Spatiotemporally mapping temperature dynamics of lysosomes and mitochondria using cascade organelle-targeting upconversion nanoparticles.

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.

Single Small Extracellular Vesicle (sEV) Quantification by Upconversion Nanoparticles.

Cancer-derived small extracellular vesicles (sEVs) are potential circulating biomarkers in liquid biopsies. However, their small sizes, low abundance, and heterogeneity in molecular makeups pose major technical challenges for detecting and characterizing them quantitatively. Here, we demonstrate a single-sEV enumeration platform using lanthanide-doped upconversion nanoparticles (UCNPs). Taking advantage of the unique optical properties of UCNPs and the background-eliminating property of total internal reflection fluorescence (TIRF) imaging technique, a single-sEV assay recorded a limit of detection 1.8 × 106 EVs/mL, which was nearly 3 orders of magnitude lower than the standard enzyme-linked immunosorbent assay (ELISA). Its specificity was validated by the difference between EpCAM-positive and EpCAM-negative sEVs. The accuracy of the UCNP-based single-sEV assay was benchmarked with immunomagnetic-beads flow cytometry, showing a high correlation (R2> 0.99). The platform is suitable for evaluating the heterogeneous antigen expression of sEV and can be easily adapted for biomarker discoveries and disease diagnosis.

Upconversion nanoparticle-assisted single-molecule assay for detecting circulating antigens of aggressive prostate cancer.

Sensitive and quantitative detection of molecular biomarkers is crucial for the early diagnosis of diseases like metabolic syndrome and cancer. Here we present a single-molecule sandwich immunoassay by imaging the number of single nanoparticles to diagnose aggressive prostate cancer. Our assay employed the photo-stable upconversion nanoparticles (UCNPs) as labels to detect the four types of circulating antigens in blood circulation, including glypican-1 (GPC-1), leptin, osteopontin (OPN), and vascular endothelial growth factor (VEGF), as their serum concentrations indicate aggressive prostate cancer. Under a wide-field microscope, a single UCNP doped with thousands of lanthanide ions can emit sufficiently bright anti-Stokes' luminescence to become quantitatively detectable. By counting every single streptavidin-functionalized UCNP which specifically labeled on each sandwich immune complex across multiple fields of views, we achieved the Limit of Detection (LOD) of 0.0123 ng/ml, 0.2711 ng/ml, 0.1238 ng/ml, and 0.0158 ng/ml for GPC-1, leptin, OPN and VEGF, respectively. The serum circulating level of GPC-1, leptin, OPN, and VEGF in a mixture of 10 healthy normal human serum was 25.17 ng/ml, 18.04 ng/ml, 11.34 ng/ml, and 1.55 ng/ml, which was within the assay dynamic detection range for each analyte. Moreover, a 20% increase of GPC-1 and OPN was observed by spiking the normal human serum with recombinant antigens to confirm the accuracy of the assay. We observed no cross-reactivity among the four biomarker analytes, which eliminates the false positives and enhances the detection accuracy. The developed single upconversion nanoparticle-assisted single-molecule assay suggests its potential in clinical usage for prostate cancer detection by monitoring tiny concentration differences in a panel of serum biomarkers.

Stable and Highly Efficient Antibody-Nanoparticles Conjugation.

Functional ligands and polymers have frequently been used to yield target-specific bio-nanoconjugates. Herein, we provide a systematic insight into the effect of the chain length of poly(oligo (ethylene glycol) methyl ether acrylate) (POEGMEA) containing polyethylene glycol on the colloidal stability and antibody-conjugation efficiency of nanoparticles. We employed Reversible Addition-Fragmentation Chain Transfer (RAFT) to design diblock copolymers composed of 7 monoacryloxyethyl phosphate (MAEP) units and 6, 13, 35, or 55 OEGMEA units. We find that when the POEGMEA chain is short, the polymer cannot effectively stabilize the nanoparticles, and when the POEGMEA chain is long, the nanoparticles cannot be efficiently conjugated to antibody. In other words, the majority of the carboxylic groups in larger POEGMEA chains are inaccessible to further chemical modification. We demonstrate that the polymer containing 13 OEGMEA units can effectively bind up to 64% of the antibody molecules, while the binding efficiency drops to 50% and 0% for the polymer containing 35 and 55 OEGMEA units. Moreover, flow cytometry assay statistically shows that about 9% of the coupled antibody retained its activity to recognize B220 biomarkers on the B cells. This work suggests a library of stabile, specific, and bioactive lanthanide-doped nanoconjugates for flow cytometry and mass cytometry application.

