Toward a Quantitative Relationship between Nanoscale Spatial Organization and Hybridization Kinetics of Surface Immobilized Hairpin DNA Probes.

Hybridization of DNA probes immobilized on a solid support is a key process for DNA biosensors and microarrays. Although the surface environment is known to influence the kinetics of DNA hybridization, so far it has not been possible to quantitatively predict how hybridization kinetics is influenced by the complex interactions of the surface environment. Using spatial statistical analysis of probes and hybridized target molecules on a few electrochemical DNA (E-DNA) sensors, functioning through hybridization-induced conformational change of redox-tagged hairpin probes, we developed a phenomenological model that describes how the hybridization rates for single probe molecules are determined by the local environment. The predicted single-molecule rate constants, upon incorporation into numerical simulation, reproduced the overall kinetics of E-DNA sensor surfaces at different probe densities and different degrees of probe clustering. Our study showed that the nanoscale spatial organization is a major factor behind the counterintuitive trends in hybridization kinetics. It also highlights the importance of models that can account for heterogeneity in surface hybridization. The molecular level understanding of hybridization at surfaces and accurate prediction of hybridization kinetics may lead to new opportunities in development of more sensitive and reproducible DNA biosensors and microarrays.

Seeding the Self-Assembly of DNA Origamis at Surfaces.

Unlike supramolecular self-assembly methods that can organize many distinct components into designer shapes in a homogeneous solution (e.g., DNA origami), only relatively simple, symmetric structures consisting of a few distinct components have been self-assembled at solid surfaces. As the self-assembly process is confined to the surface/interface by mostly nonspecific attractive interactions, an open question is how these interfacial interactions affect multicomponent self-assembly. To gain a mechanistic understanding of the roles of the surface environment in DNA origami self-assembly, here we studied the oligonucleotide-assisted folding of a long single-stranded DNA (ssDNA scaffold) that was end-tethered to a dynamic surface, which could actively regulate the DNA-surface interactions. The results showed that even weak surface attractions can lead to defective structures by inhibiting the merging of multiple domains into complete structures. A combination of surface anchoring and deliberate regulation of DNA-surface interactions allowed us to depart from the existing paradigm of surface confinement via nonspecific interactions and enabled DNA origami folding to proceed in a solution-like environment. Importantly, our strategy retains the key advantages of surface-mediated self-assembly. For example, surface-anchored oligonucleotides could sequence-specifically initiate the growth of DNA origamis of specific sizes and shapes. Our work enables information to be encoded into a surface and expressed into complex DNA surface architectures for potential nanoelectronic and nanophotonic applications. In addition, our approach to surface confinement may facilitate the 2D self-assembly of other molecular components, such as proteins, as maintaining conformational freedom may be a general challenge in the self-assembly of complex structures at surfaces.

Single Molecule Profiling of Molecular Recognition at a Model Electrochemical Biosensor.

The spatial arrangement of target and probe molecules on the biosensor is a key aspect of the biointerface structure that ultimately determines the properties of interfacial molecular recognition and the performance of the biosensor. However, the spatial patterns of single molecules on practical biosensors have been unknown, making it difficult to rationally engineer biosensors. Here, we have used high-resolution atomic force microscopy to map closely spaced individual probes as well as discrete hybridization events on a functioning electrochemical DNA sensor surface. We also applied spatial statistical methods to characterize the spatial patterns at the single molecule level. We observed the emergence of heterogeneous spatiotemporal patterns of surface hybridization of hairpin probes. The clustering of target capture suggests that hybridization may be enhanced by proximity of probes and targets that are about 10 nm away. The unexpected enhancement was rationalized by the complex interplay between the nanoscale spatial organization of probe molecules, the conformational changes of the probe molecules, and target binding. Such molecular level knowledge may allow one to tailor the spatial patterns of the biosensor surfaces to improve the sensitivity and reproducibility.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or   l-tryptophan were selectively recognized by previously identified dopamine or l-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though, slightly greater than the previously determined solution dissociation constant. Using prefunctionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with l-3,4-dihydroxyphenylalanine, l- threo-dihydroxyphenylserine, and l-5-hydroxytryptophan enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons and future identification and characterization of novel aptamers targeting neurotransmitters or other important small molecules.

Small-Molecule Patterning via Prefunctionalized Alkanethiols.

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.

Patterning of supported gold monolayers via chemical lift-off lithography.

