vaish, Amit
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 21, 2011
- Committee Members:
- Paul S Weiss, Dissertation Advisor/Co-Advisor
Peter J Butler, Committee Chair/Co-Chair
Paul S Weiss, Committee Chair/Co-Chair
Anne Andrews, Committee Chair/Co-Chair
William O Hancock, Committee Member
Joan Marie Redwing, Committee Member - Keywords:
- Chemical Patterning
Capture Surfaces
Biosenosors
Self-Assembled Monolayers
Receptor Proteins
Small Molecules - Abstract:
- Many important biological processes are mediated by the interactions between small diffusible molecules and large biomolecules. To study these interactions in a selective and controlled manner, it is important to develop materials capable of targeting interactions at the molecular level. In this thesis, the principles of bioengineering, biochemistry, neuroscience, and materials chemistry were integrated with nanotechnology-based strategies for the design, synthesis, and characterization of small-molecule-functionalized materials. The nanoengineered, neurotransmitter-functionalized platforms developed here are capable of selectively recognizing and capturing large- biomolecule binding partners while having relatively low nonspecific binding. One of the key challenges in the fabrication of neurotransmitter-functionalized surfaces is to position isolated small-probe molecules on protein-resistant matrices to provide optimal access to large binding partners. Nanotechnology-based insertion techniques were utilized to create neurotransmitter-functionalized surfaces using self-assembly of molecules with biological relevance. In particular, insertion-directed self-assembly was employed to isolate tether molecules terminating in a chemically targetable functional group for probe conjugation within a matrix of an oligo(ethylene glycol) self-assembled monolayer (SAM) to resist protein binding. Methods were developed to modify substrates with serotonin (5 HT), a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic free serotonin in solution, an alternate coupling chemistry was devised to preserve the core chemical epitope of 5-HT by attaching its amino acid precursor, 5 hydroxytryptophan (5-HTP), via its ancillary carboxyl group to inserted tether molecules. Quartz crystal microgravimetry (QCM) was used to demonstrate that 5-HT- and 5-HTP-functionalized surfaces selectively capture their cognate antibodies while having low nonspecific recognition of other antibodies. However, only 5-HTP-functionalized surfaces selectively bind recombinant receptors that natively recognize free serotonin. By contrast, serotonin-functionalized surfaces fail to bind membrane-associated serotonin receptors. It is inferred that recognition of small molecules by large biomolecules, which has evolved to distinguish between small-molecule ligands with highly similar structures in solution, requires preservation of all functional groups of free molecules. A fluorescence spectroscopy-based technique was also developed to study binding of proteins in a competitive environment on neurotransmitter-functionalized surfaces. Additionally, to broaden the scope of microcontact insertion printing for patterning hydrophilic tether molecules, the surface energy of polymeric stamps was tuned by oxygen plasma treatment to enable printing of polar tether molecules to fabricate bioactive small-molecule microarrays (SMMs). The binding of biomolecules on these patterned surfaces was detected by fluorescence microscopy and atomic force microscopy. Substrates patterned with small-molecule probes selectively captured their large-molecule binding partners in a competitive environment. These advances provide new avenues for chemically patterning small molecules and fabricating SMMs with highly specific molecular recognition capabilities.