Application of Exonuclease III-Aided Target Recycling in Flow Cytometry: DNA Detection Sensitivity Enhanced by Orders of Magnitude

DNA-functionalized microspheres in conjugation with flow cytometry detection are widely used for high-throughput nucleic acid assays. Although such assays are rapid and capable of simultaneous analysis of multiple nucleic acid analytes in a single test, the intrinsic limitation in sensitivity remains challenging. Here we report a simple, highly sensitive, and reproducible method based on Exonuclease III-aided target recycling technique applied for DNA quantification in flow cytometry. By loading a high density of Cy5-labeled probe DNA on microspheres (15 μm), we achieved hitherto unreported DNA detection limit of 3.2 pM in flow cytometry bead assay, enhancing the sensitivity by a factor of over 56.8 compared to the conventional direct hybridization bead assay. Furthermore, we evaluated multiplexing capability by simultaneous detections of two target DNAs with FAM and Cy5 reporter conjugated probes. Therefore, the novel Exonuclease III-amplified flow cytometry bead assay has great potential for the rapid, sensitive, and accurate detection and quantification of nucleic acids in clinical diagnosis and biomedical research

Taking Plasmonic Core–Shell Nanoparticles Toward Laser Threshold

The first experimental demonstration of lasing plasmonic nanoparticles in 2009 ignited interest in active plasmonic structures with optical gain. However, the understanding of lasing in plasmonic nanoparticles is largely incomplete, and even less is known about their characteristics as they are taken toward the lasing threshold. Here we present a computational method and predictions of the lasing wavelength and threshold gain for spherical core–shell nanostructures with a metal core and a gain medium in the shell. We demonstrate that light scattering provides a simple diagnostics method to establish how far a specific nanoparticle is from reaching the lasing threshold. We also show that these structures can enhance the electric field by a factor of over 1500 (at 99.9% of threshold gain) and beyond, taking biosensing with these “smart dust” nanoparticles into the single molecule sensitivity regime

New Class of Tetradentate β-Diketonate-Europium Complexes That Can Be Covalently Bound to Proteins for Time-Gated Fluorometric Application

Luminescent lanthanide complexes that can be covalently bound to proteins have shown great utility as biolabels for highly sensitive time-gated luminescence bioassays in clinical diagnostics and biotechnology discoveries. In this work, three new tetradentate β-diketonate–europium complexes that can be covalently bound to proteins to display strong and long-lived Eu³⁺ luminescence, 1,2-bis­[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BHHBCB-Eu³⁺), 1,2-bis­[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BPPBCB-Eu³⁺), and 1,2-bis­[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BTBBCB-Eu³⁺), have been designed and synthesized as biolabels for time-gated luminescence bioassay applications. The luminescence spectroscopy characterizations of the aqueous solutions of three complex-bound bovine serum albumin reveal that BHHBCB-Eu³⁺ has the strongest luminescence with the largest quantum yield (40%) and longest luminescence lifetime (0.52 ms) among the complexes, which is superior to the other currently available europium biolabels. The BHHBCB-Eu³⁺-labeled streptavidin was prepared and used for both the time-gated luminescence immunoassay of human prostate specific antigen and the time-gated luminescence microscopy imaging of a pathogenic microorganism Cryptosporidium muris. The results demonstrated the practical utility of the new Eu³⁺ complex-based biolabel for time-gated luminescence bioassay applications

High-Contrast Luminescent Immunohistochemistry Using PEGylated Lanthanide Complexes

Immunohistochemistry (IHC) using fluorescent probes provides high resolution with multiplexing capability, but the imaging contrast is limited by the brightness of the fluorescent probe and the intrinsic autofluorescence background from tissues. Herein, we improved the contrast by high-density labeling of long-lifetime lanthanide complexes and time-gated imaging. As the large (∼280 nm) Stokes shift of lanthanide complexes effectively prevents the issue of concentration quenching, we succeeded in conjugating seven europium complexes to an eight-arm hydrophilic poly(ethylene glycol) (PEG) linker for signal amplification with improved water solubility to the level of up to 10 mg/mL. Moreover, we demonstrated that both human epidermal growth factor receptor 2 (HER2) in a formalin-fixed paraffin-embedded (FFPE) tissue section and cytokeratin 18 (CK18) in a frozen section can be resolved with the enhanced contrast by 2-fold and 3-fold, respectively. Furthermore, we show that the PEGylation of multiple lanthanide complexes is compatible with tyramide signal amplification (TSA). This work suggests new opportunities for sensitive imaging of low-abundance biomarkers in a tissue matrix

Multicolor Barcoding in a Single Upconversion Crystal

We report the synthesis of luminescent crystals based on hexagonal-phase NaYF₄ upconversion microrods. The synthetic procedure involves an epitaxial end-on growth of upconversion nanocrystals comprising different lanthanide activators onto the NaYF₄ microrods. This bottom-up method readily affords multicolor-banded crystals in gram quantity by varying the composition of the activators. Importantly, the end-on growth method using one-dimensional microrods as the template enables facile multicolor tuning in a single crystal, which is inaccessible in conventional upconversion nanoparticles. We demonstrate that these novel materials offer opportunities as optical barcodes for anticounterfeiting and multiplexed labeling applications

