A method is described that demonstrates a new technique for rapid and high‐throughput single molecule sequencing. This sequencing technique is based on the successive enzymatic degradation of fluorescently labeled single DNA molecules, and the detection and identification of the released monomer molecules according to their sequential order in a microstructured channel.
The detection technique is evolved from confocal fluorescence microscopy, with two different laser sources to excite the individual mononucleotides that are either labeled with tetramethylrhodamine (TMR) or Cyanine5 (Cy5). The handling of DNA which is immobilized on carrier beads, and the detection of the cleaved monomers is performed in optically transparent and biochemically inert microstructures (glass or PMMA) with detection channels of 7 μ × 10 μm.
The projected rate of sequencing is ≈100 bases min−1, dependent solely on the rate of the enzymatic DNA cleavage.
A rapid prototyping method for the fabrication of microstructures in polymer substrates has been developed, using a combination of laser direct-write patterning of silicon membrane contact masks and vacuum-ultraviolet (VUV) pulsed laser ablation. The process allows the flexible and rapid prototyping of microreactors and microchannel systems with submicrometer resolution. Thanks to the high photon energy of the VUV (7.9 eV) irradiation, PMMA and also other polymers can be ablated without leaving behind carbonated and/or fluorescent surfaces. Fluences of ≅ 100 mJ/cm2 lead to etch rates of ≅ 140 nm/pulse and result in very smooth surfaces with a roughness in the nanometer range on a micrometer lateral scale. The structured substrates are sealed with a PMMA foil by means of a newly developed low temperature technique without adhesives.
Smooth polymer surfaces can be generated by VUV laser ablation at 157 nm. This has been demonstrated for poly-methyl-metacrylate (PMMA) by atomic force microscopy (AFM) analysis of laser-exposed surfaces. At excimer laser fluences below 30 mJ/cm2 the mean roughness (Ra) remains below 10 nm for both single and 10 shot exposures.A detailed characterization of the surface nano-morphology distribution carried out by the “power spectral density” (PSD) analysis, shows that the surface is smoothened by the VUV laser for lateral scales smaller than 20 μm.
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