Voltammetry is uniquely suited for the study of the dynamics of intercellular communication due to its high temporal and spatial resolution and excellent sensitivity. Voltammetry techniques are widely used for in vivo
measurements in anesthesized1–3
and awake animals,4,5 ex vivo
measurements in brain slices6–8
and synaptosomes,9–11 in vitro
measurements from peripheral cells,12,13
and for interrogating neurotransmitter release from single cells.14,15
Moreover, voltammetric methods, including amperometry, have been used to investigate a variety of neurochemicals such as dopamine,4,16,17
choline and acetylcholine26
with the aim of understanding chemical neurotransmission and its role in normal and altered brain function.
Measurements of neurotransmitters by voltammetry are predominantly carried out using carbon fiber microelectrodes (CFMs).27–30
These microelectrodes are widely used due to the commercial availability of small-diameter carbon fibers (5–30 µm) and readily accessible fabrication methods. Discrete measurements, even in highly heterogeneous tissues or distinct brain nuclei have been made using CFMs.1,2,16,31,32
Furthermore, small-diameter CFMs produce considerably less tissue damage than microdialysis probes having diameters >200 µm.33–35
Nonetheless, the investigation of biogenic amines and other biological signaling molecules using CFMs suffers from problems associated with poor chemical selectivity and sensor fouling.12,36,37
Hydroxyl groups on carbon fiber surfaces, in combination with an sp2
-hybridized composition, contribute to the adsorption of small polar neurotransmitters and metabolites, their oxidation products, and large biomolecules.38–41
Irreversible adsorption leads to fouling of electrode surfaces, which results in uncertainty in identifying and quantifying analytes.
To compensate for fouling, post-calibration of CFMs is often carried out.18,31,32,42
However, the trajectories of electrode responses over the course of experiments are unknown.2,43,44
Post calibration alone or averaging of pre- and post-calibration is likely to lead to over- or underestimation of analyte concentrations depending on the sensitivity of an electrode at the time of each measurement. Problems associated with precise and accurate detection are further compounded by low transmitter concentrations and interference by other high concentration electroactive compounds found in complex biological environments.45,46
Moreover, dopamine, norepinephrine, serotonin, ascorbate (ASC), and neurotransmitter metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA), all oxidize in a narrow potential window complicating selective neurotransmitter detection. Methods such as principal regression analysis have been used to distinguish some analytes among pairs of electroactive species.47
However, this approach has not gained widespread use due to difficulties associated with evaluating complex calibration sets, i.e.
, calibrations of different neurochemicals, and the probability that electrode sensitivities to and redox potentials of various components change during an experiment due to the adsorption of oxidation products20,48,49
The most commonly used method for optimizing CFMs involves the application of selectively permeable electrode coatings that allow diffusion of analytes of interest to electrode surfaces, while simultaneously minimizing the adsorption/detection of interferents. Many different materials have been reported to enhance selectivity and to reduce fouling at CFMs. These include Nafion,45
base-hydrolyzed cellulose acetate (BCA),51
and carbon nanotubes.2,54
Nafion is commonly used due to its ease of deposition on CFM surfaces and its cation selective permeability. Previous studies have also suggested that Nafion has fouling resistant properties.55
Base-hydrolyzed cellulose has been used as a fouling resistant coating material for CFMs due to its ability to exclude large biomolecules.51
The highly biocompatible nature of fibronectin, in addition to its chemical conductivity, has resulted in its use as a coating material for biosensors,50,56
although fibronectin has not previously been investigated as a surface modification for CFMs.
Although many different coatings have been employed in voltammetry studies, there is little information directly comparing these materials and specifically with regard to monoamine neurotransmitter detection. Consequently, we systematically investigated three CFM coatings: Nafion, base-hydrolyzed cellulose acetate, and fibronectin. We compared dip-coating and electro-deposition for the application of Nafion. We also investigated three different hydrolysis times for BCA. Our goals were to evaluate the effects of these different coating materials and protocols on CFM sensitivity, selectivity, and biofouling with the aim of determining performance in the context of monoamine neurotransmitter sensing by fast-cyclic voltammetry (FCV).