We designed and fabricated large arrays of polymer pens having sub-20 nm tips to perform chemical lift-off lithography (CLL). As such, we developed a hybrid patterning strategy called polymer-pen chemical lift-off lithography (PPCLL). We demonstrated PPCLL patterning using pyramidal and v-shaped polymer-pen arrays. Associated simulations revealed a nanometer-scale quadratic relationship between contact line widths of the polymer pens and two other variables: polymer-pen base line widths and vertical compression distances. We devised a stamp support system consisting of interspersed arrays of flat-tipped polymer pens that are taller than all other sharp-tipped polymer pens. These supports partially or fully offset stamp weights thereby also serving as a leveling system. We investigated a series of v-shaped polymer pens with known height differences to control relative vertical positions of each polymer pen precisely at the sub-20 nm scale mimicking a high-precision scanning stage. In doing so, we obtained linear-array patterns of alkanethiols with sub-50 nm to sub-500 nm line widths and minimum sub-20 nm line width tunable increments. The CLL pattern line widths were in agreement with those predicted by simulations. Our results suggest that through informed design of a stamp support system and tuning of polymer-pen base widths, throughput can be increased by eliminating the need for a scanning stage system in PPCLL without sacrificing precision. To demonstrate functional microarrays patterned by PPCLL, we inserted probe DNA into PPCLL patterns and observed hybridization by complementary target sequences.Keywords: alkanethiols; Chemical patterning; DNA hybridization; microcontact printing; nanolithography; soft lithography;

We report a facile, high-throughput soft lithography process that utilizes nanoscale channels formed naturally at the edges of microscale relief features on soft, elastomeric stamps. Upon contact with self-assembled monolayer (SAM) functionalized substrates, the roof of the stamp collapses, resulting in the selective removal of SAM molecules via a chemical lift-off process. With this technique, which we call self-collapse lithography (SCL), sub-30 nm patterns were achieved readily using masters with microscale features prepared by conventional photolithography. The feature sizes of the chemical patterns can be varied continuously from ∼2 μm to below 30 nm by decreasing stamp relief heights from 1 μm to 50 nm. Likewise, for fixed relief heights, reducing the stamp Young’s modulus from ∼2.0 to ∼0.8 MPa resulted in shrinking the features of resulting patterns from ∼400 to ∼100 nm. The self-collapse mechanism was studied using finite element simulation methods to model the competition between adhesion and restoring stresses during patterning. These results correlate well with the experimental data and reveal the relationship between the line widths, channel heights, and Young’s moduli of the stamps. In addition, SCL was applied to pattern two-dimensional arrays of circles and squares. These chemical patterns served as resists during etching processes to transfer patterns to the underlying materials (e.g., gold nanostructures). This work provides new insights into the natural propensity of elastomeric stamps to self-collapse and demonstrates a means of exploiting this behavior to achieve patterning via nanoscale chemical lift-off lithography.Keywords: Chemical lift-off lithography; nanolithography; self-collapse; soft lithography;

Monitoring dopamine and norepinephrine (or other structurally similar neurotransmitters) in the same brain region necessitates selective sensing. In this Viewpoint, we highlight electrochemical and optical strategies for advancing simultaneous real-time measurements of dopamine and norepinephrine transmission. The potential for DNA aptamers as recognition elements in the context of field-effect transistor sensing for selective and simultaneous neurotransmitter monitoring in vivo is also discussed.Keywords: Biosensors; CNiFERs; fast-scan cyclic voltammetry; frontal cortex; psychiatric disorders;

