Simultaneous serotonin and dopamine monitoring across timescales by rapid pulse voltammetry with partial least squares regression.

Many voltammetry methods have been developed to monitor brain extracellular dopamine levels. Fewer approaches have been successful in detecting serotonin in vivo. No voltammetric techniques are currently available to monitor both neurotransmitters simultaneously across timescales, even though they play integrated roles in modulating behavior. We provide proof-of-concept for rapid pulse voltammetry coupled with partial least squares regression (RPV-PLSR), an approach adapted from multi-electrode systems (i.e., electronic tongues) used to identify multiple components in complex environments. We exploited small differences in analyte redox profiles to select pulse steps for RPV waveforms. Using an intentionally designed pulse strategy combined with custom instrumentation and analysis software, we monitored basal and stimulated levels of dopamine and serotonin. In addition to faradaic currents, capacitive currents were important factors in analyte identification arguing against background subtraction. Compared to fast-scan cyclic voltammetry-principal components regression (FSCV-PCR), RPV-PLSR better differentiated and quantified basal and stimulated dopamine and serotonin associated with striatal recording electrode position, optical stimulation frequency, and serotonin reuptake inhibition. The RPV-PLSR approach can be generalized to other electrochemically active neurotransmitters and provides a feedback pipeline for future optimization of multi-analyte, fit-for-purpose waveforms and machine learning approaches to data analysis.

Detecting DNA and RNA and Differentiating Single-Nucleotide Variations via Field-Effect Transistors.

We detect short oligonucleotides and distinguish between sequences that differ by a single base, using label-free, electronic field-effect transistors (FETs). Our sensing platform utilizes ultrathin-film indium oxide FETs chemically functionalized with single-stranded DNA (ssDNA). The ssDNA-functionalized semiconducting channels in FETs detect fully complementary DNA sequences and differentiate these sequences from those having different types and locations of single base-pair mismatches. Changes in charge associated with surface-bound ssDNA vs double-stranded DNA (dsDNA) alter FET channel conductance to enable detection due to differences in DNA duplex stability. We illustrate the capability of ssDNA-FETs to detect complementary RNA sequences and to distinguish from RNA sequences with single nucleotide variations. The development and implementation of electronic biosensors that rapidly and sensitively detect and differentiate oligonucleotides present new opportunities in the fields of disease diagnostics and precision medicine.

Scalable Fabrication of Quasi-One-Dimensional Gold Nanoribbons for Plasmonic Sensing.

Plasmonic nanostructures have a wide range of applications, including chemical and biological sensing. However, the development of techniques to fabricate submicrometer-sized plasmonic structures over large scales remains challenging. We demonstrate a high-throughput, cost-effective approach to fabricate Au nanoribbons via chemical lift-off lithography (CLL). Commercial HD-DVDs were used as large-area templates for CLL. Transparent glass slides were coated with Au/Ti films and functionalized with self-assembled alkanethiolate monolayers. Monolayers were patterned with lines via CLL. The lifted-off, exposed regions of underlying Au were selectively etched into large-area grating-like patterns (200 nm line width; 400 nm pitch; 60 nm height). After removal of the remaining monolayers, a thin In2O3 layer was deposited and the resulting gratings were used as plasmonic sensors. Distinct features in the extinction spectra varied in their responses to refractive index changes in the solution environment with a maximum bulk sensitivity of ∼510 nm/refractive index unit. Sensitivity to local refractive index changes in the near-field was also achieved, as evidenced by real-time tracking of lipid vesicle or protein adsorption. These findings show how CLL provides a simple and economical means to pattern large-area plasmonic nanostructures for applications in optoelectronics and sensing.

Kappa Opioid Receptors Drive a Tonic Aversive Component of Chronic Pain.

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the κ opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative real-time-PCR, florescence in situ hybridization, Western blotting and GTPgS autoradiography an upregulation of expression and the function of κ opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared with surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward-related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KORloxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.SIGNIFICANCE STATEMENT We show that KORs are sufficient to drive the tonic-aversive component of chronic pain; the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high comorbidity with chronic pain) and substance abuse. Indeed, coexisting psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).

Large-Area, Ultrathin Metal-Oxide Semiconductor Nanoribbon Arrays Fabricated by Chemical Lift-Off Lithography.

