Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli

A device is developed for low-voltage electroporation using field constriction at a micro-orifice, and the application to the real-time measurement of single cell response is demonstrated. The device consists of a pair of electrodes separated by an insulator film having a regularly arranged array of micro-fabricated orifices with a typical diameter of 1?2 ?m. Cells are immobilized at the orifices by aspiration, and a pulse voltage is applied. The field lines, being unable to penetrate the insulator, go into the orifices and create a field constriction. This means that most voltage drop occurs in the vicinity of the orifice, and is imposed locally on the membrane in contact with the orifice. Hence, electroporation can be achieved regardless of the cell size, shape or orientation. The experimental verification is made with human monocytes, and uptake of a fluorescence dye is observed with pulses as low as 1 V, and almost 100% yield is achieved at 1.5 V. Then the dynamic response of a myocyte to external stimuli is measured. When the substrates for the metabolic cycle are fed by the method, a clear increase in fluorescence emission from the resultant NADH is observed.