Near‐infrared time‐resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices

Abstract

High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate)(PMMA) substrates, brand Plexiglas offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tauf = 930 ps) and IRD700 (tauf = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50 degrees C. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.

Surface modification of poly(methyl methacrylate) microfluidic devices for high‐resolution separations of single‐stranded DNA

“…4, the pristine channel resulted in an average migration time of 258 (614) s when averaged over the 20 runs. The average plate number generated over these 20 runs was 3.76610 5 plates/m, comparable to results reported earlier [32].…”

“…In previous work, we attempted to separate ssDNA fragments in an unmodified PMMA microdevice [32]. Although PMMA proved to be a suitable substrate for fluorescence detection due to its low autofluorescence level, we were unable to obtain resolution between fragments with single basepair differences required for DNA sequencing, which was attributed to potential analyte/wall interactions as indicated by peak tailing in the electropherograms and/or an inhomogeneous EOF.…”

“…In our initial attempts to separate ssDNAs in a PMMA microdevice, we were unable to achieve single base resolution as required for DNA sequencing [32]. We suspected that changes in the surface charge density on the walls of pristine PMMA due to either analyte adsorption or inhomogeneities in the surface charged groups contributed to low plate number generation.…”

Interfacing capillary gel microfluidic chips with infrared laser desorption mass spectrometry

“…The substantial electroosmotic flow (EOF) as well as potential interactions between the polypeptides and the channel walls can induce band spreading over the distance of the microchannel. The effect of solute-wall interactions and a biopolymer's electoosmotic flow has been reported for DNA separations in polymer microchips [33][34][35][36]. Dispersion of the analytes over a large distance in the microchannel translates into less material under the laser at any given position, which adversely affects the achievable detection limit.…”

Experimental and Numerical Study on PDMS Collapse for Fabrication of Micro/Nanochannels

“…This well developed technique has great potential to fabricate plastic micro/nanochannels with low cost, large scale and high throughput. However, most of the commercially thermoplastic materials used in nanoimprinting suffer from a high auto-fluorescence when excited by ultraviolet radiation [22,23]. Auto-fluorescence background interferes with on-chip optical detection, which making plastic micro/nano devices usually very difficult to measure the fluorescent molecules in the channels.…”