Three-Dimensional-Printed Electrochemical Multiwell Plates for Monitoring Food Intolerance from Intestinal Organoids.

Common symptoms of food intolerance are caused by chemical components within food that have a pharmacological activity to alter the motility of the gastrointestinal tract. Food intolerance is difficult to diagnose as it requires a long-term process of eliminating foods that are responsible for gastrointestinal symptoms. Enterochromaffin (EC) cells are key intestinal epithelium cells that respond to luminal chemical stimulants by releasing 5-HT. Changes in 5-HT levels have been shown to directly alter the motility of the intestinal tract. Therefore, a rapid approach for monitoring the impact of chemicals in food components on 5-HT levels can provide a personalized insight into food intolerance and help stratify diets. Within this study, we developed a three-dimensional (3D)-printed electrochemical multiwell plate to determine changes in 5-HT levels from intestinal organoids that were exposed to varying chemical components found in food. The carbon black/poly-lactic acid (CB/PLA) electrodes had a linear range in physiological concentrations of 5-HT (0.1-2 µM) with a limit of detection of 0.07 µM. The electrodes were stable for monitoring 5-HT overflow from intestinal organoids. Using the electrochemical multiwell plate containing intestinal organoids, increases in 5-HT were observed in the presence of 0.1 mM cinnamaldehyde and 10 mM quercetin but reduction in 5-HT levels was observed in 1 mM sorbitol when compared to control. These changes in the presence of chemicals commonly found in food were verified with ex vivo ileum tissue measurements using chromatography and amperometry with boron-doped diamond electrodes. Overall, our 3D electrochemical multiwell plate measurements with intestinal organoids highlight an approach that can be a high-throughput platform technology for rapid screening of food intolerance to provide personalized nutritional diet.

3D Printed Electrochemical Sensors.

Three-dimensional (3D) printing has recently emerged as a novel approach in the development of electrochemical sensors. This approach to fabrication has provided a tremendous opportunity to make complex geometries of electrodes at high precision. The most widely used approach for fabrication is fused deposition modeling; however, other approaches facilitate making smaller geometries or expanding the range of materials that can be printed. The generation of complete analytical devices, such as electrochemical flow cells, provides an example of the array of analytical tools that can be developed. This review highlights the fabrication, design, preparation, and applications of 3D printed electrochemical sensors. Such developments have begun to highlight the vast potential that 3D printed electrochemical sensors can have compared to other strategies in sensor development.

Ex Vivo Electrochemical pH Mapping of the Gastrointestinal Tract in the Absence and Presence of Pharmacological Agents.

Ex vivo pH profiling of the upper gastrointestinal (GI) tract (of a mouse), using an electrochemical pH probe, in both the absence and presence of pharmacological agents aimed at altering acid/bicarbonate production, is reported. Three pH electrodes were first assessed for suitability using a GI tract biological mimic buffer solution containing 0.5% mucin. These include a traditional glass pH probe, an iridium oxide (IrOx)-coated electrode (both operated potentiometrically), and a quinone (Q) surface-integrated boron-doped diamond (BDD-Q) electrode (voltammetric). In mucin, the time scale for both IrOx and glass to provide a representative pH reading was in the ∼100's of s, most likely due to mucin adsorption, in contrast to 6 s with the BDD-Q electrode. Both the glass and IrOx pH electrodes were also compromised on robustness due to fragility and delamination (IrOx) issues; contact with the GI tissue was an experimental requirement. BDD-Q was deemed the most appropriate. Ten measurements were made along the GI tract, esophagus (1), stomach (5), and duodenum (4). Under buffer only conditions, the BDD-Q probe tracked the pH from neutral in the esophagus to acidic in the stomach and rising to more alkaline in the duodenum. In the presence of omeprazole, a proton pump inhibitor, the body regions of the stomach exhibited elevated pH levels. Under melatonin treatment (a bicarbonate agonist and acid inhibitor), both the body of the stomach and the duodenum showed elevated pH levels. This study demonstrates the versatility of the BDD-Q pH electrode for real-time ex vivo biological tissue measurements.

