Amine-functionalized carbon-fiber microelectrodes for enhanced ATP detection with fast-scan cyclic voltammetry

Abstract

Here, we provide evidence that functionalizing the carbon-fiber surface with amines significantly improves direct electrochemical adenosine triphosphate (ATP) detection with fast-scan cyclic voltammetry (FSCV). ATP is an important extracellular signaling molecule throughout the body and can function as a neurotransmitter in the brain. Several methods have been developed over the years to monitor and quantitate ATP signaling in cells and tissues; however, many of them are limited in temporal resolution or are not capable of measuring ATP directly. FSCV at carbon-fiber microelectrodes is a widely used technique to measure neurotransmitters in real-time. Many electrode treatments have been developed to study the interaction of cationic compounds like dopamine at the carbon surface yet studies investigating how to improve anionic compounds, like ATP, at the carbon fiber surface are lacking. In this work, carbon-fibers were treated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) which reacts with carboxylic acid groups on the carbon surface followed by reaction with ethylenediamine (EDA) to produce NH2-functionalized carbon surfaces. Overall, we a 5.2 ± 2.5-fold increase in ATP current with an approximately 9-fold increase in amine functionality, as analyzed by X-ray Photoelectron Spectroscopy, on the carbon surface was observed after modification with EDC-EDA. This provides evidence that amine-rich surfaces improve interactions with ATP on the surface. This study provides a detailed analysis of ATP interaction at carbon surfaces and ultimately a method to improve direct and rapid neurological ATP detection in the future.

Figure 1.

ATP is not easily oxidized at unmodified carbon-fiber microelectrodes using fast-scan cyclic voltammetry. (A) Oxidation scheme for ATP (I, II, III, denotes three oxidation products). The waveform for FSCV detection of ATP starts at a −0.4 V holding potential, scanned to a 1.45 V switching potential, and back at a rate of 400 V/s and 10 Hz frequency. (B) An example of a “good” false-color plot for 1 μM ATP and the (C) Current vs. voltage (CV). The primary and secondary oxidation peaks are identified. The primary peak is on the back scan (red), and secondary oxidation peak appears on the forward scan (blue) similar to prior work detecting adenosine with FSCV.

Figure 2.

Simplified schematic of the modification procedure for EDC/EDA-modified carbon-fibers.

Figure 3.

The oxidation pretreatment method impacts the increase in oxidative ATP current after EDC/EDA modification. (A) Treating fibers with nitric acid prior to EDC/EDA modification significantly impacts ATP detection more than O₂ plasma and electrochemical preconditioning (one-way ANOVA with Bonferroni post-test, p < 0.0001, n = 4–16). (B) The temperature of the 70% HNO₃ reflux affects surface oxidation which ultimately impacts the degree of EDC/EDA modification. A temperature of 110 °C is optimal for functionalizing the surface with enough carboxyl groups and results in significantly more oxidative ATP current after EDC/EDA modification (one-way ANOVA with Bonferroni post-test, p < 0.0001, n = 7) (C) Example cyclic voltammograms for 5 μM ATP before EDC/EDA (HNO₃-treated fiber, red) and after (dotted black trace) demonstrating a clear increase in current.

Figure 4

The temperature of nitric acid treatment impacts the degree of carboxyl functionality on the carbon fiber surface. (A) The XPS survey of carbon fibers without (bare CF, black) acid treatment and after acid treatment at varying temperatures. (B-E) C1s XPS peaks for (B) bare carbon fiber and (C-E) acid-treated carbon fibers treated at varying temperatures. An increase in oxide functionality is observed as a function of acid temperature.

Figure 5.

The EDC/EDA modification procedure introduces amine groups on the carbon-fiber surface. (A) Survey of an acid-treated carbon-fiber after EDC/EDA modification. A clear N1s peak is observed. (B) Deconvoluting the N1s peak demonstrates that 87% of the N1s peak is amine groups.

Fig 6.

Functional group type on the electrode surface impacts detection. Average detected current for 5 μM ATP is lower on COOH-modified electrodes compared to bare carbon-fibers (A); however, the average current for DA is higher on COOH-modified electrodes (B). The difference in average current for both ATP and DA are not significantly different (unpaired t-test, p < 0.05, n = 7). The ratio of current before and after EDC/EDA modification shows a 5.2 ± 2.3 -fold increase in ATP with a 0.97± 0.1 -fold change in DA demonstrating that amines do not significantly impact DA interaction at the electrode (C). The difference in current enhancement is significantly different (nonpaired-test, p <0.0001, n = 7).

Figure 7.

EDC/EDA modified carbon-fibers are significantly more sensitivity to ATP. (A) Calibration curves for an unmodified CF (black) compared to a EDC/EDA-modified CF (red). The EDC/EDA-modified carbon-fibers increased sensitivity to ATP by 2.7-fold more than unmodified carbon fiber (n = 16 modified, n = 4 unmodified). (B) Example CV’s comparing 300 nM ATP before and after EDC/EDA modification. 300 nM is undetectable at bare carbon-fibers.

Figure 8.

Detection of ATP within brain tissue using the EDC/EDA modified electrodes. Exogenous deposition is indicated by the blue arrow on the false color plot (top). The current vs. time trace is shown above the color plot. 150 μM ATP was used for deposition within tissue. Both the primary (1°, 1.2 V) and secondary (2°, 1.0 V) oxidation peaks are present on the CV for ATP (bottom). Picospritzing often causes extra peaks due to the pressure ejection near the surface of the electrode.