Small-Molecule Patterning via Prefunctionalized Alkanethiols

Abstract

Interactions between small molecules and biomolecules are important physiologically and for biosensing, diagnostic, and therapeutic applications. To investigate these interactions, small molecules can be tethered to substrates through standard coupling chemistries. While convenient, these approaches co-opt one or more of the few small-molecule functional groups needed for biorecognition. Moreover, for multiplexing, individual probes require different surface functionalization chemistries, conditions, and/or protection/deprotection strategies. Thus, when placing multiple small-molecules on surfaces, orthogonal chemistries are needed that preserve all functional groups and are sequentially compatible. Here, we approach high-fidelity small-molecule patterning by coupling small-molecule neurotransmitter precursors, as examples, to monodisperse asymmetric oligo(ethylene glycol)alkanethiols during synthesis and prior to self-assembly on Au substrates. We use chemical lift-off lithography to singly and doubly pattern substrates. Selective antibody recognition of pre-functionalized thiols was comparable to or better than recognition of small molecules functionalized to alkanethiols after surface assembly. These findings demonstrate that synthesis and patterning approaches that circumvent sequential surface conjugation chemistries enable biomolecule recognition and afford gateways to multiplexed small-molecule functionalized substrates.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates

“…25,28,29,34 Nonspecific binding was minimized through the use of hydroxyl tri(ethylene glycol)-terminated undecanethiol (TEG) in the functionalized and unfunctionalized regions. 27,30,31 We have previously characterized small-molecule-functionalized substrates using infrared spectroscopy, 26,27 X-ray photoelectron spectroscopy, and electrochemistry.…”

“…27,30,31 We have previously characterized small-molecule-functionalized substrates using infrared spectroscopy, 26,27 X-ray photoelectron spectroscopy, and electrochemistry. 25 Nucleic acids were conjugated with fluorophores on their 5’-ends (Figure 1). The “correct” dopamine aptamer sequence was a 57-base DNA homolog 35 of an RNA aptamer originally isolated by SELEX by Mannironi and co-workers (Figure 1A).…”

“…Pre-functionalized thiols that display more consistent biomolecule recognition, 25 were utilized to create multiplexed substrates. Multiplexed substrates enabled measurements of K d values for multiple targets in situ .…”

“…23–25 Optimization entailed (1) spacing small-molecules for improved recognition by larger biomolecules via dilute solution deposition of tethering thiols during the preparation of mixed self-assembled monolayers (SAMs) or through the insertion of tethers into naturally existing or engineered defects in SAMs. 26–29 (2) We utilized the amino acid precursors of neurotransmitters for tethering via the additional carboxyl moiety to render amino groups more accessible for molecular recognition.…”

“…Using small-molecule tethered substrates, binding partners including antibodies 25,27 and G-protein-coupled receptors, 23,24 were selectively captured and sorted from mixtures to their respective targets. We developed soft - lithography patterning methods 32,33 to create regions of target contrast, which enabled visualization and quantification via fluorophore-labeled binding partners relative to nonspecific binding on the same substrates.…”