Fast serotonin voltammetry as a versatile tool for mapping dynamic tissue architecture: I. Responses at carbon fibers describe local tissue physiology.

It is important to monitor serotonin neurochemistry in the context of brain disorders. Specifically, a better understanding of biophysical alterations and associated biochemical functionality within subregions of the brain will enable better of understanding of diseases such as depression. Fast voltammetric tools at carbon fiber microelectrodes provide an opportunity to make direct evoked and ambient serotonin measurements in vivo in mice. In this study, we characterize novel stimulation and measurement circuitries for serotonin analyses in brain regions relevant to psychiatric disease. Evoked and ambient serotonin in these brain areas, the CA2 region of the hippocampus and the medial prefrontal cortex, are compared to ambient and evoked serotonin in the substantia nigra pars reticulata, an area well established previously for serotonin measurements with fast voltammetry. Stimulation of a common axonal location evoked serotonin in all three brain regions. Differences are observed in the serotonin release and reuptake profiles between these three brain areas which we hypothesize to arise from tissue physiology heterogeneity around the carbon fiber microelectrodes. We validate this hypothesis mathematically and via confocal imaging. We thereby show that fast voltammetric methods can provide accurate information about local physiology and highlight implications for chemical mapping. Cover Image for this issue: doi: 10.1111/jnc.14739.

Determination of tryptophan metabolism from biological tissues and fluids using high performance liquid chromatography with simultaneous dual electrochemical detection.

Serotonin and kynurenine are formed following metabolism of the essential amino acid tryptophan. Both molecules play important biological roles and the balance of how tryptophan metabolism varies to either the serotonin or kynurenine pathway may provide key insight into the inflammatory status of the biological region. At present complex chromatographic methods are utilised which predominately focus on either monitoring analytes in the serotonin or kynurenine pathway rather than both. Our study develops a simple yet robust methodology for the monitoring of tryptophan metabolism. We utilised isocratic reverse phase high-performance liquid chromatography with simultaneously dual electrochemical detection. This approach allowed for separation of co-eluted analytes and identification of analytes from both pathways within 14 minutes. For all analytes, limits of detection were <35 nM. No crosstalk was observed when dual simultaneous detection was conducted in a radial flow cell. Responses from the hippocampus, blood and ileum mucosa highlighted that each region had a varying ratio of serotonin to kynurenine pathway, indicating varied approaches to tryptophan metabolism. The developed method can monitor how the metabolism of tryptophan varies between the two pathways which can provide insight into the inflammatory state of reach region with age and disease.

Voltammetric evidence for discrete serotonin circuits, linked to specific reuptake domains, in the mouse medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is an important brain region, that controls a variety of behavioral and functional outputs. As an important step in characterizing mPFC functionality, in this paper we focus on chemically defining serotonin transmission in this area. We apply cutting-edge analytical methods, fast-scan cyclic voltammetry (FSCV) and fast-scan controlled adsorption cyclic voltammetry (FSCAV), pioneered in our laboratory, for the first real-time in vivo analysis of serotonin in the mPFC. In prior in vivo work in the substantia nigra, pars reticulata, we found that our sub-second measurements of a single evoked serotonin release were subject to two clearance mechanisms. These mechanisms were readily modeled via Uptake 1, mediated by the serotonin transporters (SERTs), and Uptake 2, mediated by monoamine transporters (dopamine transporters (DATs), norepinephrine transporters (NETs), and organic cation transporters (OCTs)). Here in the mPFC, for the first time to our knowledge, we observe two release events in response to a single stimulation of the medial forebrain bundle (MFB). Of particular note is that each response is tied to a discrete reuptake profile comprising both Uptake 1 and 2. We hypothesize that two distinct populations of serotonin axons traverse the MFB and terminate in different domains with specific reuptake profiles. We test and confirm this hypothesis using a multifaceted pharmacological, histological and mathematical approach. We thus present evidence for a highly elaborate biochemical organization that regulates serotonin chemistry in the mPFC. This knowledge provides a solid foundation on which to base future studies of the involvement of the mPFC in brain function and behavior.

