The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Ligand-Receptor Interaction Triggers Hopping and Sliding Motions on Living Cell Membranes.

Exploring the surface-capturing and releasing processes of nanocargo on the living cell membrane is critical for understanding the membrane translocation process. In this work, we achieve total internal reflection scattering (TIRS) illumination on a commercial dark-field optical microscope without the introduction of any additional optical components. By gradually reducing the diaphragm size in the excitation light path, the angle of the incident beam can be well manipulated. Under optimal conditions, the excitation light can be totally reflected at the glass/water interface, resulting in a thin layer of evanescent field for TIRS illumination. Due to the exponential decay feature of the evanescent field, the displacement of the nanocargo along the vertical direction can be directly resolved in the intensity track. With this method, we selectively monitor the dynamics of the transferrin-modified nanocargo on the living cell membrane. Transition between confined diffusion and long-range searching is involved in the binding site recognition process, which exhibits non-Gaussian and nonergodic-like behavior. More interestingly, 2D fast sliding and 3D hopping motions are also distinguished on the fluidic cell membrane, which is essentially modulated by the strength of ligand-receptor interactions, as revealed by the free-energy profiles. These heterogeneous and dynamic interactions together control the diffusion mode of the nanocargo on the lipid membrane and, thus, determine the cellular translocation efficiency.

Nanoparticle-Coupled Single-Molecule Kinetic Fingerprinting for Enzymatic Activity Detection.

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.

Shining at the Tips: Anisotropic Deposition of Pt Nanoparticles Boosting Hot Carrier Utilization for Plasmon-Driven Photocatalysis.

Bimetallic nanostructures are a promising candidate for plasmon-driven photocatalysis. However, knowledge on the generation and utilization of hot carriers in bimetallic nanostructures is still limited. In this work, we explored Pt position-dependent photocatalytic properties of bimetallic Au nanobipyramids (Au NBPs) with single-molecule fluorescence imaging. Compared with all-deposited core-shell nanostructures (aPt-Au NBPs), single-molecule imaging and simulation results show that the end-deposited bimetallic nanostructures (ePt-Au NBPs) can maintain a strong electromagnetic (EM) field and further promote the generation and transfer of energetic hot electrons for photocatalysis. Even though the Pt lattice is more stable than Au, the strong EM field at the sharp tips can boost lattice vibration, where enhanced spontaneous surface restructuring for active reaction site generation takes place. Significantly enhanced catalytic efficiency from ePt-Au NBPs is observed in contrast to that of Au NBPs and aPt-Au NBPs. These microscopic evidences offer valuable guidelines to design plasmon-based photocatalysts, particularly for bimetallic nanostructures.

Rational Design of Novel Lipophilic Aggregation-Induced Emission Probes for Revealing the Dynamics of Lipid Droplets during Lipophagy and Ferroptosis.

Lipid droplets (LDs), as crucial organelles, play a significant role in some physiological processes. Monitoring the concentration of LDs and dynamic behaviors between LDs and other organelles during some physiological processes is important for studying their biological function and medical diagnosis. Herein, we report a series of aggregation-induced emission (AIE) probes AIE-Cbz-LD-Cn (n = 1, 3, 5, 7, OMe) based on the conjugation of quinoline-malononitrile (QM) and carbazole for tracking the dynamic changes of LDs and studying the association between LDs and lysosome/endoplasmic reticulum (ER). To our great delight, AIE-Cbz-LD-C3, AIE-Cbz-LD-C5, and AIE-Cbz-LD-C7 could aggregate in LDs accurately and light up the LDs with good photostability. Among them, AIE-Cbz-LD-C7 was used to visualize the interplay between LDs and lysosomes during lipophagy due to the excellent LD-specificity. We also succeeded in tracking the number of newborn LDs generated near the endoplasmic reticulum regions revealing that the number increased considerably during ferroptosis by using AIE-Cbz-LD-C7, which supplies useful evidence for the hypothesis that LDs generate from the ER. We expect the probe AIE-Cbz-LD-C7 would be a practical tool for tracking the physiological and pathological processes contacted with LDs.

Near-Infrared Photooxygenation Theranostics Used for the Specific Mapping and Modulating of Amyloid-ß Aggregation.

The photooxygenation of amyloid-ß (Aß) protein is considered a promising strategy against Alzheimer's disease (AD). The inhibition of Aß aggregation or depolymerization of Aß aggregates can effectively alleviate and improve the condition of AD. Herein, we report a series of "off-on" near-infrared quinolinium photosensitizers (QM20-QM22) based on D-π-A structures using a target-sensing catalyst activation (TaSCAc) strategy. They exhibit turn-on fluorescence when bonded to Aß aggregates and generate singlet oxygen to achieve the specific imaging and photooxygenation of Aß aggregates, leading to attenuated Aß aggregates, enhancing their clearance through the microglial lysosomal pathway, decreasing their neurotoxicity. This study will shed light on the development of the photooxygenation of misfolded proteins for the treatment of neurodegenerative diseases.

