Real-time detection of melatonin using fast-scan cyclic voltammetry

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Real-time detection of melatonin using fast-scan cyclic voltammetry Austin L. Hensley, Adam R. Colley, and Ashley Elizabeth Ross Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01976 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Analytical Chemistry

Real-time detection of melatonin using fast-scan cyclic voltammetry Austin L. Hensley, Adam R. Colley, and Ashley E. Ross* *Corresponding author 312 Clifton Ave 404 Crosley Tower Cincinnati, OH 45221-0172 Office #: 513-556-9314 Email: [email protected] Keywords: immunomodulator, indolamine, electrochemistry, lymphocytes, carbon-fiber microelectrodes

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Abstract: Melatonin is an important hormone whose functions span from regulating circadian rhythm in the brain to providing anti-inflammatory properties in the immune system. Melatonin secretion from the pineal gland is known; however, the mechanism of melatonin signaling in the immune system is not well understood. The lymph node is the hub of the immune system and melatonin secretion from lymphocytes was proposed to be an important source specifically for regulating cytokine secretion. Methods exist to quantify the concentration of melatonin within biological samples; however, they often suffer from either a lack of selectivity for melatonin over common biological interferences or temporal resolution which is not amenable to measuring real-time signaling dynamics. Here, we have characterized an electrochemical method for optimal melatonin detection with subsecond resolution using fast-scan cyclic voltammetry at carbonfiber microelectrodes. The oxidation peaks detected for melatonin were at 1.0 V, 1.1 V and 0.6 V. Evidence for electrode fouling of the tertiary peak was present, therefore an optimized waveform was developed scanning from 0.2 V to 1.3 V at 600 V/s. The optimized waveform eliminated the detection of fouling products on the electrode with a 24 ± 10 nM limit of detection. Melatonin was distinguished between biological interferences and co-detection with the major synthetic precursor, serotonin, was possible. This method was used to detect melatonin in live lymph node slices and provides the first real-time measurements within the lymph node using FSCV. Real-time detection of melatonin dynamics could provide useful information on the mechanism of immunomodulation during inflammatory disease.


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Introduction:

Melatonin is a small signaling molecule which has been shown to regulate circadian rhythm1,2, mitochondrial homeostasis3–5, oxidative stress6,7, and immunity8–10. For decades, the source of melatonin was mostly attributed to the pineal gland; however, melatonin synthesis machinery and the receptors, MT1 and MT2, have been discovered in many tissues11 and cells including the retina12, skin13–15, spleen16, lymphocytes17, and gastrointestinal tract9,18. Immunederived melatonin has become an increasingly important source, specifically because of its suspected role in buffering the immune system.8

8

Mast cells, which are recruited to the lymph

node during infection19, can degranulate and release melatonin20 to modulate inflammation. Melatonin may act as an immunostimulant during basal or immunosuppression to pre-activate the immune system or may act as an anti-inflammatory during a chronic intensified immune response.8,21–24 Despite the growing evidence for melatonin-modulated immunity, few methods exist to measure melatonin secretion directly in real-time with high specificity. The ability to measure melatonin signaling in real-time during inflammation could provide useful information about the mechanism of immunomodulation. This paper describes a new analytical method to detect melatonin using fast-scan cyclic voltammetry (FSCV) coupled to carbon-fiber microelectrodes which can be used to directly measure melatonin signaling within the immune system in real-time. Generating an immune response is dependent on a precise spatial arrangement of cells within the lymph node25,26 yet techniques to quantitate analytes, like melatonin, within intact immune tissue are limited. Maintaining the integrity of the immune tissue during detection is beneficial for fully understanding the mechanism of immunity. Homogenized tissue samples and cell lysates have been analyzed for melatonin using HPLC27–29; however, spatially resolved detection

is not possible and signaling kinetics cannot be measured. In vivo sampling of

melatonin using microdialysis coupled to HPLC in the brain30 provides improved spatial



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resolution but is not amenable to sampling within tiny immune organs like the lymph node due to the large probe size (50-200 µm). Likewise, immunoassays offer excellent limits of detection but do not provide temporally or spatially resolved analysis in intact tissue and often suffer from cross-reactivity from structurally similar analytes.29,31 Methods which allow spatially and temporally resolved detection within intact tissues are necessary for understanding the mechanisms of melatonin signaling. Electrochemical methods to detect melatonin have been proposed.32–35 Melatonin is an electroactive indolamine, and is easily oxidized at carbon-based electrodes.32 Slow scan cyclic voltammetry at glassy carbon electrodes was used to propose an oxidation scheme for melatonin32; however, glassy carbon electrodes are large in size which aren’t amenable to intissue implantation within small organs. In addition, slow scan techniques do not provide the necessary temporal resolution for detection of rapid fluctuations in tissue. Similarly, square wave voltammetry at boron-doped diamond electrodes have been used to detect melatonin in urine samples but this technique suffers from similar temporal and spatial limitations.34 Recently, amperometry at boron-doped diamond electrodes was used to co-detect melatonin and serotonin signaling within excised mouse colon samples18,36. Amperometry has excellent temporal resolution and sensitivity; however, selectivity is limited from interferences at similar oxidizing potentials. Fast-scan cyclic voltammetry at carbon-fiber microelectrodes is a technique which provides subsecond temporal resolution with increased analyte selectivity due to a fingerprint cyclic voltammogram and has been extensively used to study neurotransmission in the brain.37–39 Carbon-fiber microelectrodes are 7-µm in diameter which permits spatially discrete regions of tissue to be sampled from with limited tissue damage.40 To our knowledge, electrochemical detection of melatonin has not been characterized using FSCV. Here, we present an optimized method to detect melatonin with subsecond temporal resolution using FSCV at carbon-fiber microelectrodes. Melatonin oxidation products have been shown to electropolymerize in solution32 which can lead to strongly adsorbed products that foul 4 ACS Paragon Plus Environment