Nanoribbon- and nanowire-based field-effect transistors (FETs) have attracted significant attention due to their high surface-to-volume ratios, which make them effective as chemical and biological sensors. However, the conventional nanofabrication of these devices is challenging and costly, posing a major barrier to widespread use. We report a high-throughput approach for producing arrays of ultrathin (∼3 nm) In2O3 nanoribbon FETs at the wafer scale. Uniform films of semiconducting In2O3 were prepared on Si/SiO2 surfaces via a sol-gel process prior to depositing Au/Ti metal layers. Commercially available high-definition digital versatile discs were employed as low-cost, large-area templates to prepare polymeric stamps for chemical lift-off lithography, which selectively removed molecules from self-assembled monolayers functionalizing the outermost Au surfaces. Nanoscale chemical patterns, consisting of one-dimensional lines (200 nm wide and 400 nm pitch) extending over centimeter length scales, were etched into the metal layers using the remaining monolayer regions as resists. Subsequent etch processes transferred the patterns into the underlying In2O3 films before the removal of the protective organic and metal coatings, revealing large-area nanoribbon arrays. We employed nanoribbons in semiconducting FET channels, achieving current on-to-off ratios over 107 and carrier mobilities up to 13.7 cm2 V-1 s-1. Nanofabricated structures, such as In2O3 nanoribbons and others, will be useful in nanoelectronics and biosensors. The technique demonstrated here will enable these applications and expand low-cost, large-area patterning strategies to enable a variety of materials and design geometries in nanoelectronics.

Self-Collapse 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.

Polymer-Pen Chemical Lift-Off Lithography.

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.

Double-Sided Opportunities Using Chemical Lift-Off Lithography.

We discuss the origins, motivation, invention, development, applications, and future of chemical lift-off lithography, in which a specified pattern of a self-assembled monolayer is removed, i.e., lifted off, using a reactive, patterned stamp that is brought into contact with the monolayer. For Au substrates, this process produces a supported, patterned monolayer of Au on the stamp in addition to the negative pattern in the original molecular monolayer. Both the patterned molecular monolayer on the original substrate and the patterned supported metal monolayer on the stamp are useful as materials and for further applications in sensing and other areas. Chemical lift-off lithography effectively lowers the barriers to and costs of high-resolution, large-area nanopatterning. On the patterned monolayer side, features in the single-nanometer range can be produced across large (square millimeter or larger) areas. Patterns smaller than the original stamp feature sizes can be produced by controlling the degree of contact between the stamp and the lifted-off monolayer. We note that this process is different than conventional lift-off processes in lithography in that chemical lift-off lithography removes material, whereas conventional lift-off is a positive-tone patterning method. Chemical lift-off lithography is in some ways similar to microtransfer printing. Chemical lift-off lithography has critical advantages in the preparation of biocapture surfaces because the molecules left behind are exploited to space and to orient functional(ized) molecules. On the supported metal monolayer side, a new two-dimensional material has been produced. The useful important chemical properties of Au (vis-à-vis functionalization with thiols) are retained, but the electronic and optical properties of bulk Au or even Au nanoparticles are not. These metal monolayers do not quench excitation and may be useful in optical measurements, particularly in combination with selective binding due to attached molecular recognition elements. In contrast to materials such as graphene that have bonding confined to two dimensions, these metal monolayers can be straightforwardly patterned-by patterning the stamp, the initial monolayer, or the initial substrate. Well-developed thiol-Au and related chemistries can be used on the supported monolayers. As there is little quenching and photoabsorption, spectroscopic imaging methods can be used on these functionalized materials. We anticipate that the properties of the metal monolayers can be tuned by varying the chemical, physical, and electronic connections made by and to the supporting molecular layers. That is, the amount of charge in the layer can be determined by controlling the density of S-Au (or other) connections and the molecular backbone and functionality, which determine the strength with which the chemical contact withdraws charge from the metal. This process should work for other coinage-metal substrates and additional systems where the binding of the outermost layers to the substrate is weaker than the molecule-substrate attachment.

A Model For Teaching Advanced Neuroscience Methods: A Student-Run Seminar to Increase Practical Understanding and Confidence.

Neuroscience doctoral students must master specific laboratory techniques and approaches to complete their thesis work (hands-on learning). Due to the highly interdisciplinary nature of the field, learning about a diverse range of methodologies through literature surveys and coursework is also necessary for student success (hands-off learning). Traditional neuroscience coursework stresses what is known about the nervous system with relatively little emphasis on the details of the methods used to obtain this knowledge. Furthermore, hands-off learning is made difficult by a lack of detail in methods sections of primary articles, subfield-specific jargon and vague experimental rationales. We designed a student-taught course to enable first-year neuroscience doctoral students to overcome difficulties in hands-off learning by introducing a new approach to reading and presenting primary research articles that focuses on methodology. In our literature-based course students were encouraged to present a method with which they had no previous experience. To facilitate weekly discussions, "experts" were invited to class sessions. Experts were advanced graduate students who had hands-on experience with the method being covered and served as discussion co-leaders. Self-evaluation worksheets were administered on the first and last days of the 10-week course and used to assess students' confidence in discussing research and methods outside of their primary research expertise. These evaluations revealed that the course significantly increased the students' confidence in reading, presenting and discussing a wide range of advanced neuroscience methods.

