Innovative Approaches and Novel Materials for Resolving NeurochemicalEvents in Real-Time Using Fast-Scan Cyclic Voltammetry

Andreas C. Schmidt and Leslie A. Sombers, Department of ChemistryCollaborations with the Department of Biomedical Engineering and Materials Science Engineering, North Carolina State University

Carbon-fiber ultramicroelectrodes are the preferred sensing substrate for thereal-time detection of in vivo neurotransmitter release using fast-scan cyclic voltammetry. The application of this technology to dopaminergic studies of neurological disease states has significantly advanced our understanding of molecular mechanisms underlying these problems; however, far less work has been done to significantly advance the detection capabilities of the technique itself over recent years. This research sought to broaden the abilities of fast-scan cyclic voltammetry through the usage of innovative new materials, such as microelectrodes made purely of carbon nanotube yarns, as well as through the development of analyte specific waveforms that allow for a reliable detection of difficult to detect neuropeptergic fluctuations in real-time. This work has improved the sensitivity, selectivity, reproducibility and reliability of fast-scan cyclic voltammetry and has been successfully tested in living brain tissue.

http://www.yamato-net.co.jp/english/products/bio/mapanalyzer.htm

Modified Sawhorse Wavefrom advantages: Able to selectively distinguish mENK in complex mixtures containing common in-vivo interferents Significantly reduces fouling - more reproducible results Decreases the current contributed by interferents Increases the current from analyte of interest Increases the separation of peak potentials, facilitating analyte identification CNTy-D offer higher senesitivity and selectivity Customizable size offers a variety of future approaches Superior electrode material to traditionally used fibers The combination of these approaches provide a foundation for studying a variety of tyrosine- containing peptides, previously impossible to reproducibly detect using an electroanalytical approach in living tissue

-0.4 1.3 Potential (V)

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rent

(nA)

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Sample Loop

BufferedElectrolyte

Waste

Injection Port

Waste

ElectrochemicalCell

Working Electrode

ReferenceElectrode

6-PortHPLC Valve

Bipotentiostator

Universal Electrochemical

Instrument

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400 V/s

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100 msec8.5 msec

NH3+

OH

OHDopamine (DA)

NH3+

O

ODopamine-o-quinione

-2 e-

+2 e-

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1.2 V

0.6 V

0.0

Modified Sawhorse Waveform (MSW)3 msec

26.5 msec

•Holding Potential: -0.2V•Peak Potential: +1.2V•Period: 26.5 msec

•Applied Frequency: 10Hz

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•Holding Potential: -0.2V•Peak Potential: +1.2V•Period: 7.0 msec

•Applied Frequency: 10Hz

• Broad peaks• Evidence of electrode fouling (extended signal) (*); causes reproducibility problems

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Conventional Triangular Waveform (TW)

Carbon-Fiber Microelectrode (CFME)300μM length; 7μM diameter

Mixture of Common Interferents

• The peak from dopamine (above) dominates• All other interferents contribute more current• Cannot distinguish mENK peak(s)

• Single peak• No current detected at potentials below our analyte’s oxidation

• Defined peaks• No evidence of fouling

*

• Dopamine (above) signal is minimized• Improved peak resolution from intereferents• Easy to pick out mENK peak for analysis

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• Many peaks in CV• Current contribution throughout entire potential range

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Colorplot of mENK 1µM mENK

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Colorplot of mENK

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1 μM dopamine10 μM ascorbic acid+0.1 pH shift

500nM mENK1 μM dopamine10 μM ascorbic acid+0.1 pH shift

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Predicted vs. Actual Concentrationof mENK in complex mixture

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750nM mENK mixed withcommon interferents: A. 500 nM Dopamine B. 10 μM Ascrobic Acid C. +0.1 pH shift

Using Principal ComponentRegression (PCR), mENKwas accurately quantifiedin a complex mixture ofvarying concentrations of the interferents

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Reproducible measurements are onlypossible with the MSW waveform.

Linear calibration plot showssensitivity of 13nA/1μM

Cyclic Voltammogram (CV)

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NH3+

OH

OHDopamine (DA)

NH3+

O

ODopamine-o-quinione

-2 e-

+2 e-

linear voltammogram

y-axis: potential (V)x-axis: time (sec)z-axis: current (nA)aligned with time

Chemical Selectivity (MSW)

Right: Voltammograms for several analytes commonly encountered in brain tissue.

1µM mENK

Mixture of Common Interferents1 μM dopamine only 1 μM dopamine only

Reproducibility and Sensitivity (MSW)

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400V/s 800V/s

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CNTy

-DCo

nven

tiona

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Ty-D

Conv

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Dopamine

Dopamine

H2O2 Adenosine

AdenosineH2O2

Serotonin

Serotonin

Ascorbic Acid

Ascorbic Acid

DOPAC

DOPAC

DA (1

)

HPO

(100)

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osine

(2)

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onin

(1)

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rbic A

cid (2

00)

DOPA

C (30

)0

50pA/µm2 Current Density:Other Analytes

25pA/µm2

n.s.*** *** * *

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HPO

(100)

Aden

osine

(2)

Serot

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(1)

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rbic A

cid (2

00)

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C (30

)0.0

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CNTy-DConventionalcarbon-fiber

Correlation Coeff.vs. Dopamine

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Left: CV of M-ENK detection combining the MSW and CNTy-D

Right: CNTy-D electrodes are more sensitive than carbon-fiber microelectrodes (CFME) for M-ENK detection.

Opioid Peptides exist at very low concentrations, �uctuate rapidly, oxidizeat higher potentials than interferents such as dopamine, and foul the electrode surface upon oxidation making electrochemical measurementsdi�cult and irreproducible.

Research Goals: 1. Create a custom waveform that cleans the electrode surface, increases selectivity, and reduces the contribution from other analytes. 2. Use Carbon nanotubes to create an electrode that is more sensitive and selective than traditional carbon �bers.

Research Challenge

Interpreting the Data with Colorplots

Electroanalytical Technique

Introduction

Analytical Equipment

How does an Analyte-Specific Waveform Compare to a Conventional Waveform?

Carbon Nanotube Yarn - Disk (CNTy-D) Electrodes

Performance Assessment

Combining the New Technologies

AcknowledgementsDr. McCarty (BME)Dr. Roberts (Chemistry)Lars Dunaway (Chemistry)Dr. Zhu, Dr. Wang (MSE) - CNT yarnChuck Mooney (AIF) - Imaging

Conclusions

NIH R01-NS076772 to LASNSF CAREER CHE7151264

Schmidt, A. C. et al. ACS Nano. 2013, 7 (9), 7864-7873

Schmidt, A. C. et al. ACS Nano. 2013, 7 (9), 7864-7873

3-D colorplot

Middle: (A) Representative voltammograms for physiological DA concentrations. (B) Calibration curve. (C) Anodic current increases linearly with scan rate when detecting 1µM dopamine using CNTy-D electrodes. (D) Voltammograms collected at scan rates ranging from 100 - 800 V/s.

Left: Scanning electron micrographs of a CNTy-D electrodes. (A) Spun MWNTs from a continuous yarn. (B) A single CNTy-D electrode. (C) Detailed image of CNT surface and glass seal of the CNTy-D electrode.

Top: (A) CNTy-D electrodes are more capable of separating various analytes due to sharper peaks. (B) CNTy-D electrodes are also more sensitive for the detection of a variety of other analytes.

Bottom: Results collected in living brain tissue. (A) Current over time at the oxidation potential of dopamine (inset is the collected dopamine CV). (B) Colorplot of in tissue electrical stimulation result.