DNA‐Coated Upconversion Nanoparticles for Sensitive Nucleic Acid FRET Biosensing

Abstract

Despite the significant advancements of developing upconversion nanoparticles (UCNPs) for high performance biosensing and bioimaging, the development of DNA‐functionalized UCNPs with thin coatings, efficient surface passivation, and fully functional DNAs for hybridization sensing in biological media remains extremely challenging. Here, a straightforward concept of labeling DNA to a thin polysulfonate polymer layer on UCNPs is presented. Both UCNPs and DNA preserve their full functionality as demonstrated by Förster resonance energy transfer (FRET) hybridization assays with a Cy3‐conjugated complementary DNA. The important bioanalytical benefits of the UCNP‐DNA nanohybrids are demonstrated by their implementation into rapid and wash‐free microRNA hybridization FRET assays. Using a simple fiber‐coupled USB‐spectrometer and 980 nm diode laser excitation in a 96‐well microplate, ratiometric FRET from UCNP‐to‐Cy3 can quantify miR20a in a 0.01–10 × 10−9 m concentration range with a detection limit of 30 × 10−12 m (4.5 fmol of miR20a), which is more than 20‐fold lower compared to quantum dot‐based FRET assays. The functional thin‐coating DNA‐UCNP nanohybrids have a strong potential for translating UCNPs into advanced DNA‐based biosensing, bioimaging, and high‐throughput and point‐of‐care clinical diagnostics.

Optimizing Upconversion Nanoparticles for FRET Biosensing

“…Therefore, the UCNP surface and Cy3.5 were in principle separated only by the thin PSS shell (∼2 nm) and the C 3 spacer (∼1 nm) . UCNPs prepared with those thin PSS shells and bioconjugated with DNA previously showed good stability without significant aggregation over up to 2 weeks and were successfully applied in DNA and RNA FRET assays. , The UCL kinetics (at 540 nm) of all DNA 1 -functionalized UCNPs (UCNP@PSS-DNA 1 ) were analyzed in HEPES buffer using both 980 and 808 nm excitation (Figure S7 and Table S3). Long 80 μs excitation pulses were used to reach intensity saturation and measure only the UCL decays of the different UCNPs, which were fitted with monoexponential functions (see Materials and Methods for details) …”

“…The implementation of a model system as similar as possible to real-scenario FRET assays is a fundamental step to gain useful insights into the real working principles of such systems. For this reason, using a recently developed DNA-UCNP bioconjugation strategy for functional FRET assays, 9 we attached probe DNAs (DNA 1 ) on the UCNP surface. These probes were complementary to target DNAs labeled with Cyanine 3.5 dyes (DNA 2 -Cy3.5).…”

“…Lanthanoid-doped upconversion nanoparticles (UCNPs) can convert near-infrared excitation into visible emission, leading to background-free photoluminescence. , This photophysical feature, combined with high photostability, the absence of photobleaching, and low toxicity, , has resulted in a broad application of UCNPs in biosensing, − bioimaging, − and theranostics. − The most commonly employed UCNPs consist of a NaYF 4 host matrix doped with Yb 3+ ions (sensitizers) that absorb at around 980 nm and Er 3+ (or Tm 3+ , Ho 3+ ) ions (activators), which produce the upconversion luminescence (UCL) in the ultraviolet–visible–near-infrared (UV–vis–NIR) spectral regions. − Due to the very low overall UCL quantum yields, UCNPs require high-power excitation. This requirement can become a serious disadvantage, because the absorption of water in the 980 nm region can result in heating and damage of biological samples and limits the light penetration depth. − One possibility to overcome this problem is the use of Nd 3+ ions as sensitizers .…”

Understanding FRET in Upconversion Nanoparticle Nucleic Acid Biosensors

“…Taking into account that our UCNPs were not optimized and that our assays were based on simple steadystate measurements without the necessity of time-resolved instrumentation, these very similar LODs are rather impressive and suggest that significantly lower concentrations may become quantifiable with optimized UCNP architectures and material compositions. 40 S6. In conclusion, thin PAA coatings (∼1 nm), direct attachment of DNA via EDC/sulfo-NHS chemistry, and close UCNP-dye distances resulted in the best UCNP-to-dye FRET DNA hybridization assay performance as demonstrated by sensitive quantification of miR-20a.…”

Recent Progress in Lanthanide-Doped Inorganic Perovskite Nanocrystals and Nanoheterostructures: A Future Vision of Bioimaging

“…Both Ln-doped NPs and DNA preserved their full functionality, as demonstrated by Förster resonance energy transfer hybridization assays with Cy3-conjugated complementary DNA. Ratiometric FRET from Ln-doped NP to Cy3 demonstrated that these nanohybrids are able to quantify miR20a in the 0.01-10 × 10 −9 M concentration range with a detection limit of 30 × 10 −12 M (4.5 fmol of miR20a) [90].…”