Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;
The
tethering of molecular beacon oligonucleotide detection probes
to surface-patterned poly(ethylene glycol) (PEG) microgels has enabled
the integration of molecular beacons into a microarray format. The
microgels not only localize the probes to specific surface positions
but also maintain them in a waterlike environment. Here we extend
the concept of microgel tethering to include dielectric microlenses.
We show that streptavidin-functionalized polystyrene microspheres
(3 μm diameter) can be colocalized with molecular beacons using
biotinylated PEG gels in patterns ranging from pseudocontinuous microgel
pads with lateral dimensions on the order of tens of micrometers to
individual microgels with lateral dimensions on the order of 400–500
nm. We use a simplex assay based on Influenza A detection to study
the lensing behavior. The microspheres increase the effective numerical
aperture of the collection optics, and we find that a tethered microsphere
increases the peak intensity collected from hybridized beacons between
1.5 and 10 times depending on the specific pattern size and areal
density of microgels. The highest signal increase occurs when a single
microsphere is tethered to a single isolated microgel. The tethering
is highly self-directed and occurs in the individual-microgel case
only when the microgel is close to the optic axis of the microsphere.
This alignment minimizes spherical aberration and maximizes coupling
of emitted fluorescent intensity into the collection optics.
In contrast to photolithography where particular wavelengths of light can couple to specific photochemistries, electron-beam lithography can drive competing chemistries. To separate surface-grafting, cross-linking, and chemical functionality, we studied the effects of 2 keV electrons on thin films of poly(ethylene glycol) end-functionalized with hydroxyls (PEG-OH) or biotins (PEG-B). Similarities in the dose-dependent thickness changes of the patterned PEGs indicate that surface grafting and cross-linking primarily involve the ethylene oxide main chain. While higher doses create thicker patterns with more biotin, the concurrent increase in thiol reactivity indicates that crosslinking competes with biotin degradation. The dose window for optimal e-beam patterning of biotinylated PEG is very narrow. Biotin is entirely consumed at higher doses. Its modified functionality is reactive with 5-((2-(and-3)-S-(acetylmercapto) succinoyl) amino) (SAMSA). This effect creates a dose-dependent orthogonal functionality that can be patterned from a single precursor thin film.
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