A novel biosensor with high signal-to-noise ratio for real-time measurement of dopamine levels in vivo

Abstract

Fast-scan cyclic voltammetry (FSCV) is an established method for measuring dopamine (DA) levels in the brain in real time. However, it is difficult to discriminate DA from other monoamines such as serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE). We report a novel DA-specific biosensor consisting of a carbon-fiber electrode coated with an ion-exchange membrane, a layer containing monoamine oxidase B, and a cellulose membrane. We performed FSCV using the probe to monitor the amount of DA in vitro and in vivo. First, we measured currents in vitro in phosphate-buffered saline as we added one micromole each of DA, 5-HT, and NE. The results confirmed that the biosensor selectively detected DA. Next, we implanted the probe in the striatum of male rats to investigate whether it could selectively detect changes in the DA content in vivo. The probe detected both the tonic change induced by methamphetamine administration and the phasic change induced by electrical stimulation of the medial forebrain bundle. In contrast, the electrode in the 6-hydroxydopamine-lesioned striatum did not respond to systemic selective serotonin or serotonin/norepinephrine reuptake inhibitors, confirming its selectivity. Furthermore, the probe in the striatum could still detect changes in the DA level 1 week after electrode implantation. The results suggest that the novel biosensor can measure real-time changes in DA levels in vivo with a relatively high signal-to-noise ratio.

Keywords: electrode; fast-scan cyclic voltammetry; monoamine oxidase.

Figure 1

(a,b) Photographs of the electrode (a) and its tip (b). (c) Schematic of the recording electrode. A carbon microfiber is sealed in a glass capillary with 100 μm of its length exposed, and attached to the implantation connector. The exposed tip is coated with a double membrane comprising Nafion and monoamine oxidase (MAO)‐B–diluted cellulose

Figure 2

Discriminative capacity of the novel electrode with and without the double membrane in vitro. (a,b) I‐T plots for the addition of dopamine (DA), serotonin (5‐HT), and norepinephrine (NE) using (a) the bare electrode and (b) the coated electrode. (c,d) Representative, background‐subtracted cyclic voltammograms of DA, 5‐HT, and NE by (c) the noncoated and (d) the coated electrode. (e) Relative amplitude of the currents for DA, 5‐HT, and NE at the peak for DA. Student t test. * p < .001. Values are shown as mean ± standard error of the mean (SEM)

Figure 3

Confirmation of the efficiency and stability of the monoamine oxidase (MAO)‐B coating on the probe in vitro. (a–c) I‐T plots for the addition of dopamine (DA), serotonin (5‐HT), and norepinephrine (NE) using (a) the MAO‐B–free double membrane–coated electrodes; (b) the MAO‐B–impregnated, non–cross‐linked membrane–coated electrodes; and (c) the MAO‐B–impregnated, cross‐linked membrane–coated electrodes. (d–f) Representative, background‐subtracted cyclic voltammograms of DA, 5‐HT, and NE by (d) the MAO‐B–free membrane–coated electrodes, (e) the non–cross‐linked MAO‐B–coated electrodes, and (f) the cross‐linked MAO‐B–coated electrode. (g) Relative amplitudes of the currents for monoamines, other neurotransmitters, precursors, and metabolites of DA, corresponding to the peak for DA. TYR = tyrosine; DOPAC = 3,4‐dihydroxyphenylacetic acid; L‐DOPA = L‐3,4‐dihydroxyphenylalanine; EPI = epinephrine; Ach = acetylcholine; GABA = 4‐aminobutanoic acid. One‐way ANOVA with post hoc Tukey test. * p < .05. ** p < .001. Values are shown as mean ± standard error of the mean (SEM)

Figure 4

Chronic measurements in vitro. (a) I‐T and (b) I‐V representative plots for the addition of dopamine (DA), serotonin (5‐HT), and norepinephrine (NE) using the cross‐linked, monoamine oxidase (MAO)‐B–impregnated membrane–coated electrodes 7 days after the initial measurements. (c) The absolute amplitudes of the currents for monoamines, other neurotransmitters, DA precursors, and metabolites at the peak for DA. Values are shown as mean ± standard error of the mean (SEM)

Figure 5

Positive‐control experiments in vivo. (a,b) Fast‐scan cyclic voltammetry (FSCV) in the rat striatum following administration of methamphetamine: (a) the I‐T plot and (b) the I‐V plot at 1,800 s post administration. (c,d) FSCV analysis during stimulation of the medial forebrain bundle: (c) the I‐T plot and (d) the I‐V plot at current peak

Figure 6

Negative‐control experiments in vivo. The rats were prepared by lesioning the substantia nigra. Fast‐scan cyclic voltammetry (FSCV) analyses were carried out following the administration of (a,b) a serotonin selective reuptake inhibitor or (c,d) a serotonin/norepinephrine reuptake inhibitor

Figure 7

Chronic measurements in vivo. (a) I‐T and (b) I‐V plots obtained in rat striatum by fast‐scan cyclic voltammetry (FSCV) assay with the novel biosensor, shortly after administration of methamphetamine at 7 days post implantation