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Clinical applications of nanophosphors have gained extensive interest in research areas such as bioimaging and targeted drug delivery. The development of nontoxic semiconductor quantum dots (QDs), which can replace the conventional fluorescent probes, can bring significant developments in the bioimaging industry. This work reports the synthesis of monoclinic Y2O3:Eu QDs, without and with surface functionalization using PEG/folic acid at low temperature and its application in live cancer cell imaging. The synthesized quantum dots show sharp absorption in the short UV region and an intense red emission at 614 nm. Concentration-dependent optical properties are studied in detail, and color purity is measured. Transmission electron microscopy substantiates the monoclinic structure, crystalline nature, and the lower particle dimensions essential for the biological applications. The surface-modified sample is characterized for its structural and luminescence properties. Biocompatibility was ensured by performing MTT Assay on L6 skeletal muscle cell lines (normal) and MCF 7 cell lines (cancer) for the samples without and with surface modification, respectively. Fluorescence detection experiments on SKMEL cells using an uncapped sample prove the suitability of the material as a fluorescent probe. The effect of surface functionalization on imaging results was established by carrying out fluorescence detection experiments on MCF 7 cells using PEG-folic acid-functionalized sample, which resulted in enhanced cell uptake, specific binding, and bright fluorescence emission. Thus, this work authenticates the suitability of the material to be used as a reliable nanophosphor and an efficient fluorescent probe for imaging cancer cells. [ABSTRACT FROM AUTHOR]

Despite the promising potential of carbon dots (CDs) as a photoluminescent nanomaterial in advancing spectral analysis techniques for the detection of various harmful heavy metal ions such as Hg2+, Cu2+, Cd2+, and Pb2+, the fundamental challenge of effectively eliminating the interference of transition metal ions in multi-ion systems persists. In this study, we present straightforward, efficient, and versatile manganese-(II)-enhanced nitrogen and sulfur codoped carbon dots (Mn-(II)-N,SCDs) specifically designed for the highly selective and sensitive detection of Hg2+ ions. Mn-(II)-N,SCDs exhibited uniform particle size (∼2.0 nm) and demonstrated excellent fluorescence performance, characterized by high fluorescence intensity and quantum yield (QY = 48.71%). The incorporation of Mn2+ not only enhances the fluorescence characteristics but also serves to effectively block the surplus transition metal ion binding sites on the surface of carbon dots, thereby leading to a heightened selective response to Hg2+. Furthermore, the synthesized Mn-(II)-N,SCDs also exhibited low cytotoxicity and efficient cellular uptake, enabling fluorescence imaging of living cells. Importantly, the developed fluorescence sensor exhibited a highly specific response to Hg2+ ions even in the presence of other metal ions in phosphate-buffered solution (PBS), with a low detection limit of 0.29 nM (S/N = 3). The efficacy of the probe was successfully demonstrated through the determination of Hg2+ in live cells and natural coastal water samples. [ABSTRACT FROM AUTHOR]

The low stability and high toxicity of CsPbBr3 perovskite nanocrystals (PNCs) greatly limit their application in the biological field. Here, six different CsPbBr3 PNCs were prepared by ligand-assisted reprecipitation (LARP) and hot injection using oleic acid (OA), oleamine (OAm), (3-Aminopropyl) triethoxysilane (APTES) modification and SiO2 coating, respectively. Different ligands, shells, and synthesis methods give CsPbBr3 PNCs diverse optical properties, stability, and biocompatibility. Among them, OA-OAm-APTES-CsPbBr3@SiO2 has the highest photoluminescence quantum yield (PLQY) (80.74%) and higher stability (fluorescence intensity remains 70% after 15 days in an atmospheric environment). OA-OAm-APTES-CsPbBr3@SiO2 (30 μg/mL) was cocultured with liver cancer cells for 12 h, and the survival rate was 80%, indicating high biocompatibility. Under the observation of the microplate reader, bright green fluorescence appeared in the cytoplasm, indicating that OA-OAm-APTES-CsPbBr3@SiO2 has great application potential in the field of bioimaging. [ABSTRACT FROM AUTHOR]

Vesicle entrapment remains a major bottleneck for the application of fluorescent nanoprobes (FNPs) with excellent optical properties in the field of super-resolution imaging of subcellular structures in living cells. Here, we developed a cell-penetrating peptide, fR8, which enables efficient cytoplasmic delivery of 3.5 nm FNPs instead of being trapped in vesicles, with a delivery efficiency of up to 75.6%. Further, we have successfully combined fusion protein technology with FNPs-fR8 to achieve high specificity and stable labeling of FNPs to various subcellular structures within live cells. Utilizing the advantages of FNPs, such as high brightness and resistance to photobleaching, FNPs modified with fR8 and the HaloTag ligand can be used for long-term super-resolution imaging of subcellular structures in living cells, allowing us to study the dynamic interactions between them. This work provides a practical tool for applying FNPs to live-cell super-resolution imaging and expands the application of super-resolution imaging technology in live-cell studies. [ABSTRACT FROM AUTHOR]

Carbonized polymer dots (CPDs) have attracted great interest as a nanomaterial in biomedical applications, especially in imaging and photodynamic therapy (PDT). However, CPDs with an aggregation-induced effect for type I PDT has been rarely reported. In this work, N,N′-dimethylurea and citric acid were used as precursors to prepare CPDs (YCPDs) with aggregation-induced effect in one pot by the solvothermal method. The YCPDs have a yellow emission of 550 nm and a high quantum yield of 68%, which lays a solid foundation for cell imaging. In addition, YCPDs can produce •O2– under white light irradiation and obtain •OH through electron transfer or hydrogen atom abstraction. In vitro results show that YCPDs possess great cytocompatibility but exert severe cytotoxicity to HeLa cells with only 2% of cell viability when exposed to ultralow-power-intensity (2.5 mW/cm2) light irradiation. Therefore, YCPDs, as a promising type I PDT nanomaterial, have a great potential for cancer therapy. [ABSTRACT FROM AUTHOR]

Upconversion nanoparticles (UCNPs) co-doped with lanthanide ions have recently attracted significant attention as fluorescent probes for super-resolution microscopy (SRM). This is due to the advantages of UCNPs over other fluorescence probes, such as fluorescent proteins, owing to their unique optical properties, limited photobleaching, and sharp emissions. However, the concurrent emission of ultraviolet (UV) wavelength radiation by UCNPs and the potential for cell photodamage, which may limit useful live-cell analysis, have been overlooked. Here, UCNPs synthesized with eight commonly used combinations of Yb/Tm and Yb/Tm/Gd dopants were excited by either pulsed- or continuous-wave (CW) lasers to evaluate their UV emission. The ratio of emitted UV-A and UV-B was measured relative to blue emission at 475 nm, which is traditionally used for imaging during SRM. We demonstrate that most UCNP samples emit UV light and that the dopant concentration has a key role in generating UV emissions. In addition, the use of pulsed or CW lasers for excitation can lead to a large variation in the amount of UV emitted. This work highlights the importance of considering upconversion dopant composition and concentration, as well as analyzing the emission of synthesized UCNPs before their use to prevent unwanted cell photodamage during live-cell imaging by SRM. Moreover, it established a need to improve the visible light emission of UCNPs with respect to UV emission for SRM applications. [ABSTRACT FROM AUTHOR]

The variations of the microenvironment in cells are always associated with changes in the concentrations of some active species. However, visual detection of these species is still a challenge. In this work, near-infrared (NIR)-emitting fluorescent Ag2S quantum dots (Ag2S QDs) were synthesized via a rapid microwave reaction under normoxic conditions, which were utilized as sensitive fluorescent nanoprobes in sensing and bioimaging of glutathione (GSH) combined with CoOOH nanosheets (CoOOH NSs). The negatively charged Ag2S QDs can easily and effectively attach to the surface of CoOOH NSs to form the Ag2S QD/CoOOH nanoprobe through electrostatic interactions, accompanied by the quenching of the fluorescence of Ag2S QDs at 685 nm via the inner-filter effect (IFE). However, upon the addition of GSH, the fluorescence of Ag2S QDs was recovered due to the reduction of CoOOH NSs into Co2+ by GSH. Meanwhile, the fluorescent emission wavelength shifts slightly, which may result from the ligand exchange reaction between GSH and d-penicillamine (DPA) to form comodified Ag2S QDs. Based on the low toxicity and high tissue penetration depth of the NIR fluorescence of the Ag2S QDs and the good biocompatibility of CoOOH NSs, the nanoprobe composed of Ag2S QDs and CoOOH NSs was successfully applied in bioimaging, which can distinguish the normal cells and cancer cells, the semiquantitative content of endogenous and exogenous GSH. The as-developed strategy not only provides a simple, convenient, cost-effective, and rapid platform for the diagnosis of clinical diseases related to GSH but shows great prospects in fluorescence-assisted surgery. [ABSTRACT FROM AUTHOR]

Poly-(ether amine) (PEA)-based fluorescent polymer carbon dots (FPCDs) have been synthesized via a simple Schiff base reaction between poly-(ether amine) (PEA) and formaldehyde followed by its hydrothermal treatment. The resulting water-soluble FPCDs are 2 nm in size and show excitation-dependent emission properties. Blue-emissive FPCDs exhibit the maximum intensity of fluorescence at 440 nm under 360 nm excitation and show a high quantum yield of ∼18%. FPCDs are used for selective Fe3+ metal ion sensing in aqueous media through the fluorescence quenching of FPCDs with a limit of detection of ∼162 nM. Nontoxic FPCDs have been used for cancer cell imaging and also for intracellular Fe3+ metal ion sensing in cancer cells. FPCDs have been used for Fe3+ ion sensing in industrial effluents, and they serve as sensors even in the presence of other competing metal ions. To determine the real application potential of FPCDs as a sensor, FPCDs are used to establish the Fe3+ metal ion content in samples of spiked blood serum with remarkable specificity, sensitivity, and accuracy. Moreover, we proposed a possible sensing mechanism wherein Fe3+ metal ions interacted with the functional groups present on the surface of FPCDs, and fluorescence quenching occurred via a static quenching mechanism along with an inner filter effect (IFE). [ABSTRACT FROM AUTHOR]

Nanomaterials that respond to intracellular signals, such as pH, have the potential for many biomedical applications, such as drug delivery, because the assembly/disassembly process can be tailored to respond to a stimulus characteristic of a specific subcellular location. In this work, two rhodamine-peptides that form stable nanotubes at physiological pH but dissociate into highly fluorescent monomers within the acidified interior of endosomal/lysosomal cellular compartments have been developed. The rhodamine dipeptide conjugates, NH 2 -KK(RhB)-NH 2 ( RhB-KK ) and NH 2 -EK(RhB)-NH 2 ( RhB-KE ) with rhodamine B chromophores appended at the ε-amino position of a lysine residue, were shown to assemble into well-defined nanotubes at pH values above ∼4-5 and to dissociate into a fluorescent monomer state at lower pH values. The pH dependence of the assembly process was investigated using circular dichroism (CD) and fluorescence spectroscopy along with transmission electron microscopy (TEM), atomic force microscopy (AFM), and confocal imaging. Although the ring opening/closing transition of the rhodamine chromophore took place at pH 4.1 for both peptides, the onset of assembly began at pH 4.6 for RhB-KE and at a comparatively more basic pH (5.8) for RhB-KK . Accordingly, the rhodamine-peptides interconverted between three pH-dependent states: an open-ring, monomeric state (λ max 580 nm, λ ex 550 nm) at pH values at or below ∼4.6; a closed-ring, nanotube form that exhibits AIEE (λ max 460 nm, λ ex = 330 nm) at higher pH values; a closed-ring, nonemissive monomeric state that emerged below the critical micelle concentrations (CMC). The pH-responsive features of the peptides were evaluated by live-cell imaging in three cancer cell lines using confocal laser scanning microscopy (CLSM). Visualizing the cells after incubation with either RhB-KE or RhB-KK produced CLSM images with a punctate appearance in the Texas Red channel that colocalized with the lysosomes. These experiments indicate that the nanotubes were rapidly trafficked into the acidic lysosomal compartments within the cells, which induced dissociation into a monomeric, open state. Uptake inhibition studies suggested that cellular uptake was mediated by either caveolae- or clathrin-mediated endocytosis, depending on the cell line studied.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Owing to the biological significance of Cl - in cells, several chemical fluorescent probes and biosensors have been constructed to monitor this anion in the cytosol and subcellular organelles. However, a fluorescent probe for the selective detection of nuclear Cl - has not been described thus far. In the current study, we developed the first nuclear Cl - -selective biosensor, Cl-YFP-NLS, whose fluorescence was effectively quenched by this anion, and demonstrated that it is an efficient and powerful tool for determining the levels of nuclear Cl - . The results of cell studies using Cl-YFP-NLS as the probe suggested that the level of Cl - in the nucleus is lower than that in the cytosol. In addition, Cl-YFP-NLS along with lysosomal (Lyso-MQAE) and mitochondrial Cl - -selective fluorescent probes (Mito-MQAE) were utilized to determine the effects of various substances on the levels of Cl - in subcellular organelles. The results showed that lysosomotropic agents decrease the lysosomal Cl - concentration and increase the levels of mitochondrial and nuclear Cl - . Also, observations suggested that substances capable of inducing mitochondrial outer membrane permeabilization without inducing lysosomal membrane permeabilization increase mitochondrial and nuclear Cl - concentrations but they do not affect the level of lysosomal Cl - . Moreover, a substance directly disrupting nuclear pore complexes increased the level of nuclear Cl - and did not change the levels of lysosomal and mitochondrial Cl - . Finally, nucleus-affecting substances that cause deoxyribonucleic acid damage and activate p53 and Bax increased the levels of mitochondrial and nuclear Cl - without influencing the level of lysosomal Cl - .

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying endoplasmic reticulum (ER) dynamics in living cells. However, the lack of high-brightness, high-photostability, and stable labeling probes makes long-term super-resolution imaging of the ER still challenging. Herein, we reported a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA) that exhibits excellent brightness, photostability, and biocompatibility. GNP-Atto565-fR8-CA can simultaneously load multiple Atto565 dye molecules, significantly improving its brightness. Modifying the cell-penetrating peptide fR8 enables GNP-Atto565-fR8-CA to be efficiently delivered into the cytoplasm, overcoming the challenge of their easy entrapment in vesicles. Fluorescent labeling of ER proteins via Halo tags enables high specificity and stable labeling of GNP-Atto565-fR8-CA to the ER. The SIM super-resolution imaging results showed that GNP-Atto565-fR8-CA can track and observe the long-term dynamic process of the ER, and can also be used for long-term super-resolution imaging of the dynamic interactions between the ER and other organelles. This work offers a practical tool to study live-cell ER ultrastructure and dynamics.

Sulfur quantum dots (SQDs) are emerging fluorescent nanomaterials, whereas most of the methods for synthesizing SQDs are limited to thermal synthesis. In this study, we report the first case of a light-driven strategy for facile synthesis of SQDs and further applied the SQDs for fluorescence cell imaging. The light-driven synthesis strategy only utilized Na 2 S as the sulfur source and nano-TiO 2 as the photosensitizer. Under ultraviolet illumination, the nano-TiO 2 photosensitizer generated a large number of • O 2- and • OH to oxidize S 2- to S x 2- and further to elemental sulfur, which could be obtained as monodispersed SQDs after etching by H 2 O 2 . The prepared SQDs exhibit excellent tunable photoluminescence properties, superior stability, and a uniform small size, with particle diameters in the range of 0.5-4 nm, and the fluorescence absolute quantum yield is as high as 27.8%. Meanwhile, the prepared SQDs also exhibited extreme biocompatibility and stability, and we further applied it for intracellular imaging and Hg 2+ sensing with satisfactory results. In comparison to the widely reported thermal synthesis, the light-driven synthesis method is greener and simpler, opening a new way for the preparation of biocompatible SQDs.

The combination of closed bipolar electrodes (cBPE) with electrochemiluminescence (ECL) imaging has demonstrated remarkable capabilities in the field of bioanalysis. Here, we established a cBPE-ECL platform for ultrasensitive detection of alkaline phosphatase (ALP) and two-dimensional imaging of epidermal growth factor receptor (EGFR). This cBPE-ECL system consists of a high-density gold nanowire array in anodic aluminum oxide (AAO) membrane as the cBPE coupled with ECL of highly luminescent cadmium selenide quantum dots (CdSe QDs) luminophores to achieve cathodic electro-optical conversion. When an enzyme-catalyzed amplification effect of ALP with 4-aminophenyl phosphate monosodium salt hydrate (p-APP) as the substrate and 4-aminophenol (p-AP) as the electroactive probe is introduced, a significant improvement of sensing sensitivity with a detection limit as low as 0.5 fM for ALP on the cBPE-ECL platform can be obtained. In addition, the cBPE-ECL sensing system can also be used to detect cancer cells with an impressive detection limit of 50 cells/mL by labeling ALP onto the EGFR protein on A431 human epidermal cancer cell membranes. Thus, two-dimensional (2D) imaging of the EGFR proteins on the cell surface can be achieved, demonstrating that the established cBPE-ECL sensing system is of high resolution for spatiotemporal cell imaging.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.

Biomolecule-stabilized metal nanoclusters (MNCs) exhibit enormous potential as unique luminescent materials in various applications. However, synthesis of highly stable and bio-friendly fluorescent MNCs is still a challenge. Here, we report a facile synthesis of red-emitting silver nanoclusters (LYS–AgNCs) within a dithiothreitol-reduced lysozyme (LYS) scaffold. The nanoclusters exhibit a uniform size distribution, excellent water solubility, and superior photoluminescence properties featuring a quantum yield of 6.1%, a massive (280 nm) Stokes shift, and solid-state emission, and pH stability augments their applicability in dual-mode sensing platforms for Cu2+ and vitamin B12 (VB12). Two contrasting fluorescence (FL)-quenching mechanisms are responsible for the sensing; Cu2+-induced FL quenching occurs via a multifaceted mechanism involving both static and dynamic quenching, whereas the inner filter effect and Förster resonance energy transfer are mainly responsible for VB12-induced FL quenching. An inexpensive portable paper strip is fabricated using LYS–AgNCs for on-site field application, enabling instrument-free fast and visual detection of Cu2+ and VB12. Moreover, LYS–AgNCs also possess favorable biocompatibility against human cervical cancer cells (HeLa), making them a suitable nanoprobe for cell imaging and an efficient agent for detecting Cu2+ and VB12 inside live cells as well. Finally, we demonstrate the applicability of LYS–AgNCs for real-sample analysis of VB12 with a satisfactory outcome. [ABSTRACT FROM AUTHOR]

Zinc oxide nanoparticles (ZnO NPs) as promising and nontoxic fluorescent labels are currently developed for cell imaging owing to their excellent biocompatibility. However, the low intensity of the emission band and stability of ZnO NPs have restricted their further applications. In this work, 5 mol % rare-earth metal erbium (Er)-doped ZnO nanoparticles with poly-(ethylene glycol) (PEG) surface modification (Er-doped ZnO/PEG NPs) are fabricated by the sol–gel method. Their fluorescence characteristics and biodegradation behavior are investigated. The results indicate that the visible photoluminescence of Er-doped ZnO/PEG NPs has been dramatically enhanced by 631.6% compared with that of bare ZnO NPs. After being stored for one month, particle size variation is less than 1 nm, and fluorescence performance can be well retained. Furthermore, the degradation and cytotoxicity results confirm the degradable and biocompatible characteristics of Er-doped ZnO/PEG NPs, thus extending their potential applications for cell imaging. [ABSTRACT FROM AUTHOR]

We report the incorporation of salicylaldehyde derivatives onto the hydrazine-tagged amyloidogenic peptides by forming photoisomerizable hydrazones. These hydrazones with positive photochromism are photostable under physiological conditions and enable photoswitching without the addition of external reductants or high-power irradiation. By applying superresolution microscopy, we were able to distinguish polymorphic nanoscopic structures of the hydrazone-incorporated peptides in vitro under different buffer conditions. Moreover, the additive-free condition in our platform allows the exploration of detailed amyloid aggregate morphologies in live cells. [ABSTRACT FROM AUTHOR]

In practice, the application of the majority of carbon dots (CDs) has been limited in biomedical and bioimaging because of insufficient excitation/emission in near-infrared (NIR) regions. Near-infrared carbon dots (NIR-CDs) benefit from distinctive merits of low toxicity, strong biological penetration, minor susceptibility to the endogenous substances, and ameliorated NIR excitation/emission. Herein, multifunctional dual-emissive near-infrared carbon dots (dNIR-CDs) with intense NIR excitation/emission are synthesized and subsequently utilized for the construction of a ratiometric nanosensor for rapid and sensitive detection of lysozyme (LYZ). When LYZ was exposed to the nanosensor, fluorescence quenching occurred quickly at the emission peak of 680 nm by the dynamic quenching mechanism, while the fluorescence intensity of the emission peak of 460 nm remained unchanged. The developed nanosensor exhibited a wide linear response within the range of 0.03–10 μM and a reproducible detection limit at the level down to 7 nM for LYZ. By virtue of the high selectivity and sensitivity of the nanosensor, LYZ can be detected at trace levels in complicated human urine samples. Furthermore, the high throughput of the nanosensor for cell imaging is demonstrated. [ABSTRACT FROM AUTHOR]

A ratiometric optical dual-mode probe was first designed for the detection of doxorubicin (DOX) based on 11-mercaptoundecanoic acid-functionalized carbon dots (MUA-CDs). The carbon dots (CDs) were synthesized via hydrothermal carbonization of m-phenylenediamine and p-aminobenzoic acid, and then it was reacted with 11-mercaptoundecanoic acid by the amide reaction to obtain the MUA-CDs. The as-synthesized MUA-CDs have a high fluorescence quantum yield (59.4%) with bright green emission. Based on a specific electrostatic interaction and the fluorescence resonance energy transfer (FRET) between MUA-CDs and DOX, a ratiometric optical dual-mode probe was constructed for the detection of DOX with good selectivity, high sensitivity, and excellent anti-interference performance. When DOX was mixed the MUA-CDs, the fluorescence of the MUA-CDs at 513 nm decreased, while the fluorescence at 590 nm increased. With this information, a ratiometric fluorescent method was established to determine DOX with a linear range and limit of detection (LOD) of 0.25–19.96 and 0.66 μM and 50.33–80.88 and 0.23 μM, respectively. Meanwhile, the color of the MUA-CDs varied from colorless to dark yellow in the presence of DOX, and therefore, a ratiometric colorimetric method was applied to determine DOX in a range of 2.50–29.80 μM with a LOD of 0.75 μM. Finally, the as-constructed probe was successfully used to determine DOX in serum, urine, and live cells with satisfactory results. This work will contribute to the specific design of functional CDs and broaden the application of functional CDs in analytical detection and biological imaging. [ABSTRACT FROM AUTHOR]

A simple and fast-responsive fluorescent nanoprobe has been developed for fluorescence imaging of miR-630 during apoptosis in A549 cells. This nanoprobe is composed of gold nanoparticles (AuNPs) modified with thiol-terminated DNA hybridized with a TAMRA labeled DNA, resulting in fluorescence quenching due to the nanometal surface energy transfer (NSET) between the surface of AuNPs and TAMRA. The hybridization of miR-630 and DNA-coupled TAMRA liberates the TAMRA-modified DNA from AuNPs, resulting in the recovery of fluorescence. Based on the specific recognition and fluorescence turn-on phenomenon, the nanoprobe was successfully used for the detection of miR-630 in a wide range of 0.5–120 nM with a detection limit (LOD) of 0.36 nM. Furthermore, this fluorescent nanoprobe with high selectivity, good nuclease stability, and biocompatibility was applied for real-time monitoring the dynamic expression level of miR-630 in the process of apoptosis in A549 cells successfully. Therefore, by monitoring the expression of miR-630 in A549 cells during treatment with different drugs, this proposed method with the superiorities of simplicity, rapidness, and high selectivity might be prospective to understand the cell apoptosis-associated process and evaluating the effect of drug stimulation for further screening of anti-cancer drugs. [ABSTRACT FROM AUTHOR]

Recently emerging perovskite quantum dots (PQDs) with extremely high (near 100%) fluorescence quantum yield (FQY) may have attractive applications in sensing and bioimaging. However, practical applications of PQDs as signal probes in aqueous media, especially in long-time bioimaging, have been greatly limited by the well-known poor stability of PQDs in water. In this work, a single intact CsPbBr3 PQD nanocrystal was well encapsulated with a SiO2 shell using a simple and highly successful coating strategy. In the synthesis, polyvinyl pyrrolidone (PVP) instead of classic oleic acid (OA) and oleylamine (OAm) was used as the ligand and surfactant to increase the affinity between PQDs and SiO2, significantly increasing the coating efficiency of PQD single nanocrystals. Meanwhile, easily hydrolyzed trimethoxysilane (TMOS) was adopted as the silicon source to shorten the hydrolysis time of silane, which obviously decreased the degradation of PQDs during SiO2 coating. The obtained PVP-PQD@SiO2 core–shell nanoparticles were uniform in size, maintained a high FQY of PQDs, and exhibited excellent air and ultraviolet (UV) stability. Moreover, PVP-PQD@SiO2 core–shell nanoparticles could be easily modified with trimethoxyoctadecylsilane (C18) and lecithin, in turn, to assemble a lipid layer on their surfaces, yielding very well water-dispersed, long-term water-stable, highly fluorescent, and low-cytotoxic PVP-PQD@SiO2@C18-PC nanoprobes. It was demonstrated that these good properties of the obtained PVP-PQD@SiO2@C18-PC nanoprobes enabled their successful application in cell imaging. [ABSTRACT FROM AUTHOR]

With their intricate design, nanoparticles (NPs) have become indispensable tools in the quest for precise cellular targeting. Among various NPs, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging the surface-enhanced Raman scattering (SERS) effect. Their multiplexing abilities for the simultaneous identification of multiple biomarkers are important in the rapidly evolving landscape of diverse cellular phenotypes and biomolecular profiling. However, the challenge is ensuring that SERS NPs can effectively target specific cells and biomarkers among intricate cell types and biomolecules with high specificity. In this study, we improve the functionalization of SERS NPs, optimizing their targeting efficiency in cellular applications for ca. 160 nm NP-based probes. Spherical SERS NPs, conjugated with antibodies targeting epidermal growth factor receptor and human epidermal growth factor receptor 2, were incubated with cells overexpressing these proteins, and their specific binding potential was quantified at each stage by using flow cytometry to achieve optimal targeting efficiency. We determined that maintaining an average of 3.5 × 10 5 thiols per NP, 300 antibodies per NP, 18,000 NPs per cell, conducting a 15 min staining incubation at 4 °C in a shaker, and using SM(PEG) 12 as a cross-linker for the NP conjugation were crucial to achieve the highest targeting efficiency. Fluorescence and Raman imaging were used with these parameters to observe the maximum ability of these NPs to efficiently target suspended cells. These highly sensitive contrast agents demonstrate their pivotal role in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.

Nitrogen-(N) and sulfur-(S) codoped carbon dots (N/S-CDs) are facilely developed by a one-pot hydrothermal process from thiourea and o-phenylenediamine as carbon precursors. The as-fabricated N/S-CDs reveal a high quantum yield of 26% and a large Stokes shift up to 106 nm under the excitation of 450 nm with good water dispersibility as well as antiphotobleaching. With addition of Fe3+, fluorescence quenching happens with a good linearity within 0.3–70 μM and a low detection limit of 0.19 μM. However, the quenched fluorescence could be rapidly recovered with the addition of monohydrogen phosphate ([HPO4]2–) due to formation of the stable complex [Fe-(HPO4)]+ by a competitive reaction between the N/S-CDs-Fe3+ complex and [HPO4]2–, a good linearity is in the range of 0.3–60 μM with a low detection limit of 0.27 μM. Thus, a significant "on–off–on" fluorescence response is performed with obvious color change from yellow-dark brown-yellow. In addition, the practical application and the confocal microscopic images all suggest that the N/S-CDs could target both Fe3+ and [HPO4]2– in living cells, implying that the N/S-CDs possess the diverse applications for Fe3+ and [HPO4]2– recognitions in biomedical fields. [ABSTRACT FROM AUTHOR]

rRNAs are prevalent in living organisms. They are produced in nucleolus and mitochondria and play essential cellular functions. In addition to the primary biofunction in protein synthesis, rRNAs have been recognized as the emerging signaling molecule and drug target for studies on nucleolus morphology, mitochondrial autophagy, and tumor cell malignancy. Currently, only a few rRNA-selective probes have been developed, and most of them encounter the drawbacks of low water solubility, poor nuclear membrane permeability, short emission wavelength, low stability against photobleaching, and high cytotoxicity. These unfavorable properties of rRNA probes limit their potential applications. In the present study, we reported a new rRNA-selective and near-infrared fluorescent turn-on probe, 4MPS-TO , capable of tracking rRNA in live human cancer cells. The real-time monitoring performance in nucleolus morphology and mitochondrial autophagy is demonstrated in HeLa cells. The probe shows great application potential for being used as a rRNA-selective, sensitive, and photostable imaging tool in chemical biology study and drug screening.

Fluorescent probes are widely studied for metal ion detection because of their multiple favorable properties such as high sensitivity and selectivity, quick response, naked eye detection, and in situ monitoring. However, optical probes that can effectively detect the Cu(I) level in cell interiors are rare due to the difficulty associated with selectively and sensitively detecting this metal ion in a cell environment. Therefore, we designed and synthesized three water-soluble probes ( 1 - 3 ) with a 1,3,5-triazine core decorated by three substituents: a hydrophobic alkyl chain, a hydrophilic maltose, and a rhodamine B hydrazine fluorophore. Among the probes, probe 1 , which has an octyl chain and a branched maltose group, was the most effective at sensing Cu + in aqueous solution. Upon addition of Cu + , this probe showed a dramatic color change from colorless to pink in daylight and displayed an intense yellow fluorescence emission under 365 nm light. The limit of detection and dissociation constant ( K d ) of this probe were 20 nM and 1.1 × 10 -12 M, respectively, which are the lowest values reported to date. The two metal ion-binding sites and the aggregation-induced emission enhancement effect, endowed by the branched maltose group and the octyl chain, respectively, are responsible for the high sensitivity and selectivity of this probe for Cu + detection, as demonstrated by 1 H NMR, dynamic light scattering, and transmission electron microscopy studies. Furthermore, the probe successfully differentiated the Cu(I) level of cancer cells from that of the normal cells. Thus, the probe holds potential for real-time monitoring of Cu(I) level in biological samples and bioimaging of cancer cells.

Advancements in cell coculture systems with porous membranes have facilitated the simulation of human-like in vitro microenvironments for diverse biomedical applications. However, conventional Transwell membranes face limitations in low porosity (ca. 6%) and optical opacity due to their large thickness (ca. 10 μm). In this study, we demonstrated a one-step, large-scale fabrication of freestanding polymer ultrathin porous (PUP) membranes with thicknesses of hundreds of nanometers. PUP membranes were produced by using a gap-controlled bar-coating process combined with polymer blend phase separation. They are 20 times thinner than Transwell membranes, possessing 3-fold higher porosity and exhibiting high transparency. These membranes demonstrate outstanding molecular permeability and significantly reduce the cell-cell distance, thereby facilitating efficient signal exchange pathways between cells. This research enables the establishment of a cutting-edge in vitro cell coculture system, enhancing optical transparency, and streamlining the large-scale manufacturing of porous membranes.

The translational and rotational dynamics of anisotropic optical nanoprobes revealed in single particle tracking (SPT) experiments offer molecular-level information about cellular activities. Here, we report an automated high-speed multidimensional SPT system integrated with a deep learning algorithm for tracking the 3D orientation of anisotropic gold nanoparticle probes in living cells with high localization precision (<10 nm) and temporal resolution (0.9 ms), overcoming the limitations of rotational tracking under low signal-to-noise ratio (S/N) conditions. This method can resolve the azimuth (0°-360°) and polar angles (0°-90°) with errors of less than 2° on the experimental and simulated data under S/N of ∼4. Even when the S/N approaches the limit of 1, this method still maintains better robustness and noise resistance than the conventional pattern matching methods. The usefulness of this multidimensional SPT system has been demonstrated with a study of the motions of cargos transported along the microtubules within living cells.

Utilizing the mercury (Hg 2+ )-triggered deprotection of thioacetals to aldehyde groups, we constructed a water-soluble triphenylamine (TPA)-based polythioacetal PTA-TPA with thioacetal groups in the backbones for efficient sensing of Hg 2+ in aqueous solutions. PTA-TPA is conveniently prepared by polycondensation of 3, 6-dioxa-1,8-octanedithiol (DODT) with 4-( N , N -diphenylamino) benzaldehyde (TPA-CHO) using thiol-terminated mPEG2k-SH as a capping agent. The interaction of Hg 2+ with PTA-TPA activates the aggregation-induced emission (AIE) process of TPA-CHO molecules, which makes the emission enhanced, and the emission color changes to sky blue, while other metal ions do not interfere with the sensing process. PTA-TPA can be used as a highly selective and ultrafast detection system for Hg 2+ with a low detection limit (LOD) of 9.88 nM and a fast response of less than 1 min. In addition, the prepared test strips report the presence of Hg 2+ with an LOD as low as 1 × 10 -5 M. Intracellular imaging applications have demonstrated that PTA-TPA acts as a biocompatible fluorescent probe for efficient Hg 2+ sensing in HeLa cells. Overall, the PTA-TPA fluorescence probes have the characteristics of easy synthesis, cost-effective, ultrafast detection speed, high selectivity, and high sensitivity, which can be used in practical applications.

Hydrogels, three-dimensional hydrophilic polymeric networks with high water retaining capacity, have gained prominence in wound management and drug delivery due to their tunability, softness, permeability, and biocompatibility. Electron-beam polymerized poly(ethylene glycol) diacrylate (PEGDA) hydrogels are particularly useful for phototherapies such as antimicrobial photodynamic therapy (aPDT) due to their excellent optical properties. This work takes advantage of the transparency of PEGDA hydrogels to investigate bacterial responses to aPDT at the single-cell level, in real-time and in situ . The photosensitizer methylene blue (MB) was loaded in PEGDA hydrogels by using two methods: reversible loading and irreversible immobilization within the 3D polymer network. MB release kinetics and singlet oxygen generation were studied, revealing the distinct behaviors of both hydrogels. Real-time imaging of Escherichia coli was conducted during aPDT in both hydrogel types, using the Min protein system to report changes in bacterial physiology. Min oscillation patterns provided mechanistic insights into bacterial photoinactivation, revealing a dependence on the hydrogel preparation method. This difference was attributed to the mobility of MB within the hydrogel, affecting its direct interaction with bacterial membranes. These findings shed light on the complex interplay between hydrogel properties and the bacterial response during aPDT, offering valuable insights for the development of antibacterial wound dressing materials. The study demonstrates the capability of real-time, single-cell fluorescence microscopy to unravel dynamic bacterial behaviors in the intricate environment of hydrogel surfaces during aPDT.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Acoustic kinetic therapy systems that target specific organelles can improve the precision of a sonosensitizer, which is a perfect combination of targeted therapy and sonodynamic therapy (SDT) and plays an important role in current acoustic kinetic therapy. In this study, we loaded PpIX, a sonosensitizer, on targeted-functional carbon dots (CDs) via an amide reaction and then generated the mitochondria-targeted system (Mit-CDs-PpIX) and nucleus-targeted system (Nuc-CDs-PpIX), respectively, to deliver the sonosensitizer. Both systems exhibited minimal cytotoxicity in the absence of ultrasound stimulation. The efficacy of the targeted SDT systems was investigated using methylthiazol tetrazolium (MTT) assays, live/dead staining, flow cytometry, etc. Compared with the free PpIX and mitochondria-targeted system, the nucleus-targeted system is more potent in killing effect under ultrasound stimulation and induces apoptosis with higher intensity. To achieve the equal killing effect, the effective concentration of Nuc-CDs-PpIX is just one third of that of Mit-CDs-PpIX.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

The biomarker 3-nitrotyrosine (3-NT) is widely recognized as an indicator of renal oxidative stress injury, making its detection crucial for the early identification of renal insufficiency. This study presents the design and synthesis of a tetraphenylstyrene imidazole derivative (TIPE-MI), which is utilized to create a supramolecular probe in conjunction with cucurbit[8]uril (Q[8]) through host-guest interactions. The resulting supramolecular self-assembly exhibits excellent optical properties and has been employed for the specific detection of 3-NT through fluorescence quenching. The introduction of 3-NT resulted in a decreased fluorescence intensity of the yellow fluorescent probe, which gradually transitioned from bright yellow to light yellow and then became colorless as the 3-NT concentration was increased. A portable detection platform was devised to augment the efficiency of detection. In order to facilitate biological applications, we have substantiated the probe's exceptional precision in detecting 3-NT in biological samples, encompassing human serum and plasma. The probe also exhibited negligible cytotoxicity. The accumulation of the probe in renal cells elicited a fluorescence signal, thereby indicating the prospective viability of this system for visual detection with renal cytocompatibility.

Carbon quantum dots (CQDs) are one of the most promising types of fluorescent nanomaterials due to their exceptional water solubility, excellent optical properties, biocompatibility, chemical inertness, excellent refractive index, and photostability. Nitrogen-containing CQDs, which include amino acid based CQDs, are especially attractive due to their high quantum yield, thermal stability, and potential biomedical applications. Recent studies have attempted to improve the preparation of amino acid based CQDs. However, the highest quantum yield obtained for these dots was only 44%. Furthermore, the refractive indices of amino acid derived CQDs were not determined. Here, we systematically explored the performance of CQDs prepared from all 20 coded amino acids using modified hydrothermal techniques allowing more passivation layers on the surface of the dots to optimize their performance. Intriguingly, we obtained the highest refractive indices ever reported for any CQDs. The values differed among the amino acids, with the highest refractive indices found for positively charged amino acids including arginine-CQDs (∼2.1), histidine-CQDs (∼2.0), and lysine-CQDs (∼1.8). Furthermore, the arginine-CQDs reported here showed a nearly 2-fold increase in the quantum yield (∼86%) and a longer decay time (∼8.0 ns) compared to previous reports. In addition, we also demonstrated that all amino acid based CQD materials displayed excitation-dependent emission profiles (from UV to visible) and were photostable, water-soluble, noncytotoxic, and excellent for high contrast live cell imaging or bioimaging. These results indicate that amino acid based CQD materials are high-refractive-index materials applicable for optoelectronic devices, bioimaging, biosensing, and studying cellular organelles in vivo. This extraordinary RI may be highly useful for exploring cellular elements with different densities.

