Microchips and Single-Photon Avalanche Diodes for DNA Separation with High Sensitivity

Modern techniques for DNA and protein analysis and separation rely on measurements of LIF and face a trend toward employing progressively smaller samples. The currently employed detectors that provide the required ultrahigh sensitivity, e.g. photomultiplier tubes (PMTs), are bulky and/or costly and delicate, whereas a key issue for the development of compact and economical instruments is the availability of miniaturized, inexpensive, and ultrasensitive photodetectors. The planar epitaxial silicon singlephoton avalanche diodes (SPADs) combine the typical advantages of microelectronics (miniaturization, ruggedness, low voltage, low power, low cost, etc.) with high sensitivity, even better than that of PMTs. The suitability of such SPADs to microchip CE has been here ascertained by developing a new apparatus with dual-wavelength LIF detection. The apparatus has been experimented in studies on the EOF suppression and on the coating stability and tested in rapid sizing of DNA fragments. The experimental results obtained in the separation of Cy5-labeled oligonucleotide demonstrate sensitivity better than 3 pM, which corresponds to less than 100 fluorescent molecules in the 50 pL illuminated volume.