The demand for new strategies to combat debilitating psychiatric and neurodegenerative diseases necessitates revolutionizing our approaches to investigate the connectivity and function of neural circuits. To elucidate how alterations in neuronal networks, which function at nanoscale synapses, contribute to brain-related disorders, it will be essential to monitor chemical neurotransmission in vivo at the length and timescales pertinent to intrinsically encoded information (Andrews, 2013). Nonetheless, current approaches for sensing neurotransmitters are far removed from these scales needed to decode chemical information processing in neurocircuitry.To address the challenges of designing ultra-small, fast, highly selective, and multiplexed biosensors, we are investigating aptamers, which have emerged as alternatives to antibodies for molecular recognition. Aptamers are synthetic single-stranded DNA or RNA sequences that fold into unique three-dimensional structures to effect specific interactions with binding targets. Yet despite their promise, the elucidation of these rare nucleotide sequences is impeded by difficulties associated with producing screening substrates having highly controlled surface chemistries and characteristics (Vaish et al, 2010). Aptamers with picomolar to femtomolar dissociation constants exist but they are limited to sequences that recognize molecules significantly larger than neurotransmitters. To unleash the full potential of aptamers for in vivo neurotransmitter biosensing, we have invested a decade of research aimed at developing materials that enable high-affinity interactions between neurotransmitters tethered to optimized biospecific surfaces and nucleic acid libraries.These substrates, termed ‘neurochips’, are fabricated so as to create enhanced environments for molecular recognition by controlling essential parameters and reducing nonspecific binding (Figure 1). Neurochips selectively capture large biomolecule binding partners including antibodies, native G-protein-coupled receptors (Vaish et al, 2010), and nucleic acid aptamers. At present, we are using neurochips to identify rare nucleotides isolated from combinatorial libraries consisting of hundreds of billions of candidate sequences based on relative affinities for small-molecule neurotransmitter targets. We have also developed micro- to nanoscale surface patterning techniques (Liao et al, 2012) and used high-throughput microfluidics (Liao et al, 2013) to create multiplexed neurotransmitter substrates. A significant advantage of multiplexed patterning is the capacity to capture and to sort different neurotransmitter-specific aptamers side-by-side while providing opportunities to determine and to compare in situ binding affinities.The discovery of neurotransmitter aptamers will enable their functional integration into nanometer-diameter field-effect transistor (FET) nanowires, which will function as neurotransmitter recording elements (Figure 1). Devices patterned with aptamer-modified FETs will be used to carry out dynamic in vivo monitoring of neurotransmission with response times on the order of milliseconds (or faster) (Kim et al, 2015). When combined with appropriate passivation to suppress biofouling, microsensors that detect dopamine with sub-second temporal resolution have been shown to function over months in vivo in rats and mice (Clark et al, 2010). Thus, neurochips will enable the development of devices that will advance the understanding of the roles of small-molecule neurotransmitters in the complex landscape of brain interneuronal communication and dysfunction. Unraveling the emergent properties of integrated chemical neurotransmission associated with neural circuits using this approach will be advantageous for uncovering processes associated with cognition, emotion, and learning and memory.During the past 3 years, AMA has received compensation from Forest Laboratories (Actavis) for work as a consultant and from the American Chemical Society for work as Associate Editor of ACS Chemical Neuroscience, in addition to income from her primary employer (University of California, Los Angeles). NN declares that, except for graduate student stipends received from the University of California, Los Angeles, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.Funding from the CalBRAIN Neurotechnology Program and the Shirley and Stefan Hatos Foundation are gratefully acknowledged.

Large numbers of women undergo antidepressant treatment during pregnancy; however, long-term consequences for their offspring remain largely unknown. Rodents exposed to serotonin transporter (SERT)-inhibiting antidepressants during development show changes in adult emotion-like behavior. These changes have been equated with behavioral alterations arising from genetic reductions in SERT. Both models are highly relevant to humans yet they vary in their time frames of SERT disruption. We find that anxiety-related behavior and, importantly, underlying serotonin neurotransmission diverge between the two models. In mice, constitutive loss of SERT causes life-long increases in anxiety-related behavior and hyperserotonemia. Conversely, early exposure to the antidepressant escitalopram (ESC; Lexapro) results in decreased anxiety-related behavior beginning in adolescence, which is associated with adult serotonin system hypofunction in the ventral hippocampus. Adult behavioral changes resulting from early fluoxetine (Prozac) exposure were different from those of ESC and, although somewhat similar to SERT deficiency, were not associated with changes in hippocampal serotonin transmission in late adulthood. These findings reveal dissimilarities in adult behavior and neurotransmission arising from developmental exposure to different widely prescribed antidepressants that are not recapitulated by genetic SERT insufficiency. Moreover, they support a pivotal role for serotonergic modulation of anxiety-related behavior.

In vivo microdialysis is widely used to investigate how neurotransmitter levels in the brain respond to biologically relevant challenges. Here, we combined recent improvements in the temporal resolution of online sampling and analysis for serotonin with a brief high-K+ stimulus paradigm to study the dynamics of evoked release. We observed stimulated serotonin overflow with high-K+ pulses as short as 1 min when determined with 2-min dialysate sampling in ventral striatum. Stimulated serotonin levels in female mice during the high estrogen period of the estrous cycle were similar to serotonin levels in male mice. By contrast, stimulated serotonin overflow during the low estrogen period in female mice was increased to levels similar to those in male mice with local serotonin transporter (SERT) inhibition. Stimulated serotonin levels in mice with constitutive loss of SERT were considerably higher yet, pointing to neuroadaptive potentiation of serotonin release. When combined with brief K+ stimulation, fast microdialysis reveals dynamic changes in extracellular serotonin levels associated with normal hormonal cycles and pharmacologic vs genetic loss of SERT function.Keywords: escitalopram; K+-stimulated overflow; Mice; serotonin transporter; sex difference

