Analyzing Spin Selectivity in DNA-Mediated Charge Transfer <i>via</i> Fluorescence Microscopy

Understanding spin-selective interactions between electrons and chiral molecules is critical to elucidating the significance of electron spin in biological processes and to assessing the potential of chiral assemblies for organic spintronics applications. Here, we use fluorescence microscopy to visualize the effects of spin-dependent charge transport in self-assembled monolayers of double-stranded DNA on ferromagnetic substrates. Patterned DNA arrays provide background regions for every measurement to enable quantification of substrate magnetization-dependent fluorescence due to the chiral-induced spin selectivity effect. Fluorescence quenching of photoexcited dye molecules bound within DNA duplexes is dependent upon the rate of charge separation/recombination upon photoexcitation and the efficiency of DNA-mediated charge transfer to the surface. The latter process is modulated using an external magnetic field to switch the magnetization orientation of the underlying ferromagnetic substrates. We discuss our results in the context of the current literature on the chiral-induced spin selectivity effect across various systems

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-<i>threo</i>-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

High-Affinity Nucleic-Acid-Based Receptors for Steroids

Artificial receptors for hydrophobic molecules usually have moderate affinities and limited selectivities. We describe three new classes of high affinity hydrophobic receptors for nonaromatic steroids based on deoxyribonucleotides, obtained through five high stringency selections coupled with tailored counter-selections. The isolation of multiple classes of high affinity steroid receptors demonstrates the surprising breadth of moderately sized hydrophobic binding motifs (<40 nucleotides) available to natural nucleic acids. Studies of interactions with analogs indicate that two classes, four-way junctions and 4XG_N motifs, comprise receptors with shapes that prevent binding of specific steroid conjugates used in counter-selections. Furthermore, they strongly prefer nonhydroxylated steroid cores, which is typical for hydrophobic receptors. The third new class accommodates hydroxyl groups in high-affinity, high-selectivity binding pockets, thus reversing the preferences of the first two classes. The high-affinity binding of aptamers to targets efficiently inhibits double-helix formation in the presence of the complementary oligonucleotides. The high affinity of some of these receptors and tailored elimination of binding through counter-selections ensures that these new aptamers will enable clinical chemistry applications

Polyserotonin Nanoparticles as Multifunctional Materials for Biomedical Applications

Serotonin-based nanoparticles represent a class of previously unexplored multifunctional nanoplatforms with potential biomedical applications. Serotonin, under basic conditions, self-assembles into monodisperse nanoparticles <i>via</i> autoxidation of serotonin monomers. To demonstrate potential applications of polyserotonin nanoparticles for cancer therapeutics, we show that these particles are biocompatible, exhibit photothermal effects when exposed to near-infrared radiation, and load the chemotherapeutic drug doxorubicin, releasing it contextually and responsively in specific microenvironments. Quantum mechanical and molecular dynamics simulations were performed to interrogate the interactions between surface-adsorbed drug molecules and polyserotonin nanoparticles. To investigate the potential of polyserotonin nanoparticles for <i>in vivo</i> targeting, we explored their nano–bio interfaces by conducting protein corona experiments. Polyserotonin nanoparticles had reduced surface–protein interactions under biological conditions compared to polydopamine nanoparticles, a similar polymer material widely investigated for related applications. These findings suggest that serotonin-based nanoparticles have advantages as drug-delivery platforms for synergistic chemo- and photothermal therapy associated with limited nonspecific interactions

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. Alternately, 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 prefunctionalized 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

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 <i>via</i> 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

Advancing Biocapture Substrates via Chemical Lift-Off Lithography

Creating small-molecule-functionalized platforms for high-throughput screening or biosensing applications requires precise placement of probes on solid substrates and the ability to capture and to sort targets from multicomponent samples. Here, chemical lift-off lithography was used to fabricate large-area, high-fidelity patterns of small-molecule probes. Lift-off lithography enables biotin–streptavidin patterned recognition with feature sizes ranging from micrometers to below 30 nm. Subtractive patterning via lift-off facilitated insertion of a different type of molecule and, thus, multiplexed side-by-side placement of small-molecule probes such that binding partners were directed to cognate probes from solution. Small molecules mimicking endogenous neurotransmitters were patterned using lift-off lithography to capture native membrane-associated receptors. We characterized patterning of alkanethiols that self-assemble on Au having different terminal functional groups to expand the library of molecules amenable to lift-off lithography enabling a wide range of functionalization chemistries for use with this simple and versatile patterning method

Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer–target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics

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