Fabrication of two-layer poly(dimethyl siloxane) devices for hydrodynamic cell trapping and exocytosis measurement with integrated indium tin oxide microelectrodes arrays

Abstract

The design, fabrication and test of a microfluidic cell trapping device to measure single cell exocytosis were reported. Procedures on the patterning of double layer template based on repetitive standard photolithography of AZ photoresist were investigated. The replicated poly(dimethyl siloxane) devices with 2.5 μm deep channels were proved to be efficient for stopping cells. Quantal exocytosis measurement can be achieved by targeting single or small clumps of chromaffin cells on top of the 10 μm × 10 μm indium tin oxide microelectrodes arrays with the developed microdevice. And about 72 % of the trapping sites can be occupied by cells with hydrodynamic trapping method and the recorded amperometric signals are comparable to the results with traditional carbon fiber microelectrodes. The method of manufacturing the microdevices is simple, low-cost and easy to perform. The manufactured device offers a platform for the high throughput detection of quantal catecholamine exocytosis from chromaffin cells with sufficient sensitivity and broad application.

Fig. 1

(A) Silicon wafer, (B) Silicon wafer coated with the first layer of AZ photoresist, (C) Transparency film with shallow channel features, (D) AZ photorisist template with patterned shallow channels, (E) template with spin-coated second layer of phototesist, (F) Transparency film with inlet and outlet channels, (G) Two-layer AZ photoresist template

Fig. 2

(A) ITO microelectrodes arrays, (B) the PDMS cell trapping device.

Fig. 3

(A) Schematic diagram of the cell trapping device, (B) Micrograph of chromaffin cell trapping at a 100 μm wide, 2.5 μm deep channel, (C) Micrograph of chromaffin cell trapping at a 50 μm wide, 2.5 μm deep channels.

Fig. 4

Chromaffin cell trapping on top of the ITO electrodes. Each square crossed a cell or small cell clumps.

Fig. 5

(A) Amperometric detection of quantal exocytosis of catecholamines from chromaffin cells using microfluidic cell trapping devices. (B) a, Expanded scale of the amperometric spikes within the dashed box in Fig. 5(A), b, Expanded view of the foot signal.

Fig. 6

(A) Histogram of the spike “half-widths” as a measure of spike duration, the half-width is defined as the time interval for each spike where the amperometric current exceeds 50% of the peak value of the spike, (B) Histograms of spike area as a measure of the catecholamine content of a single vesicle.