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2.

Human TPH2 (hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37 °C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. We report the functional implications of a polymorphic human tryptophan hydroxylase-2 gene associated with depression and bipolar disorder. The polymorphic enzyme (serine-41 converted to tyrosine) has increased activity, but decreased enzyme stability and serotonin production. Moreover, cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation of the mutant enzyme is decreased suggesting modified regulation of the S41Y variant leading to altered serotonin.

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.

Common and rare alleles of the serotonin transporter gene, SLC6A4, associated with Tourette's disorder.

To evaluate the hypothesis that functionally over-expressing alleles of the serotonin transporter (SERT) gene (solute carrier family 6, member 4, SLC6A4) are present in Tourette's disorder (TD), just as we previously observed in obsessive compulsive disorder (OCD), we evaluated TD probands (N = 151) and controls (N = 858). We genotyped the refined SERT-linked polymorphic region 5-HTTLPR/rs25531 and the associated rs25532 variant in the SLC6A4 promoter plus the rare coding variant SERT isoleucine-to-valine at position 425 (I425V). The higher expressing 5-HTTLPR/rs25531 LA allele was more prevalent in TD probands than in controls (χ(2) = 5.75; P = 0.017; odds ratio [OR], 1.35); and, in a secondary analysis, surprisingly, it was significantly more frequent in probands who had TD alone than in those who had TD plus OCD (Fisher's exact test; P = 0.0006; OR, 2.29). Likewise, the higher expressing LAC haplotype (5-HTTLPR/rs25531/rs25532) was more frequent in TD probands than in controls (P = 0.024; OR, 1.33) and also in the TD alone group versus the TD plus OCD group (P = 0.0013; OR, 2.14). Furthermore, the rare gain-of-function SERT I425V variant was observed in 3 male siblings with TD and/or OCD and in their father. Thus, the cumulative count of SERT I425V becomes 1.57% in OCD/TD spectrum conditions versus 0.15% in controls, with a recalculated, family-adjusted significance of χ(2) = 15.03 (P < 0.0001; OR, 9.0; total worldwide genotyped, 2914). This report provides a unique combination of common and rare variants in one gene in TD, all of which are associated with SERT gain of function. Thus, altered SERT activity represents a potential contributor to serotonergic abnormalities in TD. The present results call for replication in a similarly intensively evaluated sample. © 2013 Movement Disorder Society.

Males With Traumatic Lower Limb Loss Differ in Body Fat Distribution Compared to Those Without Limb Loss.

OBJECTIVES: The primary objective was to examine body fat composition in males with traumatic lower limb loss and a comparison group without limb loss. A secondary objective was to determine if there are differences in body fat composition by traumatic limb loss level. RESEARCH METHODS AND PROCEDURES: Cross-sectional analyses of baseline in-person intake data was completed at a large military medical center. Data were collected from 2011 to 2020, and analysis was conducted in 2020. Participants (n = 89) included males who sustained traumatic lower limb loss (n = 50) and an age-matched comparison group without limb loss (n = 39). Mean age of participants was 36.0 ± 13.2 years. Main outcomes measured included age, height, body mass index, weight, body fat mass and percent, android fat mass and percent, gynoid fat mass and percent, and android/gynoid percent fat ratio. Differences between groups were assessed using t-tests or Mann-Whitney U tests. Differences between limb loss levels were assessed using one-way ANOVA or Wilcoxon signed-rank test. RESULTS: Body fat percent (P = .001), gynoid fat percent (P = .010), android fat mass (P = .01), and percent (P = .02) were higher in the group with limb loss. There were no differences in body fat composition between limb loss levels (P > .05). CONCLUSION: Males with traumatic lower limb loss had a higher body fat percent compared to those without limb loss. Given higher body fat composition in individuals with limb loss and the relationship between body fat composition and cardiovascular disease risk, including body composition analysis with clinical screening could identify changes and allow for early intervention.

A functional group-guided approach to aptamers for small molecules.

Aptameric receptors are important biosensor components, yet our ability to identify them depends on the target structures. We analyzed the contributions of individual functional groups on small molecules to binding within 27 target-aptamer pairs, identifying potential hindrances to receptor isolation-for example, negative cooperativity between sterically hindered functional groups. To increase the probability of aptamer isolation for important targets, such as leucine and voriconazole, for which multiple previous selection attempts failed, we designed tailored strategies focused on overcoming individual structural barriers to successful selections. This approach enables us to move beyond standardized protocols into functional group-guided searches, relying on sequences common to receptors for targets and their analogs to serve as anchors in regions of vast oligonucleotide spaces wherein useful reagents are likely to be found.

Technology Roadmap for Flexible Sensors.

