A model system for the capture of large biomolecule targets by small-molecule probes has been developed using a combination of insertion self-assembly and chemical functionalization. Key to selective molecular recognition of small molecules is the ability to tether these such that they are readily accessible for binding. This is particularly challenging because the tether alters a significant fraction of the probe molecule. It is also critical to space small-molecule probes so that they are dilute and exposed, rather than clustered via phase separation on capture surfaces, particularly when trying to bind large-molecule targets (e.g., proteins, nucleic acids). [1][2][3][4][5] Optimal dilution avoids the effects of steric hindrance [6][7][8] and multivalent non-specific interactions. Dilute surface coverage is also important to enable strategies to prevent nonspecific binding. We have developed a general method for addressing these problems [5,[9][10][11][12] and apply these to surfaces functionalized with the small-molecule neurotransmitter serotonin (5-hydroxytryptamine; Fig. 1). Serotonin is covalently bound to tethers inserted at dilute coverage into pre-existing self-assembled monolayers (SAMs) bearing a molecular surface of oligo(ethylene glycol). We chose serotonin as our initial probe both because of its important roles as a neurotransmitter involved in anxiety and mood disorders [13] and as a prototypical small molecule for which many high-affinity binding proteins have been identified [14] but for which many more remain unknown. We demonstrate access to and specific recognition of serotonin by comparing the capture from solution of antibodies directed against serotonin versus those specific for another neurotransmitter, dopamine, or the enzyme tyrosine hydroxylase. We also show that these surfaces resist nonspecific protein adsorption of bovine serum albumin (BSA). Previous attempts by us using analogous preparations on alkylamine-functionalized sepharose beads failed because they were fraught with nonspecific binding. The assembly/synthetic strategy described produces a smallmolecule-derivatized surface capable of biospecific recognition. This approach is readily translatable to most neurotransmitters, as well as to many other small molecules. In addition to identifying selective molecular recognition elements for future biosensor applications, these serotonin-functionalized surfaces will be used in combination with mass spectrometry to identify and to characterize expression patterns of brain proteins, such as membrane-associated receptors, that selectively bind to serotonin in experiments designed to investigate the etiologies of psychiatric disorders and their treatments. [15][16][17] Serotonin-derivatized surfaces were prepared using a fourstep process. Monolayers of oligo(ethylene-glycol)-terminated alkanethiol (1) were self-assembled on Au substrates. These SAMs have been prepared previously [7,8,[18][19][20][21][22] and resist nonspecific binding of proteins (extra care is taken to minimize film ...

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.

Solid-state dye-sensitized solar cells (SS-DSCs) offer the potential to make low cost solar power a reality, however their photoconversion efficiency must first be increased. The dyes used are commonly narrow band with high absorption coefficients, while conventional photovoltaic operation requires proper band edge alignment significantly limiting the dyes and charge transporting materials that can be used in combination. We demonstrate a significant enhancement in the light harvesting and photocurrent generation of SS-DSCs due to Förster resonance energy transfer (FRET). TiO(2) nanotube array films are sensitized with red/near IR absorbing SQ-1 acceptor dye, subsequently intercalated with Spiro-OMeTAD blended with a visible light absorbing DCM-pyran donor dye. The calculated Förster radius is 6.1 nm. The donor molecules contribute a FRET-based maximum IPCE of 25% with a corresponding excitation transfer efficiency of approximately 67.5%.

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.

Chemical patterns prepared by self-assembly, combined with soft lithography or photolithography, are directly compared. Pattern fidelity can be controlled in both cases but patterning at the low densities necessary for small-molecule probe capture of large biomolecule targets is better accomplished using microcontact insertion printing (μCIP). Surfaces patterned by μCIP are used to capture biomolecule binding partners for the small molecules dopamine and biotin.

We describe the self-assembly and chemical functionalization of oligo(ethylene glycol)alkanethiol (OEG) molecules. Insertion of OEGs into n-alkanethiolate monolayer matrices depends considerably on terminal functionality, unlike insertion of n-alkanethiols. Thus, inserted fractions of OEGs cannot be inferred from related systems, yet tuning, to some extent, is possible by controlling insertion parameters. Furthermore, while the in situ reactivities of dilute inserted carboxyor amine-terminated OEGs versus n-alkanethiols protruding from the surrounding matrix are similar in amide bond formation reactions, complete monolayers of OEGs react to a greater extent compared to n-alkanethiols with similar terminal functionalities. We interpret these differences in terms of the reduced crystalline packing of terminal ethylene glycol groups of OEGs.

The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.

We have developed a method based on self-assembly of thiols on Au substrates to immobilize membrane proteins at interfaces. Using water soluble nitrilotriacetic acid (NTA)-terminated oligo(ethylene glycol) thiols, a histidine-tagged G protein-coupled membrane receptor (GPCR) was captured in a defined orientation with little nonspecific binding.