Charged Tubular Supramolecule Boosting Multivalent Interactions for the Drastic Suppression of Aß Fibrillation.

Anti-Aß therapy has dominated clinical trials for the prevention and treatment of Alzheimer's disease (AD). However, suppressing Aß aggregation and disintegrating mature fibrils simultaneously remains a great challenge. In this work, we developed a new strategy using a charged tubular supramolecule (CTS) with pillar[5]arene as the backbone and modifying amino and carboxyl groups at the tubular terminals (noted as CTS-A, CTS-A/C, and CTS-C, respectively) to suppress Aß fibrillation for the first time. According to the spectroscopic and microscopic characterizations, Aß40 fibrillation can be efficiently suppressed by CTS-A in a very low inhibitor:peptide (I:P) molar ratio (1:10). A greatly alleviated cytotoxic effect of Aß peptides after the inhibition or disaggregation process is further disclosed. The well-organized supramolecular structure drives multivalent interaction and gains enhanced efficiency on amyloid fibrillar modulation. These results open a new path for the design of supramolecules in the application of AD treatment.

Supramolecular Bioimaging through Signal Amplification by Combining Indicator Displacement Assay with Förster Resonance Energy Transfer.

Fluorescent chemosensors are powerful imaging tools in the fields of life sciences and engineering. Based on the principle of supramolecular chemistry, indicator displacement assay (IDA) provides an alternative approach for constructing and optimizing chemosensors, which has the advantages of simplicity, tunability, and modularity. However, the application of IDA in bioimaging continues to face a series of challenges, including interfering signals, background noise, and inconsistent spatial location. Accordingly, we herein report a supramolecular bioimaging strategy of Förster resonance energy transfer (FRET)-assisted IDA by employing macrocyclic amphiphiles as the operating platform. By merging FRET with IDA, the limitations of IDA in bioimaging were addressed. As a proof of concept, the study achieved mitochondria-targeted imaging of adenosine triphosphate in live cells with signal amplification. This study opens a non-covalent avenue for bioimaging with advancements in tunability, generality, and simplicity, apart from the covalent approach.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

Red-Emitting Carbon Nanodot-Based Wide-Range Responsive Nanothermometer for Intracellular Temperature Sensing.

Precise sensing of intracellular temperature can provide plenty of information on disease-related cell states and promote the development of a diagnostic method. Fluorescence-based nanothermometers, as the "noncontact" sensors, exhibit great advantages over traditional thermometers due to the dual function of imaging and sensing at the molecular level. Herein, we report a red-emitting carbon nanodots (RCDs)-based nanothermometer for intracellular temperature sensing. Results indicate that RCDs exhibit favorable temperature-responsive fluorescence property with a good linear relationship, reversibility, and reproducibility under heating and cooling treatments in a wide range from 4 to 80 °C. Meanwhile, the RCDs possess satisfactory thermal sensitivity and temperature resolution, which are superior or comparable to the current nanothermometers. The low cytotoxicity and excellent temperature-responsive fluorescence property of RCDs have also been verified in living cell studies. Therefore, the RCDs will be a promising nanothermometer for intracellular temperature sensing in diverse areas.

Fluorescent Carbon Dots for in Situ Monitoring of Lysosomal ATP Levels.

Monitoring the ATP levels in lysosomes in situ is crucial for understanding their involvement in various biological processes but remains difficult due to the interference of ATP in other organelles or the cytoplasm. Here, we report a lysosome-specific fluorescent carbon dot (CD), which can be used to detect ATP in acidic lysosomes with "off-on" changes of yellow fluorescence. These CDs were successfully applied in real-time monitoring of the fluctuating concentration of lysosomal ATP induced by drug stimulation (e.g., chloroquine, etoposide, and oligomycin). Because of the excellent specificity, these CDs are promising agents for drug screening and medical diagnostics through lysosomal ATP monitoring.

Recent advances of plasmonic nanoparticle-based optical analysis in homogeneous solution and at the single-nanoparticle level.

