Nanoelectrode array

The closed pores of the AAO substrate allow the quantitative observation of dye flux across the PSMs. On the downside, external access to the compartment below the bilayer is not possible and electrochemical gradients across the membrane can only be applied to some extent by exchanging the supernatant. The pore bottoms of AAO can readily be opened via etching, giving access to both aqueous compartments around the bilayer. This approach, however, has the distinct disadvantage that gradients can only be applied with complete coverage of the substrate by PSMs. Moreover, even with complete coverage, fluorescent dyes would diffuse quickly between the pores, losing all localization information in the process.

By incorporating metal electrodes into the substrate pores, both compartmentalization and access to the electrochemical potential across the membrane can be combined.

As the nanowires cannot be addressed individually and complete coverage of the substrate with PSMs is unfeasible at the moment, care must be taken in the choice of electrode material. When applying a potential to the electrodes, current can freely flow through open pores and distort the actual potential across PSMs. The solution to this problem is the formation of electrodes that are non-polarizable at low currents, while developing a high overpotential when exposed to significant charge flow. With this in mind, Ag/AgCl was chosen as the preferred electrode system.

The incorporation of silver wires into porous alumina via electrochemical deposition has been demonstrated while still attached to the aluminium substrate ^[159], or in the detached state.^[160,161] As only small pieces of substrate are necessary for the permeabilization assay, working with the detached AAO substrate, which can be readily split, was deemed more

105 suitable. The usual approach for forming a continuous metallization of one side of the alumina is the deposition of a thin layer of silver or gold by evaporation or sputtering.^[160–

163]

In keeping with this, 30-50 nm of silver were deposited via sputtering onto AAO substrates with 60 nm pore diameter and opened pore bottoms. This layer showed poor adhesion in the subsequent electrodeposition, however, and oxidation of the continuous Ag surface after electrodeposition impeded contacting the nanowires. The working electrode material for silver deposition was changed to gold subsequently. The thickening of the metal layer by electrodeposition of Au was found to form a robust contacting interface and short Au-wires protruding into the pores. Electrodeposition of Ag onto the Au wires resulted in well-defined, dense wires of consistent height (see Figure 4.37).

Figure 4.37: Cross-sectional SEM micrograph of silver wires embedded in AAO. Scale bar 10 µm.

The coulomb efficiency of the deposition, calculated from SEM micrographs of the resulting wires, was consistent but lower than reported for a comparable commercial electrolyte (75 %)^[159] at around 50 %. When factoring in this efficiency, the height of wire arrays could be controlled by monitoring deposited charge.

Success of each fabrication step was also observable by eye, as the optically clear AAO substrate noticeably shifted colors throughout the procedure. The sputtered gold layer

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resulted in a black appearance when seen through the substrate. Electrodeposition of gold produced a green or golden shine, which immediately changed to blue upon silver deposition.

Figure 4.38: Photographs showing AAO chips during preparation of embedded nanowires. View from backside in respect to the anodization. After gold deposition, the porous substrate appears black.

Electrodeposition generates a golden color (rim on right), which changes to blue during silver deposition (center region).

The electrochemical behavior of the incorporated silver nanowires was characterized via cyclic voltammetry in PBS saturated with AgCl. A freshly chlorinated silver wire was used as a quasi-reference electrode. The open-circuit potential of the nanowire array was found to be near 0 (± 10 mV) in reference to the silver wire.

Figure 4.39: Cyclic voltammogram of the nanoelectrode array in PBS. Scanrate 100 mV/s

107 As can be seen in Figure 4.39, the nanoelectrodes are readily oxidized and reduced, but both processes are hindered. At negative potentials, AgCl is quickly reduced and current drops as further reduction of Ag⁺ ions is diffusion limited. At positive potentials, a drop in current is also observed. This was unexpected, as the precipitation of AgCl should keep the Ag⁺ concentration constant. Due to the relatively low chloride concentration in the buffer in comparison to typical Ag/AgCl electrolytes (3 M), AgCl precipitation can potentially lead to local depletion of Cl^- at the silver surface. In conjunction with hindered diffusion in the pore from AgCl precipitate, a significant overpotential seems to be generated by this.

After extensive cycling in PBS, one sample was characterized by SEM to reveal deposits in the pores, presumably AgCl, Ag, or a mixture thereof. (see Figure 4.40). This suggests that positive currents can only be sustained for a limited time before complete silver wire oxidation. The precipitates seem to spread only towards the open pores, suggesting that the wire tips do not form a normal Ag/AgCl junction. This supports the notion of significant overpotentials for the oxidation due to diffusion limitations at high current.

Figure 4.40: Scanning electron micrograph of nanoelectrode array after repeated oxidation and reduction in PBS buffer. A : Cross-sectional overview. The right part was covered by adhesive during cyclic voltammetry. B: Detailed view of the lower boundary of the precipitates. Scale bars 1 µm.

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On the whole, the exposed nanowires showed significant polarization at moderate currents, which should allow the application of a defined transmembrane potential on PSMs even at low membrane coverage of the AAO chip.