Development and characterization of small-molecule-functionalized capture surfaces

Self-assembly and nanofabrication techniques were combined with chemistry and neuroscience principles to produce small-molecule-functionalized surfaces capable of selective molecular recognition of large-molecule binding partners. To passivate biosensing substrates, self-assembled monolayers composed of oligo(ethylene glycol)alkanethiols (OEGs), which have been shown to impart resistance to protein binding on surfaces, were formed on gold substrates. To enable functionalization of monolayers with a dilute, non-phase-separated distribution of small-molecule probes, insertion-directed self assembly was used to intercalate OEGs with different terminal functional groups into existing monolayer matrices. Carbodiimide coupling chemistry was used to derivatize the dilute functional groups with small-molecule probes. Initially, a prototypical small-molecule neurotransmitter, serotonin, was used to produce small-molecule-functionalized substrates. Each step of the surface preparation was monitored by infrared reflection absorption spectroscopy, and the resultant surfaces were shown to selectively recognize of anti-serotonin antibodies by quartz crystal microgravimmetry.;Exploiting the knowledge gained from functionalizing surfaces with serotonin, a modified chemistry was used to attach the serotonin biological precursor, 5-hydroxytryptophan (5-HTP), to similarly prepared surfaces via its ectopic carboxyl group. In addition to demonstrating selective recognition of its cognate antibody, 5-HTP surfaces were shown to be capable of selectively binding membrane-associated serotonin receptor proteins. In contrast, serotonin-functionalized surfaces fail to bind these receptors. Because native serotonin receptors bind serotonin in solution, these results suggest that all functional groups of serotonin are necessary for receptor recognition. Furthermore, 5-HTP-functionalized surfaces mimic free serotonin by presenting all functional groups of serotonin when 5-HTP is tethered through its additional carboxyl group.;To enhance the breadth of applications of such surfaces, the creation of chemically patterned surfaces was investigated. Both photolithographically assisted chemical patterning and microcontact insertion printing were used to create spatially controlled regions of inserted tether molecules. Microcontact insertion printing was shown to be better suited for patterning at the low probe densities needed for biospecific recognition. In addition to demonstrating patterned capture of proteins, new probes, dopamine and biotin, were successfully used on these surfaces. Dopamine was functionalized in situ in a similar fashion to serotonin, while prefunctionalized biotinylated OEGs were used to create biotin-functionalized surfaces. Both types of surfaces were capable of capturing binding partners with high affinity for the small-molecule probes on the surfaces in single- and mixed-protein solutions.;In addition to the biocapture properties of OEG monolayers, how insertion-directed selfassembly and chemical functionalization of OEGs compared to n-alkanethiols were investigated. Differences in protonation and monomer/dimer ratios of the carboxy group of carboxy-terminated OEG vs n-alkanethiol molecules on surfaces were found. Additionally, the fraction of insertion of OEGs into n-alkanethiolate monolayers under identical conditions was observed to vary based on the terminal functional groups of the OEG molecules, in contrast to n-alkanethiol insertion, which was independent of terminal functional group. It was also found that single-component monolayers of OEGs were more amenable to chemical reaction than n-alkanethiolate monolayers. This observation was attributed to the lower packing density of the ethylene glycol termini of the OEG molecules as compared to the n-alkanethiol molecules.;Taken together, these findings represent an increase in understanding regarding the methods for creating small-molecule-functionalized surfaces.