One-Shot Dual-Detection-Based Single-Molecule Super-Resolution Imaging Method for Real-Time Observation of Spatiotemporal Catalytic Activity Variations on the Plasmonic Gold Nanoparticle Surface.

Understanding the relationship between the surface properties of a single plasmonic nanoparticle and its catalytic performance is critical for developing highly efficient nanocatalysts. In this study, a one-shot dual-detection-based single-molecule super-resolution imaging method in the evanescent field was developed to observe real-time spatiotemporal catalytic activity on a single plasmonic gold nanoparticle (AuNP) surface. The scattering intensity of AuNPs and the fluorescence of resorufin molecules produced on the AuNP surface were obtained simultaneously to investigate the relationship between nanoparticles and catalytic reactions at a single-molecule level. Chemisorbed adsorbates (i.e., catalytic product and resorufin) changed the electron density of individual AuNPs throughout the catalytic cycle, resulting in the fluctuation of the scattering intensity of individual AuNPs, which was attributed to the electron transfer between reactant resazurin molecules and AuNPs. The increase in the electron density of individual AuNPs affected the catalytic reaction rate. Furthermore, sequential mapping of individual catalytic events at the subdiffraction limit resolution was completed for real-time surface dynamics and spatiotemporal activity variations on the single AuNP surface. The developed method can aid in understanding surface-property-dependent catalytic kinetics and facilitate the development of nanoparticle-based heterogeneous catalysts at subdiffraction limit resolution.

Organelle Proximity Analysis for Enhanced Quantification of Mitochondria-Endoplasmic Reticulum Interactions in Single Cells via Super-Resolution Microscopy.

Mitochondria (MT) and the endoplasmic reticulum (ER) maintain lipid and calcium homeostasis through membrane contacts, particularly MT-ER contacts (MERCs), spanning distances from 10 to 50 nm. However, the variation of different distance ranges and the metabolic factors influencing this variation remain poorly understood. This study employed microfluidic chip-based super-resolution microscopy in conjunction with a Moore-Neighbor tracing-incorporated organelle proximity analysis algorithm. This approach enabled precise three-dimensional localization of single-fluorescence protein molecules within narrow and irregular membrane proximities. It achieved lateral localization precision of less than 20 nm, resulting in a minimum MERC distance of approximately 8 nm in spatial and mean distances across multiple threshold ranges. Additionally, we demonstrated that the MERC distance variation was correlated with MT size rather than ER width. The proportion of each distance range varied significantly after the stimuli. Free cholesterol showed a negative correlation with various distances, while distances of 10-30 nm were associated with glucose, glutamine, and pyruvic acid. Furthermore, the 30-40 nm range was influenced by citric acid. These results underscore the role of advanced subcellular organelle analysis in elucidating the single-molecule behavior and organelle morphology in single-cell studies.

Microfluidic Engineering of Addressable Multicompartmental Microspheres for Multicellular Systems.

With the advantages of high-throughput manufacturing and customizability, on-microsphere construction of in vitro multicellular analytical systems has garnered significant attention. However, achieving a precise, biocompatible cell arrangement and spatial signal analysis in hydrogel microspheres remains challenging. In this work, a microfluidic method is reported for the biocompatible generation of addressable supersegmented multicompartmental microspheres. Additionally, these microspheres are developed as novel label-free multicellular systems. In the microfluidic approach, controllable microfluidics is used to finely tune the internal microstructure of the microspheres, and the gas ejector ensures the biocompatibility of the preparation process. As a proof of concept, six- and twenty-compartment microspheres were obtained without the addition of any biohazardous reagents. For microsphere decoding, the visualization of two basic compartments can provide clues for identifying label-free cells due to the structural regularity of the microspheres. Finally, by encapsulating cells of different types, these microspheres as multicellular systems were successfully used for cell coculture and drug testing. These biocompatible, scalable, and analyzable microspheres will open up new prospects for biomedical analysis.

Fabrication and Performance of Bubble-Containing Multicompartmental Particles: Novel Self-Orienting Carriers.

In this work, a class of bubble-containing multicompartmental particles with self-orienting capability is developed, where a single bubble is enclosed at the top of the super-segmented architecture. Such bubbles, driven by potential energy minimization, cause the particles to have a bubble-upward preferred orientation in liquid, enabling efficient decoding of their high-density signals in an interference-resistant manner. The particle preparation involves bubble encapsulation via the impact of a multicompartmental droplet on the liquid surface and overall stabilization via rational crosslinking. The conditions for obtaining these particles are systematically investigated. Methodological compatibility with materials is demonstrated by different hydrogel particles. Finally, by encapsulating cargoes of interest, these particles have found broad applications in actuators, multiplexed detection, barcodes, and multicellular systems.

Gravity-Oriented Microfluidic Device for Biocompatible End-to-End Fabrication of Cell-Laden Microgels.

Droplet microfluidics are extensively utilized to generate monodisperse cell-laden microgels in biomedical applications. However, maintaining cell viability is still challenging due to overexposure to harsh conditions in subsequent procedures that recover the microgels from the oil phase. Here, a gravity-oriented microfluidic device for end-to-end fabrication of cell-laden microgels is reported, which integrates dispersion, gelation, and extraction into a continuous workflow. This innovative on-chip extraction, driven by native buoyancy and kinetically facilitated by pseudosurfactant, exhibits 100% retrieval efficiency for microgels with a wide range of sizes and stiffnesses. The viability of encapsulated cells is perfectly maintained at ≈98% with minimal variations within and between batches. The end-to-end fabrication remarkably enhances the biocompatibility and practicality of microfluidics-based cell encapsulation and is promising to be compatible with various applications ranging from single-cell analysis to clinical therapy.

