Thin gold film-assisted fluorescence spectroscopy for biomolecule sensing.

We present a configuration for fluorescence spectroscopy that exploits the optical properties of semitransparent gold films and widely available instrumentation. This method enables monitoring of biomolecule interactions with small molecules tethered on substrates in multicomponent environments. The neurotransmitter serotonin (5-hydroxytryptamine) was covalently attached to self-assembled monolayers on thin gold films at low density to facilitate antibody recognition. Protein-binding studies were performed in a fluorescently labeled immunoassay format. We find that the use of this method enables evaluation of nonspecific binding and relative quantification of specific binding between competing binding partners. This fluorescence spectroscopy technique has the potential to assess biosensor or medical device responses in complex biological matrices.

Tuning stamp surface energy for soft lithography of polar molecules to fabricate bioactive small-molecule microarrays.

Soft-lithography-based techniques are widely used to fabricate microarrays. Here, the use of microcontact insertion printing is described, a soft-lithography method specifically developed for patterning at the dilute scales necessary for highly selective biorecognition. By carefully tuning the polar surface energy of polymeric stamps, problems associated with patterning hydrophilic tether molecules inserted into hydrophilic host self-assembled monolayers (SAMs) are surmounted. Both prefunctionalized tethers and on-chip functionalization of SAMs patterned by microcontact insertion printing enable the fabrication of small-molecule microarrays. Substrates patterned with the neurotransmitter precursor 5-hydroxytryptophan selectively capture a number of different types of membrane-associated receptor proteins, which are native binding partners evolved to recognize free serotonin. These advances provide new avenues for chemically patterning small molecules and fabricating small molecule microarrays with highly specific molecular recognition capabilities.

Native serotonin membrane receptors recognize 5-hydroxytryptophan-functionalized substrates: enabling small-molecule recognition.

Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach.