Finally, the second passivation layer on the top part of nanowire probe was etched selectively by blocking the rest of the probe, which was wrapped with polymethyl methacrylate. This anisotropic wet etching method makes the nanowire probe have a suitable structure for intracellular recording (shown in Figure 2d). The electrical properties of the nanowire MK-8776 chemical structure probes were characterized by measuring the cyclic voltammograms (CV) (Additional file 1: Figure S5 of supplementary data). CVs were measured with a Pt counter electrode and Ag/AgCl was used as a reference electrode. No decrease of current after a small peak

was observed in our nanoelectrode. Such a behavior is common in nanosize electrodes since analytes diffuse according to hemispherical diffusion in electrodes,

which leads to a higher mass transport per unit electrode surface. The sigmoidal voltammograms, which show limiting current, are characteristic of radial diffusion to cylindrical ultramicroelectrodes. Assuming that the electrode is a cylindrical S3I-201 in vivo shape, the limiting plateau currents can be determined according to the following equation [35]. (1) Here, n is the number of electrons transferred during the electrochemical process, F is Faraday’s constant, D and C are the diffusion coefficient and concentration of the electroactive species respectively, l and r are the length and radii of nanoelectrode, respectively, and t is time scale of the

CV experiment, which is represented by RT/Fv. The experimental limiting current value at our SIS3 nanoelectrode is 4.5 nA, which is similar to the theoretical limiting current value (4.21 nA/μm). The probing of neural activity was carried out using a rat clonal GH3 pituitary cell line, which has a spontaneous action potential that is known to be stimulated by a thyrotropin releasing hormone [36]. As such, it is ideal to test the feasibility of check details the nanowire probe for measuring neural activity without external stimulation to induce an action potential. Figure 3a is an SEM image of the vertical nanowire probe device before the culturing of the GH3 cells. Culturing was carried out with GH3 cells 2 days after cell plating. The standard bath solution consisted of 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 mM glucose was applied continuously into the culturing bath through a gravity-fed perfusion system during recording. Measurements were carried out at 25°C. Figure 3b is an SEM image of GH3 cultured in the same location as that shown in Figure 3a by seeding the cells of passage 10. The white circles in Figure 3b indicate the sites where the vertical nanowire probes are positioned. The image clearly shows that the nanowire probes are covered with GH3 cells. The individual probing electrode was connected to the input of a buffer.