Oligonucleotide Sensor Based on Selective Capture of Upconversion Nanoparticles Triggered by Target-Induced DNA Interstrand Ligand Reaction

Abstract

We present a sensor that exploits the phenomenon of upconversion luminescence to detect the presence of specific sequences of small oligonucleotides such as miRNAs among others. The sensor is based on NaYF4:Yb,Er@SiO2 nanoparticles functionalized with ssDNA that contain azide groups on the 3′ ends. In the presence of a target sequence, interstrand ligation is possible via the click-reaction between one azide of the upconversion probe and a DBCO-ssDNA-biotin probe present in the solution. As a result of this specific and selective process, biotin is covalently attached to the surface of the upconversion nanoparticles. The presence of biotin on the surface of the nanoparticles allows their selective capture on a streptavidin-coated support, giving a luminescent signal proportional to the amount of target strands present in the test samples. With the aim of studying the analytical properties of the sensor, total RNA samples were extracted from healthy mosquitoes and were spiked-in with a specific target sequence at different concentrations. The result of these experiments revealed that the sensor was able to detect 10–17 moles per well (100 fM) of the target sequence in mixtures containing 100 ng of total RNA per well. A similar limit of detection was found for spiked human serum samples, demonstrating the suitability of the sensor for detecting specific sequences of small oligonucleotides under real conditions. In contrast, in the presence of noncomplementary sequences or sequences having mismatches, the luminescent signal was negligible or conspicuously reduced.

Surface Functionalisation of Upconversion Nanoparticles with Different Moieties for Biomedical Applications

“…Another important role of the surface modified polymer molecules or silica shell is to provide further functionality to the UCNPs for desired operations. Design-enhanced colloidal stability [5,[9][10][11][12][13][14][15][16][17] and bio-conjugation [2,15,16,[18][19][20][21][22][23][24][25][26] are just a few examples from the extensive of significant benefits that can be gained from the functionalisation of UCNPs with functional moieties. There is a small range of common base functional moieties, including carboxyl (-COOH), amino (-NH 2 ) and thiol (-SH) groups, but their utilisation across a broad spectrum of applications differs.…”

Surface Functionalisation of Upconversion Nanoparticles with Different Moieties for Biomedical Applications

“…In a similar manner, Mendez-Gonzalez, D. et al (2017) [22] functionalised NaYF4: Yb +3 , Er 3+ UCNPs for their use in an oligonucleotide-based sensor (shown in Figure 11). The particles were coated with a dSiO2 shell where they were then subsequently functionalised with APTES to provide hydrophilicity to the UCNPs.…”

“…Oligonucleotidebased biosensor [22] Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 October 2018 doi:10.20944/preprints201810.0312.v1…”

“…Although the carboxyl moieties provide greater hydrophilicity than the amine groups, Reprinted with permission from ref. [22] . Copyright 2017…”

“…Another important role of the surface modified polymer molecules or silica shell is to provide further functionality to the UCNPs for desired operations. Design-enhanced colloidal stability [5,[9][10][11][12][13][14][15][16][17] and bio-conjugation [2,15,16,[18][19][20][21][22][23][24][25][26] are just a few examples from the extensive of significant benefits that can be gained from the functionalisation of UCNPs with functional moieties. There is a small range of common base functional moieties, including carboxyl (-COOH), amino (-NH2) and thiol (-SH) groups, but their utilisation across a broad spectrum of applications differs.…”