Implementing Morpholino-Based Nucleic Acid Sensing on a Portable Surface Plasmon Resonance Instrument for Future Application in Environmental Monitoring

Abstract

A portable surface plasmon resonance (SPR) instrument was tested for the first time for the detection of oligonucleotide sequences derived from the 16S rRNA gene of Oleispira antarctica RB-8, a bioindicator species of marine oil contamination, using morpholino-functionalized sensor surfaces. We evaluated the stability and specificity of morpholino coated sensor surfaces and tested two signal amplification regimes: (1) sequential injection of sample followed by magnetic bead amplifier and (2) a single injection of magnetic bead captured oligo. We found that the sensor surfaces could be regenerated for at least 85 consecutive sample injections without significant loss of signal intensity. Regarding specificity, the assay clearly differentiated analytes with only one or two mismatches. Signal intensities of mismatch oligos were lower than the exact match target at identical concentrations down to 200 nM, in standard phosphate buffered saline with 0.1 % Tween-20 added. Signal amplification was achieved with both strategies; however, significantly higher response was observed with the sequential approach (up to 16-fold), where first the binding of biotin-probe-labeled target oligo took place on the sensor surface, followed by the binding of the streptavidin magnetic beads onto the immobilized targets. Our experiments so far indicate that a simple coating procedure in combination with a relatively cost-efficient magnetic-bead-based signal amplification will provide robust SPR based nucleic acid sensing down to 0.5 nM of a 45-nucleotide long oligo target (7.2 ng/mL).

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“…Phosphorodiamidatemorpholino oligomers (PMO) are uncharged analogues of nucleic acids. Compared to natural nucleic acid probes, the PMO probe offers better stability and higher binding strength owing to its neutral character [ 135 ]. RNA and DNA probes are negatively charged in physiological conditions due to their phosphate groups on the nucleic acid backbone.…”

Molecularly resolved, label-free nucleic acid sensing at solid–liquid interface using non-ionic DNA analogues

“…The best capture probe for sensing nucleic acids Among the various alternative probes that have been designed and synthesized for improved sensing of nucleic acids, both PNA and MO are potentially useful. 4,76 Increased thermal stability of the MO-inclusive duplexes, solubility of MO in aqueous medium, its capacity of adopting upright orientation, and ability to resist nuclease-and protease-induced degradation, all point to potential diagnostic applications of the MO probe. Although PNA is nuclease-/protease-resistant too, and it offers nucleic acid detection capacity with high binding affinity and sequence specicity, it's lower water solubility than MO makes PNA a less attractive candidate than MO.…”

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“…SPR has been widely applied in many fields such as biochemical analysis, food inspection, environmental monitoring, etc. [ 1 , 2 , 3 , 4 , 5 , 6 ]. Conventional SPR sensing platforms are based on the Kretschmann configuration, where propagating plasmons are excited by compensating the momentum of incident light with a high-refractive-index (RI) prism.…”