Plasmonic nanoparticles with special localized surface plasmon resonance (LSPR) characters have been widely applied for optical sensing of various targets. With the combination of single nanoparticle imaging techniques, dynamic information of reactions and biological processes is obtained, facilitating the deep understanding of their principle and design of outstanding nanomaterials. In this review, we summarize the recently adopted optical analysis of diverse analytes based on plasmonic nanoparticles both in homogeneous solution and at the single-nanoparticle level. A brief introduction of LSPR is first discussed. Colorimetric and fluorimetric homogeneous detection examples by using different sensing mechanisms and strategies are provided. Single plasmonic nanoparticle-based analysis is concluded in two aspects: visualization of chemical reactions and understanding of biological processes. The basic sensing mechanisms and performances of these systems are introduced. Finally, this review highlights the challenges and future trend of plasmonic nanoparticle-based optical analysis systems.

Nanocomposites Inhibit the Formation, Mitigate the Neurotoxicity, and Facilitate the Removal of ß-Amyloid Aggregates in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is a progressive and irreversible brain disorder. Recent studies revealed the pivotal role of ß-amyloid (Aß) in AD. However, there is no conclusive indication that the existing therapeutic strategies exerted any effect on the mitigation of Aß-induced neurotoxicity and the elimination of Aß aggregates simultaneously in vivo. Herein, we developed a novel nanocomposite that can eliminate toxic Aß aggregates and mitigate Aß-induced neurotoxicity in AD mice. This nanocomposite was designed to be a small-sized particle (14 ± 4 nm) with Aß-binding peptides (KLVFF) integrated on the surface. The nanocomposite was prepared by wrapping a protein molecule with a cross-linked KLVFF-containing polymer layer synthesized by in situ polymerization. The presence of the nanocomposite remarkably changed the morphology of Aß aggregates, which led to the formation of Aß/nanocomposite coassembled nanoclusters instead of Aß oligomers. With the reduction of the pathological Aß oligomers, the nanocomposites attenuated the Aß-induced neuron damages, regained endocranial microglia's capability to phagocytose Aß, and eventually protected hippocampal neurons against apoptosis. Thus, we anticipate that the small-sized nanocomposite will potentially offer a feasible strategy in the development of novel AD treatments.

Single-Particle Tracking with Scattering-Based Optical Microscopy.

Different from traditional ensemble measurement methods, single-particle tracking (SPT) is a powerful approach to study the distribution of dynamic processes in a complex environment, providing crucial information from individual objects. This Feature summarizes the optical microscopic techniques and data analysis methods for scattering-based SPT. Some essential SPT-based applications within the cell are also delineated.

Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles.

Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy transfer (LRET) between the aptamer-modified upconversion nanoparticles (UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1, because the aptamers possess stronger affinity toward AFB1 than GNPs. Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.

Single-Particle Enumeration-Based Sensitive Glutathione S-Transferase Assay with Fluorescent Conjugated Polymer Nanoparticle.

Glutathione S-transferase (GST) is a group of multifunctional enzyme and participates in many physiological processes, such as xenobiotic biotransformation, drug metabolism, and degradation of toxic products. Herein, we demonstrate a label-free fluorescent conjugated polymer nanoparticle (FCPNPs)-based single-particle enumeration (SPE) method for the sensitive GST assay. Fluorescence resonance energy transfer (FRET) is formed between the glutathione-modified FCPNPs (FCPNPs-GSH) and polyethylenimine-capped gold nanoparticles (GNPs@PEI). Therefore, the fluorescence of FCPNPs-GSH is quenched remarkably. In the presence of GST, GNPs@PEI stay away from FCPNPs-GSH due to the specific interaction between FCPNPs-GSH and GST, leading to the inhibition of FRET. As a result, the fluorescence emission of FCPNPs-GSH is restored, which is reflected as the increase of the number of fluorescent particles in the microscopic image. By statistically counting the target concentration-dependent fluorescent particle number, accurate quantification of GST is achieved. The linear range from 0.01 to 6 µg/mL is obtained for GST assay and the limit-of-detection (LOD) is 1.03 ng/mL, which is much lower than the ensemble fluorescence spectra measurements in bulk solution. In urine sample assay, satisfactory recoveries in the range of 97.5-106.5.0% are achieved. Because of the high sensitivity and excellent specificity, this method can be extended to the detection of other disease-related biomolecules in the future.

Efficient Modulation of ß-Amyloid Peptide Fibrillation with Polymer Nanoparticles Revealed by Super-Resolution Optical Microscopy.

