Spectrally matched duplexed nucleic acid bioassay using two-colors from a single form of upconversion nanoparticle

Optical sensing can provide opportunity for simultaneous determination of multiple targets as well as for implementation of ratiometric methods that can improve accuracy and precision. Herein we report a paper-based two-color oligonucleotide detection assay with tunable sensitivity that is based on use of a single type of upconversion nanoparticle (UCNP). Water-soluble UCNPs were designed to concurrently offer green and red emission. These avidin functionalized UCNPs were adsorbed onto a cellulose support, and Cy3 was used as a green channel acceptor for Survival Motor Neuron (SMN1) target, and Cy5.5 was the red channel acceptor for the glucuronidase gene (uidA) target. Selective DNA hybridization of the labeled targets with the corresponding probe provided emission from dyes, which was the basis for concurrent quantification of both targets. The limit of detection (LOD) could be tuned by changing the relative ratio of the SMN1 and uidA probes. A higher proportion of a probe provided for a lower LOD. When the SMN1/uidA probe ratio was 1:4, the LOD for SMN1 and uidA target were 54.3 and 30.5 fmol, and when the probe ratio was 4:1, the LOD for the above targets were 22.1 and 1260 fmol, respectively. Selectivity evaluation showed that one base pair mismatched DNA for SMN1 and uidA could be discriminated in most cases. The assay showed resistance to nonspecific adsorption of interfering DNA and protein and was even functional for targets in undiluted serum. This work represents a significant step in the development of paper-based multiplexed UCNP luminescence assays.