Multicolor Barcoding in a Single Upconversion Crystal

We report the synthesis of luminescent crystals based on hexagonal-phase NaYF₄ upconversion microrods. The synthetic procedure involves an epitaxial end-on growth of upconversion nanocrystals comprising different lanthanide activators onto the NaYF₄ microrods. This bottom-up method readily affords multicolor-banded crystals in gram quantity by varying the composition of the activators. Importantly, the end-on growth method using one-dimensional microrods as the template enables facile multicolor tuning in a single crystal, which is inaccessible in conventional upconversion nanoparticles. We demonstrate that these novel materials offer opportunities as optical barcodes for anticounterfeiting and multiplexed labeling applications

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

Developing Red-Emissive Ruthenium(II) Complex-Based Luminescent Probes for Cellular Imaging

Ruthenium­(II) complexes have rich photophysical attributes, which enable novel design of responsive luminescence probes to selectively quantify biochemical analytes. In this work, we developed a systematic series of Ru­(II)-bipyrindine complex derivatives, [Ru­(bpy)_3‑<i>n</i>(DNP-bpy)_<i>n</i>]­(PF₆)₂ (<i>n</i> = 1, 2, 3; bpy, 2,2′-bipyridine; DNP-bpy, 4-(4-(2,4-dinitrophenoxy)­phenyl)-2,2′-bipyridine), as luminescent probes for highly selective and sensitive detection of thiophenol in aqueous solutions. The specific reaction between the probes and thiophenol triggers the cleavage of the electron acceptor group, 2,4-dinitrophenyl, eliminating the photoinduced electron transfer (PET) process, so that the luminescence of on-state complexes, [Ru­(bpy)_3‑<i>n</i>(HP-bpy)_<i>n</i>]²⁺ (<i>n</i> = 1, 2, 3; HP-bpy, 4-(4-hydroxyphenyl)-2,2′-bipyridine), is turned on. We found that the complex [Ru­(bpy)­(DNP-bpy)₂]²⁺ remarkably enhanced the on-to-off contrast ratio compared to the other two (37.8 compared to 21 and 18.7). This reveals a new strategy to obtain the best Ru­(II) complex luminescence probe via the most asymmetric structure. Moreover, we demonstrated the practical utility of the complex as a cell-membrane permeable probe for quantitative luminescence imaging of the dynamic intracellular process of thiophenol in living cells. The results suggest that the new probe could be a very useful tool for luminescence imaging analysis of the toxic thiophenol in intact cells

Double-Sensitive Drug Release System Based on MnO₂ Assembled Upconversion Nanoconstruct for Double-Model Guided Chemotherapy

A double-sensitive drug release system based on MnO₂ nanosheets was synthesized using a facile method for imaging guided chemotherapy in cancer cells. The upconversion nanoparticles (UCNPs) as core are used for upconversion luminescence (UCL) imaging, and the coating of mesoporous silica shows excellent ability for loading drug. The assembly with MnO₂ nanosheets can respond to low intracellular pH and GSH in cancer cells. The obtained UCNPs@mSiO₂–MnO₂ nanoparticles with excellent biocompatibility can be applied as drug carriers. As expected, the UCNPs@mSiO₂(DOX)–MnO₂ (UCDMs) nanoconstruct loading with DOX offer practical chemotherapeutic effects <i>in vitro</i>, suggesting the active drug release in cancer cells. The luminescence intensity and magnetic resonance signals increased with the decomposition of the MnO₂ nanosheets and the release of DOX under low pH and high levels of GSH. In addition, the UCDMs exhibit excellent intracellular UCL imaging, indicating that they can be used as a selective imaging agent in a cancerous environment. Thus, the UCDMs present potential application for use as theranostic agents in imaging guided therapy

One-Step Loading of Gold and Gd₂O₃ Nanoparticles within PEGylated Polyethylenimine for Dual Mode Computed Tomography/Magnetic Resonance Imaging of Tumors

We report here a facile method for one-step loading of gold (Au) and gadolinium oxide (Gd₂O₃) nanoparticles (NPs) within polyethylenimine (PEI) premodified with polyethtylene glycol (PEG) for dual mode computed tomography (CT) and magnetic resonance (MR) imaging of tumors. PEGylated PEI was used as a template to complex Au­(III) and Gd­(III) salts, followed by sodium borohydride reduction and acetylation of remaining PEI surface amines to generate the hybrid PEI@Au/Gd₂O₃ NPs. The hybrid NPs exhibit a remarkable colloidal stability and cytocompatibility and possess a high X-ray attenuation efficacy and <i>r</i>₁ relaxivity, enabling their uses for dual mode CT/MR imaging of tumors