A Temperature-Controlled Microwell Platform for Electrochemical Melting Analyses

Abstract

We have designed and fabricated a miniature device for proof-of-concept studies on biomolecular interactions using electrochemical melting analysis. A collection of temperature controlled three-electrode platforms was built on a 100-mm glass wafer, and it includes a total of eight separate probe sites. Each site consists of one 200 nm thick platinum (Pt) heater, one 200 nm gold (Au) electrode and two 200 nm Pt electrodes, as well as a 10 µL polydimethylsiloxane (PDMS) well, all used for probing electrochemical signals as a function of sample temperature. Our Pt heaters have the ability to control sample solution temperatures in the microwell from 4 °C to 120 °C. Heaters were fabricated with different geometries (width and shape) in order to select the best candidate for future device fabrication. Electrochemical melting analysis has been performed by detecting signals derived from changes in methylene blue (MB)-labeled DNA that occur during the heating process. The MB-labeled single strand(ss) DNA was added to hybridize with its matched thiolated ssDNA on Au electrode surface to form duplexes. Electron-transfer(eT) was monitored using square wave voltammetry (SWV) from the beginning of a thermal cycling process. During the heating process the eT was limited significantly when the heater temperature reached the duplex melting temperature. The dehybridized MB-labeled ssDNA left the Au electrode surface causing a current decrease. As a result, a current vs. temperature melting curve was obtained that allowed us to determine the sample DNA melting temperature. The rapid heating capability provided by the Pt heater allows the electrochemical melting measurements to be completed in 20-30 minutes, and that is significantly faster than the typical time for optical melting methods. Figure 1 illustrates the wired microsystem used in the DNA experiment, and includes an enlargement showing the configuration of the miniature electrodes and heater. We plan to build the described probes into a 3x3 array-based microchip for demonstrating the potential for high throughput DNA melting analyses to study biomolecular binding stability.