The supported monolayer of Au that accompanies alkanethiolate molecules removed by polymer stamps during chemical lift-off lithography is a scarcely studied hybrid material. We show that these Au-alkanethiolate layers on poly(dimethylsiloxane) (PDMS) are transparent, functional, hybrid interfaces that can be patterned over nanometer, micrometer, and millimeter length scales. Unlike other ultrathin Au films and nanoparticles, lifted-off Au-alkanethiolate thin films lack a measurable optical signature. We therefore devised fabrication, characterization, and simulation strategies by which to interrogate the nanoscale structure, chemical functionality, stoichiometry, and spectral signature of the supported Au-thiolate layers. The patterning of these layers laterally encodes their functionality, as demonstrated by a fluorescence-based approach that relies on dye-labeled complementary DNA hybridization. Supported thin Au films can be patterned via features on PDMS stamps (controlled contact), using patterned Au substrates prior to lift-off (e.g., selective wet etching), or by patterning alkanethiols on Au substrates to be reactive in selected regions but not others (controlled reactivity). In all cases, the regions containing Au-alkanethiolate layers have a sub-nanometer apparent height, which was found to be consistent with molecular dynamics simulations that predicted the removal of no more than 1.5 Au atoms per thiol, thus presenting a monolayer-like structure.

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

Nucleotide arrays require controlled surface densities and minimal nucleotide-substrate interactions to enable highly specific and efficient recognition by corresponding targets. We investigated chemical lift-off lithography with hydroxyl- and oligo(ethylene glycol)-terminated alkanethiol self-assembled monolayers as a means to produce substrates optimized for tethered DNA insertion into post-lift-off regions. Residual alkanethiols in the patterned regions after lift-off lithography enabled the formation of patterned DNA monolayers that favored hybridization with target DNA. Nucleotide densities were tunable by altering surface chemistries and alkanethiol ratios prior to lift-off. Lithography-induced conformational changes in oligo(ethylene glycol)-terminated monolayers hindered nucleotide insertion but could be used to advantage via mixed monolayers or double-lift-off lithography. Compared to thiolated DNA self-assembly alone or with alkanethiol backfilling, preparation of functional nucleotide arrays by chemical lift-off lithography enables superior hybridization efficiency and tunability.

Fabrication of High-Performance Ultrathin In2O3 Film Field-Effect Transistors and Biosensors Using Chemical Lift-Off Lithography.

We demonstrate straightforward fabrication of highly sensitive biosensor arrays based on field-effect transistors, using an efficient high-throughput, large-area patterning process. Chemical lift-off lithography is used to construct field-effect transistor arrays with high spatial precision suitable for the fabrication of both micrometer- and nanometer-scale devices. Sol-gel processing is used to deposit ultrathin (∼4 nm) In2O3 films as semiconducting channel layers. The aqueous sol-gel process produces uniform In2O3 coatings with thicknesses of a few nanometers over large areas through simple spin-coating, and only low-temperature thermal annealing of the coatings is required. The ultrathin In2O3 enables construction of highly sensitive and selective biosensors through immobilization of specific aptamers to the channel surface; the ability to detect subnanomolar concentrations of dopamine is demonstrated.

From the bottom up: dimensional control and characterization in molecular monolayers.

Self-assembled monolayers are a unique class of nanostructured materials, with properties determined by their molecular lattice structures, as well as the interfaces with their substrates and environments. As with other nanostructured materials, defects and dimensionality play important roles in the physical, chemical, and biological properties of the monolayers. In this review, we discuss monolayer structures ranging from surfaces (two-dimensional) down to single molecules (zero-dimensional), with a focus on applications of each type of structure, and on techniques that enable characterization of monolayer physical properties down to the single-molecule scale.

Subtractive patterning via chemical lift-off lithography.

Conventional soft-lithography methods involving the transfer of molecular "inks" from polymeric stamps to substrates often encounter micrometer-scale resolution limits due to diffusion of the transferred molecules during printing. We report a "subtractive" stamping process in which silicone rubber stamps, activated by oxygen plasma, selectively remove hydroxyl-terminated alkanethiols from self-assembled monolayers (SAMs) on gold surfaces with high pattern fidelity. The covalent interactions formed at the stamp-substrate interface are sufficiently strong to remove not only alkanethiol molecules but also gold atoms from the substrate. A variety of high-resolution patterned features were fabricated, and stamps were cleaned and reused many times without feature deterioration. The remaining SAM acted as a resist for etching exposed gold features. Monolayer backfilling into the lift-off areas enabled patterned protein capture, and 40-nanometer chemical patterns were achieved.