Intelligent DNA nanomachines are powerful and versatile molecular tools for bioimaging and biodiagnostic applications; however, they are generally constrained by complicated synthetic processes and poor reaction efficiencies. In this study, we developed a simple and efficient molecular machine by coupling a self-powered rolling motor with a lipidic nanoflare (termed RMNF), enabling high-contrast, robust, and rapid probing of cancer-associated microRNA (miRNA) in serum and living cells. The lipidic nanoflare is a cholesterol-based lipidic micelle decorated with hairpin-shaped tracks that can be facilely synthesized by stirring in buffered solution, whereas the 3D rolling motor (3D RM) is a rigidified tetrahedral DNA scaffold equipped with four single-stranded "legs" each silenced by a locking strand. Once exposed to the target miRNA, the 3D RM can be activated, followed by self-powered precession based on catalyzed hairpin assembly (CHA) and lighting up of the lipidic nanoflare. Notably, the multivalent 3D RM that moves using four DNA legs, which allows the motor to continuously and acceleratedly interreact with DNA tracks rather than dissociate from the surface of the nanoflare, yielded a limit of detection (LOD) of 500 fM at 37 °C within 1.5 h. Through the nick-hidden and rigidified structure design, RMNF exhibits high biostability and a low false-positive signal under complex physiological settings. The final application of RMNF for miRNA detection in clinical samples and living cells demonstrates its considerable potential for biomedical imaging and clinical diagnosis.

Covalent organic frameworks (COFs) have emerged as promising drug carriers due to their structural variability, inherent porosity, and customizable functions. However, most COFs used in drug delivery suffer from low cellular bioavailability and poor luminescence properties. In this study, we designed a series of size-tunable, crystalline, and red-fluorescent COF nanospheres (COFNSs) for trackable anticancer drug delivery. The semiconducting COFNSs were prepared by condensations of 1,3,5-triformylbenzene (TFB) with various dihydrazide blocks through the Schiff-base reaction, resulting in red emission at 647 nm and excellent fluorescence stability (∼100% for 1 h). Such fluorescence property allowed for systematic investigation of the cellular endocytosis pathway of COFNSs, visualization of drug delivery, and observation of the cell apoptosis process. The COFNSs exhibited high cell viability (>90%), a loading capacity of 183 wt % for the anticancer drug camptothecin (CPT), and significant enhancement in inhibiting 4T1 cancers both in vitro and in vivo as the CPT nanocarrier. This progress presents a valuable approach to design COF nanocarriers with integrated fluorescent and drug delivery functions.

The development of self-assembled organic dots (ODs) with lipophilic fluorescent properties has significant potential in various applications, especially in sensing and bioimaging. Herein, we report a one-pot gram-scale synthesis of lipophilic red-emitting oligomeric L-ODs with high quantum yield (39.3%), which is prepared by a solvothermal condensation of o-phenylenediamine and L-tartaric acid. Interestingly, as-prepared ODs show no solid and aqueous emission but intense red fluorescence in the majority of organic solvents. The titration of water indicates that ODs can be applied to the moisture detection of organic solvent. And since ODs exhibit excellent biocompatibility, they are successfully used as a lipophilic red-emitting imaging reagent. Furthermore, contrast experiment and theoretical simulation are adopted to reveal the photoluminescence mechanism of L-ODs. Thus, the hydrogen-bond-induced red-shift of the benzimidazole intermediate is thought to be the main reason for the intense red-emitting fluorescence of the prepared L-ODs. The gram-scale and simple reaction without any purification suggests that these lipophilic oligomeric ODs have a promising prospect in the application of fluorescent probe and lipophilic environment imaging. [ABSTRACT FROM AUTHOR]

8-Hydroxydeoxyguanosine (8-OHdG) is one of the main products of DNA oxidative stress injury and has been widely used for quantitative analyzing ROS levels. However, the intracellular detection of 8-OHdG is rarely reported and could provide for a novel tool for oxidative damage research. Here, a highly fluorescent silver nanocluster was synthesized by conjugating 12 cytosine bases as Ag nanocluster templates with 8-OHdG aptamer as the specific binding target (named 8-OHdG-12C), resulting in a 10-fold increase in fluorescence intensity of the as-synthesized probe. The 8-OHdG-12C@AgNCs showed a good fluorescence property and were stable for weeks. In addition, it can visualize intracellular 8-OHdG in a spatiotemporal manner. We used oxidative or reductive inducers of ROS and showed that the probe was located within cells and proportional to the cell oxidative damage induced. Together, these results indicate that 8-OHdG-12C@AgNCs hold great potential for in vivo 8-OHdG detection and may be a useful method to determine how DNA oxidative damage influences cancer cells and apoptosis. [ABSTRACT FROM AUTHOR]

We successfully prepared hybrid nanoparticles (NPs) with photofunctional interfaces between the citric acid (Cit) molecules and europium-(III) ion (Eu3+)-doped hydroxyapatite (HA) (Eu:HA) to provide bifunctional cell-labeling and cytostatic suppression. In particular, the Eu:HA NPs were synthesized in the presence of Cit, and the Cit molecules were hybridized with the Eu:HA NPs (Cit/Eu:HA). The physicochemical properties based on the interfacial Eu:HA–Cit interactions in the NPs were elucidated. The atomic structures on the Eu:HA NP surface layers were disordered by increasing the liquid–solid interfaces by the interactions between the Cit molecules and the Ca site of Eu:HA NPs. It was suggested that the Cit molecules that interacted with the Eu:HA NP surfaces sterically hindered the NP growth by the inorganic–organic interactions. Moreover, it was demonstrated that the interactions of Cit as an organic molecule and Eu:HA as an inorganic matrix were important for achieving the efficient photoluminescence properties. Thus, the efficient luminescence ability including the internal efficiency for cancer cell labeling was achieved, and simultaneously, the Cit molecular effect on the suppression of the cancer cell line growth was investigated. As a result, the luminescence enhancement with the hybridization was successfully elucidated. In particular, the low symmetry of the coordination structure for the Eu3+ ion at the Ca-(I) site provided the enhanced luminescence efficiency. Furthermore, the folate N-hydroxysuccinimidyl ester (FA-NHS) was immobilized on the Cit/Eu:HA NPs to enhance the uptake efficiency of the NPs into the cancer cells. Then, the cytocompatibility and the cell-labeling property were evaluated to investigate the effect of the NPs on the cancer cell growth suppression by the Cit molecules. Cancer cell growth suppression was successfully achieved by the interactions with the Cit/Eu:HA NPs. Furthermore, the Cit/Eu:HA NPs reacted with the cells to exhibit red-color luminescence from the cells while suppressing cancer cell growth, indicating the bifunction of cell-labeling and cytostatic suppression in one particle. The hybridized Cit molecules could significantly contribute to the tumorized cell (sphere) growth suppression. In particular, the Cit/Eu:HA NPs were effectively reacted with the spheres after a culture time of 60 h, and the luminescent labeling with following the cellular shapes could be achieved 1 h after NP addition, indicating the rapid labeling process with cytostatic suppression for interacting with the spheres. [ABSTRACT FROM AUTHOR]

