Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification.

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.

Dye-sensitized rare-earth-doped nanoprobe for simultaneously enhanced NIR-II imaging and precise treatment of bacterial infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for causing life-threatening infections that result in high morbidity and mortality rates. The development of advanced imaging and therapeutic methods for in vivo diagnosis and treatment of MRSA infections remains challenging. Here, we develop a hybrid nanoplatform based on rare-earth-doped nanoparticles (RENPs) sensitized by a moiety-engineered near-infrared (NIR) TPEO-820 dye and with a ZIF-8 layer that incorporates CysNO, a photochemically triggered nitric oxide donor. We then use the hybrid for both NIR-II bioimaging and photoactivatable treatment of MRSA-infected wounds. We show that the NIR dye sensitization leads to an 8.5-fold enhancement of the downshifting emission and facilitates deep-tissue NIR-II imaging of bacterial infections. Moreover, the sensitization strategy enhances the UV emission of RENPs by two orders of magnitude, leading to the efficiently controllable release of nitric oxide for effective disinfection of MRSA in vitro and in vivo. The hybrid nanoplatform thus offers promising opportunities for simultaneous localization and controllable treatment of MRSA. STATEMENT OF SIGNIFICANCE: Early detection and treatment of MRSA infections are crucial for reducing public health risks. It is a significant challenge that develops sensitive in vivo diagnosis and complete elimination of drug-resistant bacterial infections. Herein, a nanoplatform has been developed for photoactivatable therapy of MRSA infections and deep tissue NIR-II imaging. This platform utilizes lanthanide-doped rare earth nanoparticles (RENPs) that are sensitized by a moiety-engineered near-infrared (NIR) dye TPEO-820. The TPEO-820 sensitized RENPs exhibit 5 times increase in the release of NO concentration for MRSA treatment compared to unsensitized RENPs, enabling precise therapy of MRSA infection both in vitro and in vivo. Moreover, the platform demonstrates NIR-II luminescence in vivo, allowing for sensitive imaging in deep tissue for MRSA infection.

On-Chip Mirror Enhanced Multiphoton Upconversion Super-Resolution Microscopy.

Multiphoton upconversion super-resolution microscopy (MPUM) is a promising imaging modality, which can provide increased resolution and penetration depth by using nonlinear near-infrared emission light through the so-called transparent biological window. However, a high excitation power is needed to achieve emission saturation, which increases phototoxicity. Here, we present an approach to realize the nonlinear saturation emission under a low excitation power by a simply designed on-chip mirror. The interference of the local electromagnetic field can easily confine the point spread function to a specific area to increase the excitation efficiency, which enables emission saturation under a lower excitation power. With no additional complexity, the mirror assists to decrease the excitation power by 10-fold and facilities the achievement of a lateral resolution around 35 nm, 1/28th of the excitation wavelength, in imaging of a single nanoparticle on-chip. This method offers a simple solution for super-resolution enhancement by a predesigned on-chip device.

Thioredoxin1 is a target to attenuate diabetes­induced RPE cell dysfunction in human ARPE19 cells by alleviating oxidative stress.

Diabetes­induced cell dysfunction of the retinal pigment epithelium (RPE) contributes to the initiation and progression of diabetic retinopathy (DR). Thioredoxin 1 (Trx1) plays a key role in DR. However, the effect and mechanism of Trx1 on diabetes­induced cell dysfunction of the RPE is not fully understood during DR. In the present study, the effect of Trx1 on this process and its related mechanism were investigated. A Trx1 overexpression cell line, ARPE19­Trx1/LacZ, was constructed and treated with or without high glucose (HG). Flow cytometry was used to analyze apoptosis of these cells and the mitochondrial membrane potential was analyzed using JC­1 staining solution. A DCFH­DA probe was also used to detect the reactive oxygen species (ROS) generation. Western blotting was used to examine the expression of related proteins in ARPE­19 cells after HG treatment. The results demonstrated that the RPE layer was damaged in clinical samples. ROS formation and RPE cell dysfunction increased after HG treatment in vitro. Besides, the expression of mitochondrial­mediated apoptosis related proteins (Bax, apoptosis­inducing factor, cytochrome C, Caspase3 and Caspase9) also increased; however, overexpression of Trx1 attenuated these changes and improved the function of ARPE19 cells. These results indicated that overexpression of Trx1 alleviated diabetes­induced RPE cell dysfunction in DR by attenuating oxidative stress.