Construction of Phosphorothiolated 2-Pyrrolidinones via Photoredox/Copper-Catalyzed Cascade Radical Cyclization/Phosphorothiolation.

A photoredox/copper-catalyzed cascade radical cyclization/phosphorothiolation reaction of N-allylbromoacetamides and P(O)SH compounds has been established. A broad range of novel nonfluorine- or difluoro-substituted 2-pyrrolidinones bearing the C(sp3)-SP(O)(OR)2 moiety can be conveniently constructed in moderate to good yields under mild conditions. Importantly, most of the tested phosphorothiolated 2-pyrrolidinones showed potent inhibitory effects toward both AChE and BChE.

An electrochemiluminescence microsensor based on DNA-silver nanoclusters amplification for detecting cellular adenosine triphosphate.

Adenosine triphosphate (ATP), as the primary energy source, plays vital roles in many cellular events. Developing an efficient assay is crucial to rapidly evaluate the level of cellular ATP. A portable and integrated electrochemiluminescence (ECL) microsensor array based on a closed bipolar electrode (BPE) was presented. In the BPE unit, the ECL chemicals and oxidation/reduction were separated from the sensing chamber. The ATP aptamer was assembled with single-stranded DNA (ssDNA) in the sensing chamber. ATP capture made the aptamer disassemble from the ssDNA and facilitated DNA-templated silver nanocluster (Ag NC) generation by the target-rolling circle amplification (RCA) reaction. The guanine-rich padlock sequence produced tandem periodic cytosine-rich sequences by the RCA, inducing Ag NC generation in the cytosine-rich region of the produced DNA strands through Ag+ reduction. The in situ Ag NC generation enhanced the circuit conductivity of the BPE and promoted the ECL reaction of [Ru(bpy)2dppz]2+/tripropylamine in the anodic reservoir. On this ECL microsensor, a good linear relationship of ATP was achieved ranging from 30 to 1000 nM. The ATP content in HepG2 cells was selectively and sensitively determined without complex pretreatment. The ATP amount of 25 cells could be successfully detected when a sub-microliter sample was loaded.

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

Understanding Macrophage-Tumor Interactions: Insights from Single-Cell Behavior Monitoring in a Sessile Microdroplet System.

Interaction between tumor-associated macrophages and tumor cells is crucial for tumor development, metastasis, and the related immune process. However, the macrophages are highly heterogeneous spanning from anti-tumorigenic to pro-tumorigenic, which needs to be understood at the single-cell level. Herein, a sessile microdroplet system designed for monitoring cellular behavior and analyzing intercellular interaction, demonstrated with macrophage-tumor cell pairs is presented. An automatic procedure based on the inkjet printing method is utilized for the precise pairing and co-encapsulation of heterotypic cells within picoliter droplets. The sessile nature of microdroplets ensures controlled fusion and provides stable environments conducive to adherent cell culture. The nitric oxide generation and morphological changes over incubation are explored to reveal the complicated interactions from a single-cell perspective. The immune response of macrophages under distinct cellular microenvironments is recorded. The results demonstrate that the tumor microenvironment displays a modulating role in polarizing macrophages from anti-tumorigenic into pro-tumorigenic phenotype. The approach provides a versatile and compatible platform to investigate intercellular interaction at the single-cell level, showing promising potential for advancing single-cell behavior studies.

A biosensor method based on surfactant-mediated surface droplet evaporation for the detection of Aflatoxin B1.

Aflatoxin B1 (AFB1) is a common mycotoxin that is of significant global concern due to its impact on food safety. Herein, we innovatively develop a sensing platform to detect AFB1 based on evaporation of surfactant solutions on the hydrophobic surface, resulting in dried patterns with varied sizes. The surfactant CTAB solution produces a relatively large dried pattern due to the surface wetting. However, the reduction in the dried pattern size is found when the mixture of CTAB and AFB1 aptamer is tested, because the formation of CTAB/aptamer complex. Moreover, the dried pattern size of the mixture of CTAB, aptamer, and AFB1 increases due to the specific binding of AFB1 to its aptamer. Using this innovative strategy, the AFB1 detection can be fulfilled with a detection limit of 0.77 pg/mL. As a simple, convenient, inexpensive, and label-free method, the surfactant-mediated surface droplet evaporation-based biosensor is very promising for various potential applications.

Establishment of microneurovascular units in alginate hydrogel microspheres to reveal the anti-hypoxic effect of salidroside.

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Evaluation of aconitine cardiotoxicity with a heart-on-a-particle prepared by a microfluidic device.

A heart-on-a-particle model based on multicompartmental microgel is proposed to simulate the heart microenvironment and study the cardiotoxicity of drugs. The relevant microgel was fabricated by a biocompatible microfluidic-based approach, where heart function-related HL-1 and HUVEC cells were arranged in separate compartments. Finally, the mechanism of aconitine-induced heart toxicity was elucidated using mass spectrometry and molecular biotechnology.