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.Keywords: aptamer; biosensor; chemical lift-off lithography; dopamine; field-effect transistor; metal-oxide semiconductor; nanofabrication; nanotechnology; neurotransmitter; sensor; sol−gel chemistry;

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.Keywords: alkanethiol patterning; chemical lift-off lithography; DNA hybridization; nucleotide arrays; self-assembled monolayers;

The serotonin transporter (SERT), a primary target for many antidepressants, is expressed in the brain and also in peripheral blood cells. Although platelet SERT function is well accepted, lymphocyte SERT function has not been definitively characterized. Due to their small size, platelets often are found in peripheral blood mononuclear cell preparations aimed at isolating lymphocytes, monocytes, and macrophages. The presence of different cells makes it difficult to assign SERT expression and function to specific cell types. Here, we use flow cytometry and IDT307, a monoamine transporter substrate that fluoresces after uptake into cells, to investigate SERT function in lymphocyte and platelet populations independently, as well as simultaneously without prior isolation. We find that murine lymphocytes exhibit temperature-dependent IDT307 transport but uptake is independent of SERT. Lack of measurable SERT function in lymphocytes was corroborated by chronoamperometry using serotonin as a substrate. When we examined rhesus and human mixed blood cell populations, we found that platelets, and not lymphocytes, were primary contributors to SERT function. Overall, these findings indicate that lymphocyte SERT function is minimal. Moreover, flow cytometry, in conjunction with the fluorescent transporter substrate IDT307, can be widely applied to investigate SERT in platelets from populations of clinical significance.Keywords: chronoamperometry; depression; flow cytometry; Lymphocytes; platelets; Serotonin transporter

The complexities of the involvement of the serotonin transmitter system in numerous biological processes and psychiatric disorders is, to a substantial degree, attributable to the large number of serotonin receptor families and subtypes that have been identified and characterized for over four decades. Of these, the 5-HT1A receptor subtype, which was the first to be cloned and characterized, has received considerable attention based on its purported role in the etiology and treatment of mood and anxiety disorders. 5-HT1A receptors function both at presynaptic (autoreceptor) and postsynaptic (heteroreceptor) sites. Recent research has implicated distinct roles for these two populations of receptors in mediating emotion-related behavior. New concepts as to how 5-HT1A receptors function to control serotonergic tone throughout life were highlights of the proceedings of the 2012 Serotonin Club Meeting in Montpellier, France. Here, we review recent findings and current perspectives on functional aspects of 5-HT1A auto- and heteroreceptors with particular regard to their involvement in altered anxiety and mood states.Keywords: antidepressant; anxiety; autoreceptor; behavior; development; heteroreceptor; Serotonin

Online microdialysis is a sampling and detection method that enables continuous interrogation of extracellular molecules in freely moving subjects under behaviorally relevant conditions. A majority of recent publications using brain microdialysis in rodents report sample collection times of 20–30 min. These long sampling times are due, in part, to limitations in the detection sensitivity of high performance liquid chromatography (HPLC). By optimizing separation and detection conditions, we decreased the retention time of serotonin to 2.5 min and the detection threshold to 0.8 fmol. Sampling times were consequently reduced from 20 to 3 min per sample for online detection of serotonin (and dopamine) in brain dialysates using a commercial HPLC system. We developed a strategy to collect and to analyze dialysate samples continuously from two animals in tandem using the same instrument. Improvements in temporal resolution enabled elucidation of rapid changes in extracellular serotonin levels associated with mild stress and circadian rhythms. These dynamics would be difficult or impossible to differentiate using conventional microdialysis sampling rates.Keywords: behavior; circadian; knockout; mice; no net flux; serotonin transporter

Precise self-assembled monolayer chemistries and microfluidic technology are combined to create small-molecule biorecognition arrays. Small-molecule neurotransmitters or precursors are spatially encoded on monolayer-modified substrates. This platform enables multiplexed screening of G-protein-coupled receptors (GPCRs) from complex media via protein–ligand interactions. Preserving access to all epitopes of small molecules is critical for GPCR recognition. The ability to address multiple small molecules on solid substrates and to sort protein mixtures based on specific affinities is a critical step in creating biochips for proteomic applications.