Population Control of Upconversion Energy Transfer for Stimulation Emission Depletion Nanoscopy.

Upconverting stimulated emission depletion microscopy (U-STED) is emerging as an effective approach for super-resolution imaging due to its significantly low depletion power and its ability to surpass the limitations of the square-root law and achieve higher resolution. Though the compelling performance, a trade-off between the spatial resolution and imaging quality in U-STED has been recognized in restricting the usability due to the low excitation power drove high depletion efficiency. Moreover, it is a burden to search for the right power relying on trial and error as the underpinning mechanism is unknown. Here, a method is proposed that can easily predict the ideal excitation power for high depletion efficiency with the assistance of the non-saturate excitation based on the dynamic cross-relaxation (CR) energy transfer of upconversion nanoparticles. This allows the authors to employ the rate equation model to simulate the populations of each relevant energy state of lanthanides and predict the ideal excitation power for high depletion efficiency. The authors demonstrate that the resolution of STED with the assistance of nonsaturated confocal super-resolution results can easily achieve the highest resolution of sub-40 nm, 1/24th of the excitation wavelengths. The finding on the CR effect provides opportunities for population control in realizing low-power high-resolution nanoscopy.

Quantitative Point of Care Tests for Timely Diagnosis of Early-Onset Preeclampsia with High Sensitivity and Specificity.

Preeclampsia is a heterogeneous and multiorgan cardiovascular disorder of pregnancy. Here, we report the development of a novel strip-based lateral flow assay (LFA) using lanthanide-doped upconversion nanoparticles conjugated to antibodies targeting two different biomarkers for detection of preeclampsia. We first measured circulating plasma FKBPL and CD44 protein concentrations from individuals with early-onset preeclampsia (EOPE), using ELISA. We confirmed that the CD44/FKBPL ratio is reduced in EOPE with a good diagnostic potential. Using our rapid LFA prototypes, we achieved an improved lower limit of detection: 10 pg ml-1 for FKBPL and 15 pg ml-1 for CD44, which is more than one order lower than the standard ELISA method. Using clinical samples, a cut-off value of 1.24 for CD44/FKBPL ratio provided positive predictive value of 100 % and the negative predictive value of 91 %. Our LFA shows promise as a rapid and highly sensitive point-of-care test for preeclampsia.

Power-Dependent Optimal Concentrations of Tm3+ and Yb3+ in Upconversion Nanoparticles.

Lanthanide-doped upconversion nanoparticles (UCNPs) have enabled a broad range of emerging nanophotonics and biophotonics applications. Here, we provide a quantitative guide to the optimum concentrations of Yb3+ sensitizer and Tm3+ emitter ions, highly dependent on the excitation power densities. To achieve this, we fabricate the inert-core@active-shell@inert-shell architecture to sandwich the same volume of the optically active section. Our results show that highly doped UCNPs enable an approximately 18-fold enhancement in brightness over that of conventional ones. Increasing the Tm3+ concentration improves the brightness by 6 times and increases the NIR/blue ratio by 11 times, while the increase of Yb3+ concentration enhances the brightness by 3 times and only slightly affects the NIR/blue ratio. Moreover, the optimal doping concentration of Tm3+ varies from 2% to 16%, which is highly dependent on the excitation power density ranging from 102 to 107 W/cm2. This work provides a guideline for designing bright UCNPs under different excitation conditions.

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.

Aspect Ratio of PEGylated Upconversion Nanocrystals Affects the Cellular Uptake In Vitro and In Vivo.