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the electrode surface so a waveform to limit electrode fouling while maintaining low limits of detection was developed. The stability of the optimized waveform for melatonin detection was verified which permits its use to detect dynamic fluctuations in tissue over time. Co-detection of melatonin with the synthesis precursors serotonin or N-acetyl-serotonin was possible. Melatonin was also detected in live lymph node slices to prove its utility for in tissue analysis. Overall, this paper provides the first method to detect subsecond fluctuations in melatonin signaling in the lymph node and will be useful in the future for monitoring melatonin regulated immunity. Methods: Reagents: All reagents were purchased from Fisher Scientific (USA) unless otherwise noted. Dopamine, serotonin, histamine, and N-acetyl-serotonin (Sigma-Aldrich, St. Louis MO) were dissolved in 0.1 M HCl for 10 mM stock solutions. Melatonin and 6-hydroxymelatonin (Sigma Aldrich) were dissolved in 70% ethanol for 10 mM stock solutions. Dopamine, serotonin, histamine, N-acetyl-serotonin and melatonin stock solutions were stored at 4º C while 6hydroxymelatonin was stored at -20º C. Stock solutions were diluted daily in Tris buffer for testing. The Tris buffer consists of 15 mM Tris (hydroxymethyl) aminomethane, 1.25 mM NaH2PO4, 2.0 mM Na2SO4, 3.25 mM KCl, 140 mM NaCl, 1.2 mM CaCl2 dehydrate, and 1.2 mM MgCl2 hexahydrate at pH 7.4. For slice experiments, calibrations and data were collected using 1x DPBS (Hyclone, GE, USA). All aqueous solutions were made with deionized water (Milli-Q, Millipre, Billerica MA). Fast-scan cyclic voltammetry experiments: Fast-scan cyclic voltammograms were collected using the WaveNeuro with a 5 megaohm headstage (Pine Instruments, Durham NC). Data was collected using HDCV software (UNC-Chapel Hill, Mark Wightman) and a computer interface board (National Instruments PC1e-6363, Austin TX). For the traditional waveform, the electrode was scanned from -0.4 to 1.3 V (vs Ag/AgCl) and back with a 400 V/s scan rate and a repetition rate of 10 Hz. The modified waveform for melatonin scanned from 0.2 V to 1.3 V and back at 600 V/s and a repetition rate of 10 Hz. A 5 kHz low pass filter was used for experiments with 5 ACS Paragon Plus Environment


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scan rates greater than 400 V/s. All data was background subtracted to remove any nonFaradaic currents by averaging 10 CVs from approximately 2 seconds before the analysis point. Electrodes were calibrated using flow injection analysis as previously reported41 with a flow rate of 1 mL/min using a Fusion 200 Two-Channel Chemyx Syringe pump (Stafford, TX). 3-second injections of the compounds were made to mimic transient changes. Carbon-fiber microelectrode fabrication: Cylindrical carbon-fiber microelectrodes were fabricated from 7-µm T-650 carbon fibers (Gift from Mitsubishi Chemical Carbon Fiber and Composites Inc., Sacramento CA). Carbon-fibers were vacuum aspirated into 1.2 x 0.68 mm glass capillaries (A&M Systems, Sequim WA) and were pulled into two using a vertical Narishige PE-22 Electrode Puller (Tokyo, Japan). Extended carbon-fibers were cut 50-100 µm from the glass seal using a scalpel under a microscope (Fisher Education). Electrodes were soaked in isopropyl alcohol for at least 10 minutes prior to use and backfilled with 1M KCl. Animal experiments: All animal work was approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Female C57BL/6 mice between the ages of 6-8 weeks old (Charles River, USA) were housed in a vivarium and given food and water ab libitum. Lymph node slices were harvested and collected as previously reported.42,43 On the day of the experiment, mice were anesthetized with isoflurane (Henry Shrein, USA) and euthanized by cervical dislocation. The mesenteric lymph nodes were removed and placed in ice-cold DPBS without calcium or magnesium (Hyclone) with 2 % heat-inactivated fetal bovine serum (premium grade FBS, VWR ≤ 20 EU/mL) for approximately 2 min. To slice, the lymph nodes were embedded in 6 % low melting point agarose (Lonza, NJ USA) prepared in 1x DPBS, and placed on ice to gel. An 8 mm tissue punch (Robbins Instruments, NJ USA) was used to obtain a block of agarose containing the lymph node. The block was mounted onto the stage using superglue, and sliced to 300-µm thick using a Leica VT1000S vibratome (Chicago, IL, USA). The vibratome was set to a speed of 90 and frequency of 3. Slices were handled with a camel-hair paint brush (TedPella, CA USA) to limit damage. Slices embedded in agarose were placed in a 6-well 6 ACS Paragon Plus Environment

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culture plate containing “complete RPMI”. Complete RPMI contained: RPMI (Hyclone) supplemented with 10 % FBS, 1x L-glutamine (Gibco), 50 U/mL Pen/Strep (Gibco), 50 µM betamercaptoethanol (Gibco), 1 mM sodium pyruvate (Hyclone), 1x non-essential amino acids (Hyclone), and 20 mM HEPES (Gibco). Slices were housed in a sterile incubator set to 37 ºC with 5 % CO2 for approximately 1-hr prior to the experiment. For details on the slice experiments, see the Supplemental Information.

Statistics: All statistics were performed in GraphPad Prism 7 (GraphPad Software Inc., La Jolla CA). Statistical p values were considered significant at the 95% confidence level (p

Real-time detection of melatonin using fast-scan cyclic voltammetry

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