Vertically ordered Si needles are of particular interest for long-term intracellular recording owing to their capacity to infiltrate living cells with negligible damage and minimal toxicity. Such intracellular recordings could greatly benefit from simultaneous live cell imaging without disrupting their culture, contributing to an in-depth understanding of cellular function and activity. However, the use of standard live imaging techniques, such as inverted and confocal microscopy, is currently impeded by the opacity of Si wafers, typically employed for fabricating vertical Si needles. Here, we introduce a transparent intracellular sensing platform that combines vertical Si needles with a percolated network of Au-Ag nanowires on a transparent elastomeric substrate. This sensing platform meets all prerequisites for simultaneous intracellular recording and imaging, including electrochemical impedance, optical transparency, mechanical compliance, and cell viability. Proof-of-concept demonstrations of this sensing platform include monitoring electrical potentials in cardiomyocyte cells and in three-dimensionally engineered cardiovascular tissue, all while conducting live imaging with inverted and confocal microscopes. This sensing platform holds wide-ranging potential applications for intracellular research across various disciplines such as neuroscience, cardiology, muscle physiology, and drug screening.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.

Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC), as an enzyme-free signal amplification method, has been used in detecting and imaging a variety of biomarkers. The localization strategy can effectively increase the local concentration of the DNA reaction modules to improve the signal amplification effect. However, the localization strategy may also amplify the leak reaction of the EDC, and effective signal amplification can be limited by the unclear structure-function relationship. Herein, we utilized locked nucleic acid (LNA) modification to enhance the stability of the localized entropy-driven circuit (LEDC), which suppressed a 94.6% leak signal. The coarse-grained model molecular simulation was used to guide the structure design of the LEDC, and the influence of critical factors such as the localized distance and spacer length was analyzed at the molecular level to obtain the best reaction performance. The sensitivities of miR-21 and miR-141 detected by a simulation-guided optimal LEDC probe were 17.45 and 65 pM, 1345 and 521 times higher than free-EDC, respectively. The LEDC was further employed for the fluorescence imaging of miRNA in cancer cells, showing excellent specificity and sensitivity. This work utilizes LNA and molecular simulations to comprehensively improve the performance of a localized DNA signal amplification circuit, providing an advanced DNA probe design strategy for biosensing and imaging as well as valuable information for the designers of DNA-based probes.

Sulfur dioxide (SO 2 ) derivatives are extensively utilized as both a preservative for foods and an active gaseous signal molecule in various physiological and pathological processes, but their excessive intake would bring harmful effects on human health; so, the determination of SO 2 derivatives is of great importance. Herein, we developed a ratiometric fluorescent probe named 2-(2'-hydroxyphenyl)benzothiazole-3-ethyl-1,1,2-trimethyl-1 H -benzo[ e ]indolium (HBT-EMBI) by introducing a hemicyanine unit of EMBI to an HBT group for the detection of SO 2 derivatives via an excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) effects. The probe displays some advantages, such as a colorimetric change from purple to colorless, a ratiometric fluorescence with zero cross-talk, and a remarkably large emission shift (Δλ = 164 nm) under a single-wavelength excitation. Accordingly, the probe HBT-EMBI has been successfully employed for the colorimetric and ratiometric determination of SO 2 derivatives in real food samples and the quantitative visualization of SO 2 derivative variations in HepG2 cells.

Nanoscopic investigation of bacterial cells is essential to reveal their physiological status, impacting all cellular functions. Currently, this requires labeled probes or targeted staining procedures. Herein, we report a new bacterial feature, intracellular dynamics-resolved Rayleigh scattering (IDRS), that visualizes spatiotemporal cytoplasmic transitions in unlabeled bacteria and characterizes their real-time physiological status in 10 s. From single-bacterium IDRS signals, we discovered unique spatial patterns and their multiple transitions in Gram-negative and Gram-positive bacteria. The magnitude of IDRS signal variation highly correlated with the metabolic status of bacteria, differentiating persistent subpopulations. This is also the first report demonstrating distinct real-time metabolic conditions of unlabeled drug-resistant bacteria that are exposed to different doses of antibiotics. Our strategy opens up a way to simultaneously trace in situ metabolic and antibiotic resistance statuses, which can be applied in single-cell level control of bacterial metabolism and efficacy with a heterogeneous nature.

Luminescent supramolecular metallacycles have attracted great interest as a new promising class of sensing substrates. In this work, two tetraphenylethene (TPE)-based diimidazole and dipyrazole ligands with the aggregation-induced emission (AIE) feature were designed for the construction of supramolecular tetragonal metallacycles 1 - 4 with two 90° mononuclear [(bpy)M] 2+ or dinuclear [(bpy) 2 M 2 ] 4+ acceptors (bpy = 2,2'-dipyridine; M = Pd, Pt), in which the fluorescence can be quenched to an "off" state due to the ligand-to-metal charge transfer (LMCT). Metallacycle 1 was utilized as a fluorescence sensor for phosphate (PO 4 3- ) detection in aqueous solution by means of disassembly, leading to the release of the ligand. Additionally, the metallacycle can be regenerated through self-assembly via the introduction of Pd(II) acceptors. PO 4 3- was detected using TPE-based metallacycles over a wide concentration range, with a detection limit as low as 2.1 × 10 -8 M. Furthermore, sensor 1 also presented the semiquantitative visual detection ability for PO 4 3- in the test paper mode via fluorescence changes. The aforementioned studies not only enhance the current research on fluorescent materials but also offer a strategy for the creation of stimuli-responsive supramolecular coordination complexes.