Novel membrane-based electrochemical sensor for real-time bio-applications

Abstract

This article presents a novel membrane-based sensor for real-time electrochemical investigations of cellular- or tissue cultures. The membrane sensor enables recording of electrical signals from a cell culture without any signal dilution, thus avoiding loss of sensitivity. Moreover, the porosity of the membrane provides optimal culturing conditions similar to existing culturing techniques allowing more efficient nutrient uptake and molecule release. The patterned sensor electrodes were fabricated on a porous membrane by electron-beam evaporation. The electrochemical performance of the membrane electrodes was characterized by cyclic voltammetry and chronoamperometry, and the detection of synthetic dopamine was demonstrated down to a concentration of 3.1 pM. Furthermore, to present the membrane-sensor functionality the dopamine release from cultured PC12 cells was successfully measured. The PC12 cells culturing experiments showed that the membrane-sensor was suitable as a cell culturing substrate for bio-applications. Real-time measurements of dopamine exocytosis in cell cultures were performed, where the transmitter release was recorded at the point of release. The developed membrane-sensor provides a new functionality to the standard culturing methods, enabling sensitive continuous in vitro monitoring and closely mimicking the in vivo conditions.

Figure 1.

(a) Sketch of the three-electrode membrane-based electrochemical sensor. The sensor involves patterned deposition of a working, counter and reference electrode; (b) Sketch of the shadow mask used to deposit the reference electrode; (c) Sketch of the shadow mask used to deposit the working and the counter electrodes; (d) Membrane-sensors connected to a potentiostat by crocodile clips. (1) Reference electrode; (2) Counter electrode and (3) Working electrode; (e) SEM image of the electrode covered membrane showing black dots representing the pores. The inset shows a line scan across a single pore revealing that the pores were not blocked by the metal deposition.

Figure 2.

Cyclic voltammograms obtained with membrane-sensor. (a) Typical cyclic voltammograms in 10 mM Ferricyanide at differential potential scan rates. Inset: Current peak heights versus square root of the sweep rates (SR); (b) Examples of typical cyclic voltammograms in 0.1 M synthetic dopamine at various potential scan rates.

Figure 3.

Calibration of membrane-sensor. Plot of the average charge signals versus dopamine concentrations for chronoamperometric responses obtained by the membrane-sensor. Error bars denote standard deviation of the measurements. (a) The measurements obtained for the entire range of tested concentrations; (b) The measurements obtained at the lower concentration range (up to 25 pM). Inset: Typical chronoamperometric recording of dopamine.

Figure 3.

Calibration of membrane-sensor. Plot of the average charge signals versus dopamine concentrations for chronoamperometric responses obtained by the membrane-sensor. Error bars denote standard deviation of the measurements. (a) The measurements obtained for the entire range of tested concentrations; (b) The measurements obtained at the lower concentration range (up to 25 pM). Inset: Typical chronoamperometric recording of dopamine.

Figure 4.

PC12 cells differentiated on the membrane-sensor. Differentiation for (a) 14 days and (b) 30 days. The characteristic neuronal-like morphology is exhibited by the cells.

Figure 5.

Typical chronoamperometric current-time trace corresponding to dopamine exocytosis from differentiated PC12 cells, obtained upon induction of dopamine release on top of the membrane-sensor. Inset shows the average charge accumulated during the measurement.