Microchips and single‐photon avalanche diodes for DNA separation with high sensitivity

Abstract

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 single-photon 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.

Portable genotyping system: Four-colour microchip electrophoresis

“…In the next months we will perform a full set of DNA analysis to validate our apparatus, but since the system here presented and the apparatus described in [17] have comparable optical efficiency, we are confident that even the detection limit will be comparable.…”

“…The huge potential of SPAD detector in LIF-MCE DNA analysis was demonstrated in a previous work of our group [17] in which the use of two SPADs as detection head allowed a sensitivity limit of 3 pM with 50 pl injection volume. The apparatus here presented represents the natural evolution of the previous one in terms of compactness and type of analysis, without decreasing the overall performances.…”

“…The apparatus described in [17] employed interferential filters to separate fluorescence wavelengths and optical fibers to couple fluorescence light on the corresponding SPAD detector. For that system its possible to derive a peak collection efficiency of 1.6%, considering fluorescence emission from the microcapillary as a lambertian source and accounting for filter's transmittivity, light fiber coupling and numerical aperture of the collection lens.…”

An evaluation of the experimental approaches to detection of small ions in CE

“…The sample was directly introduced into the separation channel through a sharp inlet tip, which was placed in the sample vial, without using any injection cross. Vrouwe et al [132] used conducting glass CE microchip for online monitoring of drinking water, which enables the separation and determination of inorganic ions within less than 15 s. Rech et al [125] applied a glass CE chip in combination with dual-wavelength LIF detection for rapid sizing of DNA fragments reaching an extremely high sensitivity -down to 3 pmol/L of the analytes were detected. Ding et al [129] used a glass CE microchip coupled with an end-column electrochemiluminescence detector for fast separation and sensitive determination of the pharmaceuticals tramadol and lidocaine.…”

“…For detection of luminophore-free analytes, pre-, post-, or on-column derivatization with suitable fluorescent tagging agents is required, or a fluorescent marker has to be added to the BGE to enable their indirect fluorescent detection. Many microchip CE applications have been realized using the LIF detection [111,112,[123][124][125].…”