Biospecific Recognition of Tethered Small Molecules Diluted in Self‐Assembled Monolayers

Abstract

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 ...

Hybrid approaches to nanometer-scale patterning: Exploiting tailored intermolecular interactions

“…It is important to note that in the overlapping patterned regions, the tether molecules (which are functionalized with smallmolecule probes) are isolated such that they do not interact with other probe molecules in the surrounding region. This molecular isolation can be controlled and influenced by many factors and has been confirmed by both ensemble and localized analytical techniques (Mullen et al 2007a;Shuster et al 2008).…”

“…A patterned elastomeric stamp coated with the molecules to be patterned is brought into contact with a substrate coated with a preexisting monolayer that is not easily displaced; the molecules on the stamp insert into the defects in the preexisting monolayer where the stamp and substrate are in contact. Schematics are not to scale terminal functionalities from the preexisitng SAM are inserted into its defect sites (Weck et al 1999;Mullen et al 2007b;Shuster et al 2008). Subsequently, the terminal groups of the tether molecules are functionalized with small-molecule ligands.…”

“…This type of biospecific surface is currently being developed to separate proteins from biological mixtures (e.g., tissue homogenates, serum, etc. ), to elucidate molecular interactions between known proteins and their smallmolecule targets, and to identify unknown proteins that bind selectively to small-molecule ligands (Mullen et al 2007b;Mrksich 2008;Shuster et al 2008). Further, these surfaces are not limited to functionalization with small biomolecules, but can present other small molecules such as chemical warfare agents and toxic industrial chemicals.…”

“…These surfaces have been employed to demonstrate biospecific recognition by serotonin-specific antibodies (Shuster et al 2008). Figure 5a and b shows inserted tether molecules isolated within a oligo(ethylene glycol)-terminated SAM before and after functionalization of the tether carboxylic acid terminal group with serotonin via EDC/NHS coupling chemistry (Hermanson 1996).…”

Controlled DNA Patterning by Chemical Lift-Off Lithography: Matrix Matters

“…28,29 Insertion also prevents phase separation. 23,30À33 A recent study of DNA hybridization on Au electrodes demonstrated that surface hybridization was reduced because DNA probes tended to aggregate into domains after backfilling with alcoholterminated alkanethiols. 34 In contrast, tethered DNA molecules inserted into defect sites in preformed SAMs produced dilute coverage wherein individual probe strands were isolated from each other.…”