Optimization and miniaturization of SE-ECL for potential-resolved, multi-color, multi-analyte detection

Electrochemiluminescence (ECL) is a bioanalytical technique with numerous advantages, including the potential for high temporal and spatial resolution, a high signal-to-noise ratio, a broad dynamic range, and rapid measurement capabilities. To reduce the complexity of a multi-electrode approach, we use a single-electrode electrochemiluminescence (SE-ECL) configuration to achieve the simultaneous emission and detection of multiple colors for applications that require multiplexed detection of several analytes. This method exploits intrinsic differences in the electric potential applied along single electrodes built into electrochemical cells, enabling the achievement of distinct colors through selective excitation of ECL luminophores. We present results on the optimization of SE-ECL intensity for different channel lengths and widths, with sum intensities being 5 times larger for 6 cm vs 2 cm channels and linearly increasing with the width of the channels. Furthermore, we demonstrated for the first time that applying Alternating Current (AC) voltage within the single electrode setup for driving the ECL reactions has a dramatic effect on the emitted light intensity, with square waveforms resulting in higher intensities vs sine waveforms. Additionally, multiplexed multicolor SE-ECL on a 6.5 mm × 3.6 mm CMOS semiconductor image sensor was demonstrated for the first time, with the ability to simultaneously distinguish four different colors, leading to the ability of measuring multiple analytes.