A High-Throughput Colorimetric Microplate Assay for Determination of Plasma Arginase Activity.

Arginase, an enzyme involved in the urea cycle, is gaining attention as a critical player in numerous chronic pathologies. Additionally, increased activity of this enzyme has been shown to correlate with poor prognosis in a range of cancers. Colorimetric assays that measure the conversion of arginine to ornithine have long been used to determine the activity of arginase. However, this analysis is hindered by a lack of standardization across protocols. Here, we describe in detail a novel revision of the Chinard's colorimetric assay used to determine arginase activity. Dilution series of patient plasma are plotted to form a logistic function, from which activity can be interpolated by comparison to an ornithine standard curve. Inclusion of patient dilution series rather than a single point increases the robustness of the assay. This high-throughput microplate assay analyzes 10 samples per plate to produce highly reproducible results.

Lanthanide Ion Resonance-Driven Rayleigh Scattering of Nanoparticles for Dual-Modality Interferometric Scattering Microscopy.

Light scattering from nanoparticles is significant in nanoscale imaging, photon confinement. and biosensing. However, engineering the scattering spectrum, traditionally by modifying the geometric feature of particles, requires synthesis and fabrication with nanometre accuracy. Here it is reported that doping lanthanide ions can engineer the scattering properties of low-refractive-index nanoparticles. When the excitation wavelength matches the ion resonance frequency of lanthanide ions, the polarizability and the resulted scattering cross-section of nanoparticles are dramatically enhanced. It is demonstrated that these purposely engineered nanoparticles can be used for interferometric scattering (iSCAT) microscopy. Conceptually, a dual-modality iSCAT microscopy is further developed to identify different nanoparticle types in living HeLa cells. The work provides insight into engineering the scattering features by doping elements in nanomaterials, further inspiring exploration of the geometry-independent scattering modulation strategy.

Multi-metal nanomaterials obtained from oil/water interface as effective catalysts in reduction of 4-nitrophenol.

In this study Pt and Pd-based nanostructured thin films have been successfully fabricated by room temperature self-assembly of metal nanoparticles (NPs) at the interface between toluene and water without/with using stabilizers such as graphene oxide (GO) or aminoclay (AC). Successful formation of these thin films is investigated by transmission electron microscopy (TEM), energy dispersive analysis of X-ray (EDAX) and X-ray diffraction (XRD). Catalytic hydrogenation of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) was investigated using thin film nanocatalysts. The as synthesized nanostructured thin films exhibit high catalytic activity toward hydrogenation reaction of 4-NP. This study highlights the value of nano alloy thin films and their ability as catalyst in catalytic hydrogenation reaction.

Electrocatalytic oxidation behavior of NADH at Pt/Fe3O4/reduced-graphene oxide nanohybrids modified glassy carbon electrode and its determination.

We have developed Pt/Fe3O4/reduced-graphene oxide nanohybrids modified glassy carbon (Pt/Fe3O4/RGO/GC) electrode as a novel system for the preparation of electrochemical sensing platform. Characterization of as-made composite was determined using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM) and energy-dispersive analysis of X-ray (EDAX) where the Pt, Fe, Si, O and C elements were observed. The Pt/Fe3O4/RGO/GC electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Due to the synergistic effect between Pt, Fe3O4 and RGO, the nanohybrid exhibited excellent performance toward dihydronicotinamide adenine dinucleotide (NADH) oxidation in 0.1M phosphate buffer solution, pH7.0, with a low detection limit of 5nM.