The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a physical barrier to regulate and prevent the uptake of endogenous metabolites and xenobiotics. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. Therefore, there is considerable interest in identifying drug carriers that can maintain the biostability of therapeutic molecules and target their transport across the BBB. In this regard, upconversion nanoparticles (UCNPs) have become popular as a nanoparticle-based solution to this problem, with the additional benefit that they display unique properties for in vivo visualization. The majority of studies to date have explored basic spherical UCNPs for drug delivery applications. However, the biophysical properties of UCNPs, cell uptake and BBB transport have not been thoroughly investigated. In this study, we described a one-pot seed-mediated approach to precisely control longitudinal growth to produce bright UCNPs with various aspect ratios. We have systematically evaluated the effects of the physical aspect ratios and PEGylation of UCNPs on cellular uptake in different cell lines and an in vivo zebrafish model. We found that PEGylated the original UCNPs can enhance their biostability and cell uptake capacity. We identify an optimal aspect ratio for UCNP uptake into several different types of cultured cells, finding that this is generally in the ratio of 2 (length/width). This data provides a crucial clue for further optimizing UCNPs as a drug carrier to deliver therapeutic agents into the CNS. STATEMENT OF SIGNIFICANCE: The central nervous system (CNS) is protected by the blood-brain barrier (BBB), which acts as a highly selective semipermeable barrier of endothelial cells to regulate and prevent the uptake of toxins and pathogens. However, the BBB prevents most non-lipophilic drugs from reaching the CNS following systematic administration. The proposed research is significant because identifying the aspect ratio of drug carriers that maintains the biostability of therapeutic molecules and targets their transport across the blood-brain barrier (BBB) is crucial for designing an efficient drug delivery system. Therefore, this research provides a vital clue for further optimizing UCNPs as drug carriers to deliver therapeutic molecules into the brain.

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.

Enhancing Hybrid Upconversion Nanosystems via Synergistic Effects of Moiety Engineered NIR Dyes.

Hybrid upconversion nanosystems have been reported to improve the low absorption efficiency of lanthanide-doped upconversion nanoparticles (UCNPs). However, the low quantum yield and poor photostability of NIR dyes pose challenges for practical uses. Here, we introduce a bulky moiety, 4-(1,2,2-triphenylvinyl)-1,1'-biphenyl (TPEO), to enhance its quantum yield by suppressing the bond rotation and improve the stability by deactivating the photoinduced oxidization. Compared with the conventional IR806, the formed NIR dye, TPEO-Cy, has been characterized to deliver three times higher quantum yield and seven times better photostability. Moreover, we take advantage of the strong affinity of sulfonate chains on the TPEO-Cy to bind to the surface of UCNPs. Taking together the synergistic effect, we have achieved a 242-fold upconversion emission enhancement over the benchmark of IR806-sensitized system and an ∼800 000-fold increase than the bare UCNPs. Our design of the NIR dyes suggests a new scope to search for more efficient upconversion nanohybrids.

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.

Axial localization and tracking of self-interference nanoparticles by lateral point spread functions.

Sub-diffraction limited localization of fluorescent emitters is a key goal of microscopy imaging. Here, we report that single upconversion nanoparticles, containing multiple emission centres with random orientations, can generate a series of unique, bright and position-sensitive patterns in the spatial domain when placed on top of a mirror. Supported by our numerical simulation, we attribute this effect to the sum of each single emitter's interference with its own mirror image. As a result, this configuration generates a series of sophisticated far-field point spread functions (PSFs), e.g. in Gaussian, doughnut and archery target shapes, strongly dependent on the phase difference between the emitter and its image. In this way, the axial locations of nanoparticles are transferred into far-field patterns. We demonstrate a real-time distance sensing technology with a localization accuracy of 2.8 nm, according to the atomic force microscope (AFM) characterization values, smaller than 1/350 of the excitation wavelength.

Optimizing the Polymer Cloak for Upconverting Nanoparticles: An Evaluation of Bioactivity and Optical Performance.