ß-Amyloid peptide (Aß) aggregation is the essential hallmark of neurodegenerative disorders such as Alzheimer's disease. Efficient inhibitors are highly desired for the prevention of Aß assembly that has been considered as the primary therapeutic strategy for neurodegenerative diseases. Apart from this, visualization of the aggregates and morphology at high spatial resolution is widely considered of crucial significance on biological treatment. In this work, we have developed small-sized (with diameter of ∼4.7 nm) and positively charged fluorescent conjugated polymer nanoparticles (CPNPs) with strong inhibition effect on Aß1-40 peptides fibrillation. Interestingly, the CPNPs also possess excellent photophysical properties, including high photon counts, robust blinking, and repetitive fluorescence switching, that are especially suitable for localization-based super-resolution imaging. Spatial resolution of ∼20 nm for these blinking CPNPs is readily achieved. According to the optical microscopic results, it was found that binding of CPNPs to the terminal of seed fibrils can effectively inhibit the fibrillation process. Owing to these attractive biological and unique photophysical properties, the small-sized CPNPs show high potential in a variety of super-resolution based biological applications.

Single-Particle Enzyme Activity Assay with Spectral-Resolved Dark-Field Optical Microscopy.

In a clinical assay, enzymes are essential biomarkers for human disease diagnosis. In this work, a spectral-resolved single-particle detection (SPD) method is introduced to quantify alkaline phosphatase (ALP) activity in human serum with a supraparticle (SP) based on MnO2-modified gold nanoparticle (denoted as GNP@MnO2 SP) as the probe. In the presence of ALP, 2-phospho-l-ascorbic acid trisodium salt can be hydrolyzed into l-ascorbic acid, which serves as a good reduction agent to trigger the decomposition of the MnO2 shell on the GNP surface. Given that a trace amount of ALP exists, noticeable scattering color change can be detected at the single-particle level due to the sensitive localized surface plasmon resonance (LSPR) effect from GNPs. With spectral-resolved dark-field optical microscopy, a linear dynamic range of 0.06 to 2.48 mU/mL ( R2 = 0.99) and a very low limit of detection of 5.8 µU/mL for the ALP assay are readily achieved, which is more sensitive over the methods based on ensemble sample measurement. As a consequence, this strategy opens a new avenue for the design of an ultrasensitive detection method for disease-correlated biomarker diagnosis in the future.

Carbon Dots as Fluorescent/Colorimetric Probes for Real-Time Detection of Hypochlorite and Ascorbic Acid in Cells and Body Fluid.

Hypochlorite (ClO-) and ascorbic acid (AA) are reported to have a high correlation with oxidative stress and related diseases, so it is necessary and critical to develop sensitive and fast response sensors to investigate the dynamical variation of these redox substances, especially those sensors which can detect ClO- and AA in real time in two manners. However, it is still an unmet challenge for now. Herein, novel carbon dots (RD-CDs) which can respond to ClO- and AA rapidly, reversibly, and dynamically by fluorescence and colorimetry were synthesized. In the fluorescence manner, the constructed nanosensor possessed high selectivity toward ClO- in the range of 0.1-100 µM with a detection limit of 83 nM, and can be selectively recovered by AA. It endows this sensor with good capacity as a fluorescent probe for dynamic detection of ClO- and AA in living cells, which can be monitored by a fluorescence microscope. In the colorimetric manner, ClO- and AA can be detected by UV-vis in the range of 5-200 µM and 1-30 µM, respectively. The concentrations of ClO- and AA in humor can be measured by RD-CDs in both fluorescence and colorimetric mode. The results above-mentioned demonstrate its great potential in biosensing.

Hydrogen-Bond-Induced Emission of Carbon Dots for Wash-Free Nucleus Imaging.

Carbon dots (CDs) are emerging as powerful tools for biosensing and bioimaging because of their intrinsic properties such as abundant precursors, facile synthesis, high biocompatibility, low cost, and particularly robust tunability and stability. In this work, a new type of CDs was prepared from m-phenylenediamine and folic acid by hydrothermal method. Interestingly, the as-prepared CDs show blue emission in non-hydrogen-bonding solution, whereas robust green emission in hydrogen-bonding solution. Based on this phenomenon, a novel fluorescence sensing mechanism named as hydrogen-bonding-induced emission (HBIE) was proposed. The HBIE-CDs have large Stokes shift (141 nm) in water, good biocompatibility, and ultrasmall size, which facilitates their translocation into living cells. Very importantly, the as-prepared HBIE-CDs show strong affinity toward nucleic acid without interference from other biological species. After binding with DNA/RNA through hydrogen bond, as high as 6-fold green fluorescence enhancement of HBIE-CDs was observed. Since the nucleus is rich in DNA/RNA, these HBIE-CDs were successfully used for rapid and, especially, wash-free subcellular in situ imaging of the nucleus in living cells in a fluorescence turn on mode, which has a great practicability to be used for nucleus imaging in bioanalytical studies and clinical applications.