Plastic Microchip Electrophoresis with Analyte Velocity Modulation. Application to Fluorescence Background Rejection

Abstract

Fluorescence background interference from the device is inherent in plastic microchips, particularly with blue or UV excitation. Conventionally, microchip background has been reduced with confocal optics or circumvented with specialized long-wavelength fluorophores. We show that microchip background can be rejected with analyte velocity modulation. In this scheme, the driving voltage is modulated at low frequency, typically in the range of 7−20 Hz. Consequently, migration velocities and analyte signals are modulated at the same frequency. Microchip fluorescence is unmodulated, so that lock-in detection (synchronous demodulation) easily separates the analyte signal from background. The technique does not require a laser source. In our implementation, a blue (485 nm) LED is the light source. Simple optics are used to shape the source and focus it to a spot ∼50 μm in diameter inside a microchip. Photomultiplier detection is employed, and a lock-in amplifier is used to demodulate the signal. Apertures in the system generate a derivative response, which can be converted to conventional bands by integration. Fluorescence rejection provided by our current system lowers detection limits by ∼1 order of magnitude compared to dc measurements with the same optical train.