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 ...