A bisalicylhydrazone based fluorescence probe, bisalicyladehyde benzoylhydrazone (BS-BH), has been designed to detect Al3+. It exhibited high sensitivity and selectivity towards Al3+ in methanol-water media in physiological condition. Large stokes shifts (∼122 nm) and over ∼1000-fold enhanced fluorescence intensity were observed, which was ascribed to the formation of the two relatively independent rigid extended π conjugated systems bridged by biphenyl group when binding with Al3+. A 1:2 binding ratio between BS-BH and Al3+ was shown by Job\'s plot. Based on the fluorescence titration data, the detection limit was down to 3.50 nM and the association constant was evaluated to be 1.12 × 109 M-2. The plausible fluorescence sensing mechanism of suppressed ESIPT, inhibited PET, activated CHEF and restricted C = N isomerization was confirmed by a variety of spectral experiments and DFT / TD-DFT calculations. The reversibility of recognition of Al3+ for probe BS-BH was validated by adding Na2-EDTA. In addition, the MTT assay showed the good biocompatibility of BS-BH and BS-BH could be used for imaging Al3+ in living cells.

Amplifying DNA conjugated affinity ligands can improve the sensitivity and multiplicity of cell imaging and play a crucial role in comprehensively deciphering cellular heterogeneity and dynamic changes during development and disease. However, the development of one-step, controllable, and quantitative DNA amplification methods for multiplexed imaging of live-cell membrane proteins is challenging. Here, we introduce the template adhesion reaction (TAR) method for assembling amplifiable DNA sequences with different affinity ligands, such as aptamers or antibodies, for amplified and multiplexed imaging of live-cell membrane proteins with high quantitative fidelity. The precisely controllable TAR enables proportional amplification of membrane protein targets with variable abundances by modulating the concentration ratios of hairpin templates and primers, thus allowing sensitive visualization of multiple membrane proteins with enhanced signal-to-noise ratios (SNRs) without disturbing their original ratios. Using TAR, we achieved signal-enhanced imaging of six proteins on the same live-cell within 1-2 h. TAR represents an innovative and programmable molecular toolkit for multiplexed profiling of membrane proteins in live-cells.

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.

In this work, we developed a fast and straightforward colorimetric and photoluminescent chemosensor probe (P1), featuring bis-thiophene-thiosemicarbazide moieties as its signaling and binding unit. This probe exhibited rapid sensitivity to Hg2+ and Cu2+ ions in a semi-aqueous medium, resulting in distinct colorimetric and photoluminescent changes. In the presence of Cu2+, P1 displayed an impressive 50-fold increase in photoluminescence (PL) at 450 nm (with excitation at 365 nm). The probe P1 formed a 1:1 complex with Hg2+ and Cu2+ ions, featuring association constant values of 4.04 × 104 M-1 and 1.25 × 103 M-1, respectively. P1 has demonstrated its efficacy in the analysis of real samples, yielding promising results. Additionally, the probe successfully visualized copper ions on a mouse fibroblast cell line (NIH3T3), highlighting its potential as an intracellular probe for copper ion detection.

Hydrogen sulfide (H2S) is a pungent gas that is one of the key mediators of signal transduction in biological systems, and its presence is related to the freshness of some protein foods. Using phenothiazine derivatives as fluorophores and 2, 4-dinitrobenzene sulfonate (DNBS) fragments as reaction groups, a near-infrared (NIR) probe WX-HS for H2S identification was designed. With the addition of H2S, WX-HS appeared a strong fluorescence signal at 660 nm with short reaction time (90 s) and high sensitivity, and fluorescence state change from non-fluorescent to orange-red. In addition, WX-HS could effectively detect H2S produced during food oxidation. Based on its low cytotoxicity, the WX-HS probe further enabled the detection and imaging of H2S in A549 cells.

As one of endogenous reactive oxygen species (ROS), peroxynitrite (ONOO-) performs various functions in both pathological and physiological mechanisms. In this work, an optical and near-infrared (NIR) fluorescent probe (NX), which based on 3-dihydro-1H-xanthene and 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) group was designed and prepared to detect ONOO-. This probe revealed an obvious optical and a fluorescent response when ONOO- was present and it exhibited higher selectivity over other ROS. Especially, the dual NIR fluorescence changes at 660 and 800 nm allowed quantitative detection of ONOO- in the range of 15-40 μM, and the detection limit was 82 nM. Finally, the probe was effectively employed to visualize exogenous and endogenous ONOO- in HepG2 cells and zebrafish, respectively. All the results indicated the dual NIR-channel probe could serve as a potent detecting tools in studying ONOO- in vitro and in vivo.

Fluorescent microspheres are of significant interests due to their wide applications in biotechnology fields. However, their preparation presents several challenges, such as the need for dye labeling, the complexity of materials and often sophisticated preparation conditions. Here a simple process for hydrophilic and crosslinked polyurethane (CPU) microspheres, with carboxyl groups on the surface via one-step precipitation polymerization in 40 min, is presented. The microsphere size is easily adjusted by varying experimental conditions. CPU microspheres exhibit high thermal and pH stability with good redispersibility in water, and emit fluorescence without any modification or dye labeling. The emission mechanism is discussed. CPU microspheres are used as fluorescent probe to detect 4-nitrophenol (4-NP) based on their emission in UV light region, with excellent selectivity and sensitivity. In addition, they are reusable with detection limit unchanged after 7 cycles of reuses, a significant feature of this work. The mechanism of fluorescence detection is thoroughly explored and ascribed to the internal filtration effect. Based on the emission in visible light region, CPU microspheres are used as a model of PU microplastics (MPs) to visualize their biodistribution in HeLa and macrophage cells, as well as in zebrafish larvae, providing a reliable tracer for the visualization and tracking of PU MPs in organisms.

With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.

The 5-nitroimidazole (5-NI) class of antibiotics, such as metronidazole, ornidazole, secnidazole, and tinidazole, are widely used to prevent bacterial infection in humans and livestock industries. However, their overuse contaminates the farmed animal products and water bodies. Hence, a selective, sensitive, and cost-effective method to detect 5-NI antibiotics is the need of the hour. Herein, we report a rapid, inexpensive, and efficient sensing system to detect 5-NI drugs using an as-prepared solution of ε-poly-l-lysine (ε-PL), a naturally occurring and biodegradable homopolypeptide that has an intrinsic fluorescence via clustering-triggered emission. The low nanomolar detection limit (3.25-3.97 nM) for the aforementioned representative 5-NI drugs highlights the sensitivity of the system, outperforming most of the reported sensors alike. The resulting fluorescence quenching was found to be static in nature. Importantly, excellent recovery (100.26-104.41%) was obtained for all real samples and animal products tested. Visual detection was demonstrated by using paper strips and silica gel for practical applications. Furthermore, ε-PL could detect 5-NI antibiotics in living 3T3-L1 mouse fibroblast cells via cellular imaging. Taken together, the present work demonstrates the detection of 5-NI antibiotics using a biocompatible natural polypeptide, ε-PL, and represents a simple and inexpensive analytical tool for practical application.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.