The ability of upconversion nanoparticles (UCNPs) to convert low-energy near-infrared (NIR) light into high-energy visible-ultraviolet light has resulted in their development as novel contrast agents for biomedical imaging. However, UCNPs often succumb to poor colloidal stability in aqueous media, which can be conquered by decorating the nanoparticle surface with polymers. The polymer cloak, therefore, plays an instrumental role in ensuring good stability in biological media. This study aims to understand the relationship between the length and grafting density of the polymer shell on the physicochemical and biological properties of these core-shell UCNPs. Poly(ethylene glycol) methyl ether methacrylate block ethylene glycol methacrylate phosphate (PPEGMEMAn-b-PEGMP3) with different numbers of PEGMEMA repeating units (26, 38, and 80) was prepared and attached to the UCNPs via the phosphate ligand of the poly(ethylene glycol methacrylate phosphate) (PEGMP) block at different polymer densities. The in vitro and in vivo protein corona, cellular uptake in two-dimensional (2D) monolayer and three-dimensional (3D) multicellular tumor spheroid (MCTS) models, and in vivo biodistribution in mice were evaluated. Furthermore, the photoluminescence of single-polymer-coated UCNPs was compared in solid state and cancer cells using laser scanning confocal microscopy (LSCM). Our results showed that the bioactivity and luminescence properties are chain length and grafting density dependent. The UCNPs coated with the longest PPEGMEMA chain, grafted at low brush density, were able to reduce the formation of the protein corona in vitro and in vivo, while these UCNPs also showed the brightest upconversion luminescence in the solid state. Moreover, these particular polymer-coated UCNPs showed enhanced cellular uptake, extended in vivo blood circulation time, and more accumulation in the liver, brain, and heart.

Optical tweezers beyond refractive index mismatch using highly doped upconversion nanoparticles.

Optical tweezers are widely used in materials assembly1, characterization2, biomechanical force sensing3,4 and the in vivo manipulation of cells5 and organs6. The trapping force has primarily been generated through the refractive index mismatch between a trapped object and its surrounding medium. This poses a fundamental challenge for the optical trapping of low-refractive-index nanoscale objects, including nanoparticles and intracellular organelles. Here, we report a technology that employs a resonance effect to enhance the permittivity and polarizability of nanocrystals, leading to enhanced optical trapping forces by orders of magnitude. This effectively bypasses the requirement of refractive index mismatch at the nanoscale. We show that under resonance conditions, highly doping lanthanide ions in NaYF4 nanocrystals makes the real part of the Clausius-Mossotti factor approach its asymptotic limit, thereby achieving a maximum optical trap stiffness of 0.086 pN µm-1 mW-1 for 23.3-nm-radius low-refractive-index (1.46) nanoparticles, that is, more than 30 times stronger than the reported value for gold nanoparticles of the same size. Our results suggest a new potential of lanthanide doping for the optical control of the refractive index of nanomaterials, developing the optical force tag for the intracellular manipulation of organelles and integrating optical tweezers with temperature sensing and laser cooling7 capabilities.

Nanorods with multidimensional optical information beyond the diffraction limit.

Precise design and fabrication of heterogeneous nanostructures will enable nanoscale devices to integrate multiple desirable functionalities. But due to the diffraction limit (~200 nm), the optical uniformity and diversity within the heterogeneous functional nanostructures are hardly controlled and characterized. Here, we report a set of heterogeneous nanorods; each optically active section has its unique nonlinear response to donut-shaped illumination, so that one can discern each section with super-resolution. To achieve this, we first realize an approach of highly controlled epitaxial growth and produce a range of heterogeneous structures. Each section along the nanorod structure displays tunable upconversion emissions, in four optical dimensions, including color, lifetime, excitation wavelength, and power dependency. Moreover, we demonstrate a 210 nm single nanorod as an extremely small polychromatic light source for the on-demand generation of RGB photonic emissions. This work benchmarks our ability toward the full control of sub-diffraction-limit optical diversities of single heterogeneous nanoparticles.