Selective Detection of Oxytocin on Diamond Electrodes in Preparation for Making In Vivo Measurements

Abstract

<Introduction> It has recently been revealed that oxytocin (OT), a peptide hormone known for its role in lactation and parturition, works as a neurotransmitter as well. Due to a series of findings of positive effects on social behaviors such as trust in human, oxytocin has been of keen interest to neuroscientists. What is required now for further understanding of oxytocin science is a real-time measurement of oxytocin in vivo. On the other hand, in the region where oxytocin is secreted in the hypothalamus, another peptide, vasopressin (VP), is also secreted. Vasopressin is also a nonapeptide with a quite similar structure to oxytocin. Therefore, selective measurement of oxytocin and vasopressin is inevitable. Electrochemical detection allows a real-time measurement in millisecond order with high sensitivity. Compared to conventional electrodes, boron-doped diamond (BDD) electrode has a variety of outstanding properties such as wide potential window and low background current, which have led to a number of reports about sensitive measurement of substances which cannot be detected by other electrodes. Using BDD microelectrodes, in vivomeasurement of biomolecules have been achieved and applied to medical and physiological studies. In addition, surface termination of BDD can be changed from original state (hydrogen termination) to oxygen termination by some oxidation treatment, which enables selective measurement. In the present study, electrochemical detection of oxytocin was investigated using BDD electrodes. Two types of BDD, as-deposited (AD-BDD) and anodically-oxidized (AO-BDD), were used in order to achieve selective measurement. <Experiments> BDD electrodes were prepared by growing polycrystalline BDD thin films on silicon substrates or needle tungsten wires using microwave plasma-assisted chemical vapor deposition (MPCVD) system. Electrochemical measurements were conducted in a three-electrode system with BDD, platinum, and Ag/AgCl (KCl saturated) electrode as a working electrode, counter electrode, and reference electrode, respectively. Phosphate buffer saline (PBS) and Tris buffer were used as buffer solution after adjusted to pH 7.4. Surface transformation of BDD electrodes to oxygen termination was conducted on AD-BDD by means of anodic oxidation of 3.0 V application for 20 minutes in PBS. <Results and discussion>The electrochemical behavior of oxytocin was studied using AD-BDD. Cyclic voltammetry (CV) was performed for 0.1 mM oxytocin in 0.1 M PBS with a scan rate of 100 mV/s. Oxidation peak was observed at 0.7 V (vs. Ag/AgCl). Among the 9 amino acids constructing oxytocin, only tyrosine (Tyr) has been reported to be electrochemically oxidized. Tyrosine also gave oxidation signal at 0.7 V and the shape of voltammogram was quite similar to that of oxytocin. Consequently, it was deduced that oxidation of oxytocin is occurred at tyrosine moiety. When compared to other electrodes, BDD showed 4 times higher signal to background ratio than glassy carbon electrode. No signal was observed in the case of platinum electrode. These results showed that sensitive measurement of oxytocin is possible by using BDD electrode. Since vasopressin also contains tyrosine in its structure, it should show the oxidation signal. CV of vasopressin was compared to that of oxytocin. Exactly the same voltammograms were observed. On the other hand, AO-BDD, which has oxygen-terminated surface, showed apparent difference in voltammograms (Figure). Although the peak potential of vasopressin was maintained at the same position as AD-BDD, oxytocin showed a broad signal shifted to more positive potential region. On-set potential of tyrosine was still higher than that of oxytocin. One possible explanation to the results is the electrostatic interaction between the electrode surface and the molecules. The surface state of AO-BDD rich in C-O functional groups can be expected to have electrostatically negative state. On the other hand, tyrosine is negatively charged, oxytocin is almost neutral, and vasopressin is positively charged in the condition of pH 7.4. These facts and the results of CV well explain the mechanism of electrostatic interaction. Aiming at in vivo and selective measurement, chronoamperometry combined with flow injection analysis (FIA) by BDD microelectrodes was conducted. AD-BDD microelectrode applied at 1.0 V gave high linearity (R2 = 0.994) of the current peaks over the oxytocin concentration range from 0.1 to 10.0 μM with a detection limit of 50 nM (S/N =3). On the other hand, using AO-BDD microelectrode, selective measurement of oxytocin and vasopressin was achieved by the use of applied potential of 0.54 V, which gave the signals of only vasopressin. Consequently, the concentration of oxytocin can be obtained by subtracting the concentration of vasopressin measured on AO-BDD from that of oxytocin + vasopressin measured on AD-BDD. Figure 1