Humans rely increasingly on sensors to address grand challenges and to improve quality of life in the era of digitalization and big data. For ubiquitous sensing, flexible sensors are developed to overcome the limitations of conventional rigid counterparts. Despite rapid advancement in bench-side research over the last decade, the market adoption of flexible sensors remains limited. To ease and to expedite their deployment, here, we identify bottlenecks hindering the maturation of flexible sensors and propose promising solutions. We first analyze challenges in achieving satisfactory sensing performance for real-world applications and then summarize issues in compatible sensor-biology interfaces, followed by brief discussions on powering and connecting sensor networks. Issues en route to commercialization and for sustainable growth of the sector are also analyzed, highlighting environmental concerns and emphasizing nontechnical issues such as business, regulatory, and ethical considerations. Additionally, we look at future intelligent flexible sensors. In proposing a comprehensive roadmap, we hope to steer research efforts towards common goals and to guide coordinated development strategies from disparate communities. Through such collaborative efforts, scientific breakthroughs can be made sooner and capitalized for the betterment of humanity.

Flexible and Implantable Polyimide Aptamer-Field-Effect Transistor Biosensors.

Monitoring neurochemical signaling across time scales is critical to understanding how brains encode and store information. Flexible (vs stiff) devices have been shown to improve in vivo monitoring, particularly over longer times, by reducing tissue damage and immunological responses. Here, we report our initial steps toward developing flexible and implantable neuroprobes with aptamer-field-effect transistor (FET) biosensors for neurotransmitter monitoring. A high-throughput process was developed to fabricate thin, flexible polyimide probes using microelectromechanical-system (MEMS) technologies, where 150 flexible probes were fabricated on each 4 in. Si wafer. Probes were 150 µm wide and 7 µm thick with two FETs per tip. The bending stiffness was 1.2 × 10-11 N·m2. Semiconductor thin films (3 nm In2O3) were functionalized with DNA aptamers for target recognition, which produces aptamer conformational rearrangements detected via changes in FET conductance. Flexible aptamer-FET neuroprobes detected serotonin at femtomolar concentrations in high-ionic strength artificial cerebrospinal fluid. A straightforward implantation process was developed, where microfabricated Si carrier devices assisted with implantation such that flexible neuroprobes detected physiological relevant serotonin in a tissue-hydrogel brain mimic.

Optogenetic Stimulation of Midbrain Dopamine Neurons Produces Striatal Serotonin Release.

Targeting neurons with light-driven opsins is widely used to investigate cell-specific responses. We transfected midbrain dopamine neurons with the excitatory opsin Chrimson. Extracellular basal and stimulated neurotransmitter levels in the dorsal striatum were measured by microdialysis in awake mice. Optical activation of dopamine cell bodies evoked terminal dopamine release in the striatum. Multiplexed analysis of dialysate samples revealed that the evoked dopamine was accompanied by temporally coupled increases in striatal 3-methoxytyramine, an extracellular dopamine metabolite, and in serotonin. We investigated a mechanism for dopamine-serotonin interactions involving striatal dopamine receptors. However, the evoked serotonin associated with optical stimulation of dopamine neurons was not abolished by striatal D1- or D2-like receptor inhibition. Although the mechanisms underlying the coupling of striatal dopamine and serotonin remain unclear, these findings illustrate advantages of multiplexed measurements for uncovering functional interactions between neurotransmitter systems. Furthermore, they suggest that the output of optogenetic manipulations may extend beyond opsin-expressing neuronal populations.

Wearable aptamer-field-effect transistor sensing system for noninvasive cortisol monitoring.

Wearable technologies for personalized monitoring require sensors that track biomarkers often present at low levels. Cortisol­a key stress biomarker­is present in sweat at low nanomolar concentrations. Previous wearable sensing systems are limited to analytes in the micromolar-millimolar ranges. To overcome this and other limitations, we developed a flexible field-effect transistor (FET) biosensor array that exploits a previously unreported cortisol aptamer coupled to nanometer-thin-film In2O3 FETs. Cortisol levels were determined via molecular recognition by aptamers where binding was transduced to electrical signals on FETs. The physiological relevance of cortisol as a stress biomarker was demonstrated by tracking salivary cortisol levels in participants in a Trier Social Stress Test and establishing correlations between cortisol in diurnal saliva and sweat samples. These correlations motivated the development and on-body validation of an aptamer-FET array­based smartwatch equipped with a custom, multichannel, self-referencing, and autonomous source measurement unit enabling seamless, real-time cortisol sweat sensing.

Head-to-head comparisons of carbon fiber microelectrode coatings for sensitive and selective neurotransmitter detection by voltammetry.

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.

Thin gold film-assisted fluorescence spectroscopy for biomolecule sensing.

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.