Exonuclease III-Assisted Upconversion Resonance Energy Transfer in a Wash-Free Suspension DNA Assay

Abstract

Sensitivity is the key in optical detection of low-abundant analytes, such as circulating RNA or DNA. The enzyme Exonuclease III (Exo III) is a useful tool in this regard; its ability to recycle target DNA molecules results in markedly improved detection sensitivity. Lower limits of detection may be further achieved if the detection background of autofluorescence can be removed. Here we report an ultrasensitive and specific method to quantify trace amounts of DNA analytes in a wash-free suspension assay. In the presence of target DNA, the Exo III recycles the target DNA by selectively digesting the dye-tagged sequence-matched probe DNA strand only, so that the amount of free dye removed from the probe DNA is proportional to the number of target DNAs. Remaining intact probe DNAs are then bound onto upconversion nanoparticles (energy donor), which allows for upconversion luminescence resonance energy transfer (LRET) that can be used to quantify the difference between the free dye and tagged dye (energy acceptor). This scheme simply avoids both autofluorescence under infrared excitation and many tedious washing steps, as the free dye molecules are physically located away from the nanoparticle surface, and as such they remain "dark" in suspension. Compared to alternative approaches requiring enzyme-assisted amplification on the nanoparticle surface, introduction of probe DNAs onto nanoparticles only after DNA hybridization and signal amplification steps effectively avoids steric hindrance. Via this approach, we have achieved a detection limit of 15 pM in LRET assays of human immunodeficiency viral DNA.

Surface Functionalisation of Upconversion Nanoparticles with Different Moieties for Biomedical Applications

“…Various modification strategies exist that have tried to develop universal methods and overcome these limitations but stability in aqueous environments is still a considerable challenge [10]. The four main strategies for surface modification include: (a) ligand engineering [2,3,5,7,9,10,12,31]; (b) layer-bylayer assembly [2,3,5,7,32]; (c) ligand attraction [2,3,5,7,33]; and (d) surface polymerisation [2,3,5,6,11,13,[15][16][17][34][35][36][37][38][39]. Each modification method as well as the specific molecules selected, has its own benefits and hindrances.…”

“…A ligand exchange reaction does not necessarily involve full replacement of all the hydrophobic ligands present on the surface, however, the complete exchange of ligands is ensured if an excess of the new ligand is added to a suitable solvent at elevated temperatures, with the choice of solvent being reliant on the dynamic solvability of both the hydrophobic and new hydrophilic ligands [5]. Various hydrophilic ligands have been introduced onto the surfaces of UCNPs via ligand exchange, including ligands such as citrate [3,5,29], hexanedioic acid [3,5,29], polyethylene glycol (PEG) derivatives [5,10,29,31], 6-aminohexanoic acid [3,5,46], polyacrylic acid (PAA) derivatives [10,43,47], and phosphate derivatives [10,31,48]. Newly affixed ligands tend to require a certain length in order to be able to provide functionality that can be accessible for subsequent modification but this can be advantageously used in the design of nanoparticle stability.…”

Surface Functionalisation of Upconversion Nanoparticles with Different Moieties for Biomedical Applications

“…Various hydrophilic ligands have been introduced onto the surfaces of UCNPs via ligand exchange, including ligands such as citrate [3,5,29] , hexanedioic acid [3,5,29] , polyethylene glycol (PEG) derivatives [5,10,29,31] , 6-aminohexanoic acid [3,5,43] , polyacrylic acid (PAA) derivatives [10,44,45] and phosphate derivatives [10,31,46] . Newly affixed ligands tend to require a certain length in order to be able to provide functionality that can be accessible for subsequent modification but this can be advantageously used in the design of nanoparticle stability.…”

“…Moreover, nanoparticles intended for use in biomedical applications require uniformity in morphology and size which presents a greater difficulty in obtaining ideal samples [29] .Various modification strategies exist that have tried to develop universal methods and overcome these limitations but stability in aqueous environments is still a considerable challenge [10] . The four main strategies for surface modification include: (a) ligand engineering [2,3,5,7,9,10,12,31] ; (b) layer-by-layer assembly [2,3,5,7,32] ; (c) ligand attraction [2,3,5,7,33] ; and (d) surface polymerisation [2,3,5,6,11,13,[15][16][17][34][35][36][37][38][39] . Each modification method as well as the specific molecules selected, has its own benefits and hindrances.While there are many ways to modify the surfaces of UCNPs, this review aims at covering the two most popular methods, that being ligand engineering and silanisation.Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15…”

A Versatile Photoinduced Electron Transfer‐Based Upconversion Fluorescent Biosensing Platform for the Detection of Disease Biomarkers and Nerve Agent

“…In most of the homogeneous detection systems, the utilization of UCNPs generally depends on the fluorescence resonance energy transfer (FRET) principle, in which the UCNPs serve as the energy donors and a range of energy acceptors, including fluorescent materials and nonfluorescent materials, are modified on their surface to convert/quench the upconverting fluorescence 1a,6. In this process, the target molecules act as the bridge to access or forbid the energy transfer from UCNPs to the acceptors, leading to the decrease or recovery of the upconverting fluorescence that reflects the target concentration . The critical distance between the energy donors and acceptors in an FRET system is within 10 nm 1a,8.…”