The human serotonin transporter (SERT) gene possesses a 43-base pair (bp) insertion-deletion promoter polymorphism, the h5-HTTLPR. Genotype at this locus correlates with variation in anxiety-related personality traits and risk for major depressive disorder in many studies. Yet, the complex effects of the h5-HTTLPR, in combination with closely associated single-nucleotide polymorphisms (SNPs), continue to be debated. Moreover, although SERT is of high clinical significance, transporter function in vivo remains difficult to assess. Rhesus express a promoter polymorphism related to the h5-HTTLPR. The rh5-HTTLPR has been linked to differences in stress-related behavior and cognitive flexibility, although allelic variations in serotonin uptake have not been investigated. We studied the serotonin system as it relates to the 5-HTTLPR in rhesus peripheral blood cells. Sequencing of the rh5-HTTLPR revealed a 23-bp insertion, which is somewhat longer than originally reported. Consistent with previous reports, no SNPs in the rh5-HTTLPR and surrounding genomic regions were detected in the individuals studied. Reductions in serotonin uptake rates, cell surface SERT binding, and 5-hydroxyindoleacetic acid/serotonin ratios, but not SERT mRNA levels, were associated with the rh5-HTTLPR short allele. Thus, serotonin uptake rates are differentiable with respect to the 5-HTTLPR in an easily accessible native peripheral tissue. In light of these findings, we foresee that primary blood cells, in combination with high sensitivity functional measurements enabled by chronoamperometry, will be important for investigating alterations in serotonin uptake associated with genetic variability and antidepressant responsiveness in humans.

Chemical patterns prepared by self-assembly, combined with soft lithography or photolithography, are directly compared. Pattern fidelity can be controlled in both cases but patterning at the low densities necessary for small-molecule probe capture of large biomolecule targets is better accomplished using microcontact insertion printing (μCIP). Surfaces patterned by μCIP are used to capture biomolecule binding partners for the small molecules dopamine and biotin.

Voltammetry is widely used to investigate neurotransmission and other biological processes but is limited by poor chemical selectivity and fouling of commonly used carbon fiber microelectrodes (CFMs). We performed direct comparisons of three key coating materials purported to impart selectivity and fouling resistance to electrodes: Nafion, base-hydrolyzed cellulose acetate (BCA), and fibronectin. We systematically evaluated the impact on a range of electrode parameters. Fouling due to exposure to brain tissue was investigated using an approach that minimizes the use of animals while enabling evaluation of statistically significant populations of electrodes. We find that BCA is relatively fouling-resistant. Moreover, detection at BCA-coated CFMs can be tuned by altering hydrolysis times to minimize the impact on sensitivity losses while maintaining fouling resistance. Fibronectin coating is associated with moderate losses in sensitivity after coating and fouling. Nafion imparts increased sensitivity for dopamine and norepinephrine but not serotonin, as well as the anticipated selectivity for cationic neurotransmitters over anionic metabolites. Although Nafion has been suggested to resist fouling, both dip-coating and electrodeposition of Nafion are associated with substantial fouling, similar to levels observed at bare electrodes after exposure to brain tissue. Direct comparisons of these coatings identified unique electroanalytical properties of each that can be used to guide selection tailored to the goals and environment of specific studies.

We present a configuration for fluorescence spectroscopy that exploits the optical properties of semitransparent gold films and widely available instrumentation. This method enables monitoring of biomolecule interactions with small molecules tethered on substrates in multicomponent environments. The neurotransmitter serotonin (5-hydroxytryptamine) was covalently attached to self-assembled monolayers on thin gold films at low density to facilitate antibody recognition. Protein-binding studies were performed in a fluorescently labeled immunoassay format. We find that the use of this method enables evaluation of nonspecific binding and relative quantification of specific binding between competing binding partners. This fluorescence spectroscopy technique has the potential to assess biosensor or medical device responses in complex biological matrices.

We describe the self-assembly and chemical functionalization of oligo(ethylene glycol)alkanethiol (OEG) molecules. Insertion of OEGs into n-alkanethiolate monolayer matrices depends considerably on terminal functionality, unlike insertion of n-alkanethiols. Thus, inserted fractions of OEGs cannot be inferred from related systems, yet tuning, to some extent, is possible by controlling insertion parameters. Furthermore, while the in situ reactivities of dilute inserted carboxy- or amine-terminated OEGs versus n-alkanethiols protruding from the surrounding matrix are similar in amide bond formation reactions, complete monolayers of OEGs react to a greater extent compared to n-alkanethiols with similar terminal functionalities. We interpret these differences in terms of the reduced crystalline packing of terminal ethylene glycol groups of OEGs.

Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach.Keywords (keywords): 5-Hydroxytryptamine; chemical patterning; functionalized surfaces; membrane-associated receptors; receptor binding; self-assembled monolayers

Chemical patterns prepared by self-assembly, combined with soft lithography or photolithography, are directly compared. Pattern fidelity can be controlled in both cases but patterning at the low densities necessary for small-molecule probe capture of large biomolecule targets is better accomplished using microcontact insertion printing (μCIP). Surfaces patterned by μCIP are used to capture biomolecule binding partners for the small molecules dopamine and biotin.