Optimizing Upconversion Nanoparticles for FRET Biosensing.

Upconversion nanoparticles (UCNPs) are some of the most promising nanomaterials for bioanalytical and biomedical applications. One important challenge to be still solved is how UCNPs can be optimally implemented into Förster resonance energy transfer (FRET) biosensing and bioimaging for highly sensitive, wash-free, multiplexed, accurate, and precise quantitative analysis of biomolecules and biomolecular interactions. The many possible UCNP architectures composed of a core and multiple shells doped with different lanthanoid ions at different ratios, the interaction with FRET acceptors at different possible distances and orientations via biomolecular interaction, and the many and long-lasting energy transfer pathways from the initial UCNP excitation to the final FRET process and acceptor emission make the experimental determination of the ideal UCNP-FRET configuration for optimal analytical performance a real challenge. To overcome this issue, we have developed a fully analytical model that requires only a few experimental configurations to determine the ideal UCNP-FRET system within a few minutes. We verified our model via experiments using nine different Nd-, Yb-, and Er-doped core-shell-shell UCNP architectures within a prototypical DNA hybridization assay using Cy3.5 as an acceptor dye. Using the selected experimental input, the model determined the optimal UCNP out of all theoretically possible combinatorial configurations. An extreme economy of time, effort, and material was accompanied by a significant sensitivity increase, which demonstrated the powerful feat of combining a few selected experiments with sophisticated but rapid modeling to accomplish an ideal FRET biosensor.

Understanding FRET in Upconversion Nanoparticle Nucleic Acid Biosensors.

Upconversion nanoparticles (UCNPs) have been frequently applied in Förster resonance energy transfer (FRET) bioanalysis. However, the understanding of how surface coatings, bioconjugation, and dye-surface distance influence FRET biosensing performance has not significantly advanced. Here, we investigated UCNP-to-dye FRET DNA-hybridization assays in H2O and D2O using ∼24 nm large NaYF4:Yb3+,Er3+ UCNPs coated with thin layers of silica (SiO2) or poly(acrylic acid) (PAA). FRET resulted in strong distance-dependent PL intensity changes. However, the PL decay times were not significantly altered because of continuous Yb3+-to-Er3+ energy migration during Er3+-to-dye FRET. Direct bioconjugation of DNA to the thin PAA coating combined with the closest possible dye-surface distance resulted in optimal FRET performance with minor influence from competitive quenching by H2O. The better comprehension of UCNP-to-dye FRET was successfully translated into a microRNA (miR-20a) FRET assay with a limit of detection of 100 fmol in a 80 µL sample volume.

Isolating Nanocrystals with an Individual Erbium Emitter: A Route to a Stable Single-Photon Source at 1550 nm Wavelength.

Single-photon emitters based on individual atoms or individual atomic-like defects are highly sought-after components for future quantum technologies. A key challenge in this field is how to isolate just one such emitter; the best approaches still have an active emitter yield of only 50% so that deterministic integration of single active emitters is not yet possible. Here, we demonstrate the ability to isolate individual erbium emitters embedded in 20 nm nanocrystals of NaYF4 using plasmonic aperture optical tweezers. The optical tweezers capture the nanocrystal, whereas the plasmonic aperture enhances the emission of the Er and allows the measurement of discrete emission rate values corresponding to different numbers of erbium ions. Three separate synthesis runs show near-Poissonian distribution in the discrete levels of emission yield that correspond to the expected ion concentrations, indicating that the yield of active emitters is approximately 80%. Fortunately, the trap allows for selecting the nanocrystals with only a single emitter, and so this gives a route to isolating and integrating single emitters in a deterministic way. This demonstration is a promising step toward single-photon quantum information technologies that utilize single ions in a solid-state medium, particularly because Er emits in the low-loss fiber-optic 1550 nm telecom band.

Construction of near infrared light triggered nanodumbbell for cancer photodynamic therapy.

The application of photodynamic therapy (PDT) in deep tissue has been severely restricted by the poor photosensitizers loading and tissue-penetration of visible light for exciting the photosensitizers. How to prepare a nanocarrier with high drug loading amount and remote controllability still remains the challenge. In this article, a novel drug delivery system nanodumbbell was designed. The nanodumbbell was assembled from the hydrophobic upconverting nanoparticle (UCN) core and hydrophilic polymersome shell. The "nanodumbbell" offers possibilities to overcome the problem mentioned above. The UCN core works as a transducer to convert deeply penetrating near-infrared light to visible light to activate photosensitizers zinc (II) phthalocyanine (ZnPc) for photodynamic therapy. The polymersome lipid shell is used for loading ZnPc and protecting the whole system from nonspecific absorbance or corrosion during the transportation. The nanodumbbell is appealing because it can simultaneously achieve the high loading amount of ZnPc while avoiding UCNs aggregation. The reactive oxygen species (ROS) production test and PDT test in vitro suggested that the fluorescence emitted from the UCNs can be effectively transferred to the photosensitizers to produce cytotoxic ROS. When the UCN@lipid@polymersome nanodumbbell was decorated with targeting peptide (RGD), it presented better target specificity to cells. Our data suggest that this nanoparticle may serve as a useful nanoplatform for PDT treatment in deep-cancer therapy based on upconverting mechanism.

Multifunctional Microspheres Encoded with Upconverting Nanocrystals and Magnetic Nanoparticles for Rapid Separation and Immunoassays.

Immunoassays based on the downconversion target materials (organic dyes or quantum dots) lead to fairly strong spectral interference between the coded signal and reporter signal, which seriously affects the detection accuracy and hampers their applications. In this work, a new kind of upconverting nanocrystals encoded magnetic microspheres (UCNMMs) were designed and prepared successfully to solve the problem mentioned above. The UCNMMs were obtained by incorporating magnetic Fe3O4 nanoparticles and upconverting nanocrystals with polystyrene microspheres. Due to that upconverting nanocrystals (UCNs) and reporter signals are excitated by near-infrared and UV/visible light separately, immunoassays based on UCNMMs do not occur optical spectral interferences. Furthermore, these new functionalized UCNMMs have excellent properties in binding biomolecules and fast separating, which would have large potential applications in multiplexed assays.

Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake, and Cytotoxicity.

Although computer simulation and cell culture experiments have shown that elongated spherical particles can be taken up into cells more efficiently than spherical particles, experimental investigation on effects of these different shapes over the particle-membrane association has never been reported. Therefore, whether the higher cellular uptake of an elongated spherical particles is a result of a better particle-membrane association as suggested by some calculation works or a consequence of its influence on other cellular trans-membrane components involved in particle translocation process, cannot be concluded. Here, we study the effect of particle shape on the particle-membrane interaction by monitoring the association between particles of various shapes and lipid bilayer membrane of artificial cell-sized liposomes. Among the three shaped lanthanide-doped NaYF4 particles, all with high shape purity and uniformity, similar crystal phase, and surface chemistry, the elongated spherical particle shows the highest level of membrane association, followed by the spherical particle with a similar radius, and the hexagonal prism-shaped particle, respectively. The free energy of membrane curvature calculated based on a membrane indentation induced by a particle association indicates that among the three particle shapes, the elongated spherical particle give the most stable membrane curvature. The elongated spherical particles show the highest cellular uptake into cytosol of human melanoma (A-375) and human liver carcinoma (HepG2) cells when observed through a confocal laser scanning fluorescence microscope. Quantitative study using flow cytometry also gives the same result. The elongated spherical particles also possess the highest cytotoxicity in A-375 and normal skin (WI-38) cell lines, comparing to the other two shaped particles.