In Vivo Ambient Serotonin Measurements at Carbon-Fiber Microelectrodes.

The mechanisms that control extracellular serotonin levels in vivo are not well-defined. This shortcoming makes it very challenging to diagnose and treat the many psychiatric disorders in which serotonin is implicated. Fast-scan cyclic voltammetry (FSCV) can measure rapid serotonin release and reuptake events but cannot report critically important ambient serotonin levels. In this Article, we use fast-scan controlled adsorption voltammetry (FSCAV), to measure serotonin's steady-state, extracellular chemistry. We characterize the "Jackson" voltammetric waveform for FSCAV and show highly stable, selective, and sensitive ambient serotonin measurements in vitro. In vivo, we report basal serotonin levels in the CA2 region of the hippocampus as 64.9 ± 2.3 nM (n = 15 mice, weighted average ± standard error). We electrochemically and pharmacologically verify the selectivity of the serotonin signal. Finally, we develop a statistical model that incorporates the uncertainty in in vivo measurements, in addition to electrode variability, to more critically analyze the time course of pharmacological data. Our novel method is a uniquely powerful analysis tool that can provide deeper insights into the mechanisms that control serotonin's extracellular levels.

Regrowth of Serotonin Axons in the Adult Mouse Brain Following Injury.

It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests.

A voltammetric and mathematical analysis of histaminergic modulation of serotonin in the mouse hypothalamus.

Histamine and serotonin are neuromodulators which facilitate numerous, diverse neurological functions. Being co-localized in many brain regions, these two neurotransmitters are thought to modulate one another's chemistry and are often implicated in the etiology of disease. Thus, it is desirable to interpret the in vivo chemistry underlying neurotransmission of these two molecules to better define their roles in health and disease. In this work, we describe a voltammetric approach to monitoring serotonin and histamine simultaneously in real time. Via electrical stimulation of the axonal bundles in the medial forebrain bundle, histamine release was evoked in the mouse premammillary nucleus. We found that histamine release was accompanied by a rapid, potent inhibition of serotonin in a concentration-dependent manner. We developed mathematical models to capture the experimental time courses of histamine and serotonin, which necessitated incorporation of an inhibitory receptor on serotonin neurons. We employed pharmacological experiments to verify that this serotonin inhibition was mediated by H3 receptors. Our novel approach provides fundamental mechanistic insights that can be used to examine the full extent of interconnectivity between histamine and serotonin in the brain. Histamine and serotonin are co-implicated in many of the brain's functions. In this paper, we develop a novel voltammetric method for simultaneous real-time monitoring of histamine and serotonin in the mouse premammillary nucleus. Electrical stimulation of the medial forebrain bundle evokes histamine and inhibits serotonin release. We show voltammetrically, mathematically, and pharmacologically that this serotonin inhibition is H3 receptor mediated.

In vivo histamine voltammetry in the mouse premammillary nucleus.

Histamine plays a major role in the mediation of allergic reactions such as peripheral inflammation. This classical monoamine is also a neurotransmitter involved in the central nervous system but its role in this context is poorly understood. Studying histamine neurotransmission is important due to its implications in many neurological disorders. The sensitivity, selectivity and high temporal resolution of fast scan cyclic voltammetry (FSCV) offer many advantages for studying electroactive neurotransmitters. Histamine has previously been studied with FSCV; however, the lack of a robust Faradaic electrochemical signal makes it difficult to selectively identify histamine in complex media, as found in vivo. In this work, we optimize an electrochemical waveform that provides a stimulation-locked and unique electrochemical signal towards histamine. We describe in vitro waveform optimization and a novel in vivo physiological model for stimulating histamine release in the mouse premammillary nucleus via stimulation of the medial forebrain bundle. We demonstrate that a robust signal can be used to effectively identify histamine and characterize its in vivo kinetics.