Nitrogen rich carbon nanoparticles are known to provide higher fluorescence stokes shift, and thereby are potential candidates for fluorescent sensors. Herein, a facile one-step hydrothermal synthesis is reported for N-rich carbon nanospheres (G-CNS) from caffeine and o-phenylenediamine as precursors. The as-synthesized G-CNS showed high fluorescence with λem at 509 nm, with a highly selective fluorescence turn-off response towards Fe2+/Fe3+, rendering these carbon nanospheres as potential candidates to detect intracellular labile iron pool in live cells. The intracellular labile iron pool in iron-overloaded cells was sensed using the synthesized G-CNS. Mechanistically, the fluorescence quenching via dynamic pathway involves the formation of an excited state charge transfer process, which undergoes non-radiative decay.

Cytotoxicity assays are crucial for assessing the efficacy of drugs in killing cancer cells and determining their potential therapeutic value. Measurement of the effect of drug concentration, which is an influence factor on cytotoxicity, is of great importance. This paper proposes a cytotoxicity assay using microwave sensors in an end-point approach based on the detection of the number of live cells for the first time. In contrast to optical methods like fluorescent labeling, this research uses a resonator-type microwave biosensor to evaluate the effects of drug concentrations on cytotoxicity by monitoring electrical parameter changes due to varying cell densities. Initially, the feasibility of treating cells with ultrapure water for cell counting by a microwave biosensor is confirmed. Subsequently, inhibition curves generated by both the CCK-8 method and the new microwave biosensor for various drug concentrations were compared and found to be congruent. This agreement supports the potential of microwave-based methods to quantify cell growth inhibition by drug concentrations.

Water absorption of mid-infrared (MIR) radiation severely limits the options for vibrational spectroscopy of the analytes-including live biological cells-that must be probed in aqueous environments. While internal reflection elements, such as attenuated total reflection prisms and metasurfaces, partially overcome this limitation, such devices have their own limitations: ATR prisms are difficult to integrate with multiwell cell culture workflows, while metasurfaces suffer from a limited spectral range and small penetration depth into analytes. In this work, we introduce an alternative live cell biosensing platform based on metallic nanogratings fabricated on top of elevated dielectric pillars. For the MIR wavelengths that are significantly longer than the grating period, reflection-based spectroscopy enables broadband sensing of the analytes inside the trenches separating the dielectric pillars. Because the depth of the analyte twice-traversed by the MIR light excludes the highly absorbing thick water layer above the grating, we refer to the technique as inverted transflection spectroscopy (ITS). The analytic power of ITS is established by measuring a wide range of protein concentrations in solution, with the limit of detection in the single-digit mg mL-1. The ability of ITS to interrogate live cells that naturally wrap themselves around the grating is used to characterize their adhesion kinetic.

The viscosity within cells is a crucial microenvironmental factor, and sulfur dioxide (SO2 ) has essential functions in regulating cellular apoptosis and inflammation. Some evidence has been confirmed that changes in viscosity and overexposure of SO2 within the cell may cause detrimental effects including, but not limited to, respiratory and cardiovascular illnesses, inflammation, fatty liver, and various types of cancer. Therefore, precise monitoring of SO2 and viscosity in biological entities holds immense practical importance. Therefore, in this research, we developed a versatile fluorescent TCF-Cou that enables the dual detection of SO2 and viscosity in the living system. Probe TCF-Cou possessed a response to viscosity and SO2 through red and green emissions. The alteration of SO2 and viscosity levels in live cells and zebrafish were also monitored using probe TCF-Cou. We hope that this fluorescent probe could be a potential tool for revealing the related pathological and physiological processes through monitoring the changes in SO2 and viscosity.

H2O2 is an important signaling molecule in the body, and its levels fluctuate in many pathological sites, therefore, it can be used as a biomarker for early diagnosis of disease. Since the environment in vivo is extremely complex, it is of great significance to develop a probe that can accurately monitor the fluctuation of H2O2 level without interference from other physiological processes. Based on this, we designed and synthesized two new near-infrared H2O2 fluorescent probes, LTA and LTQ, based on the ICT mechanism. Both of them have good responses to H2O2, but LTA has a faster response speed. In addition, the probe LTA has good biocompatibility, good water solubility, and a large Stokes shift (95 nm). The detection limit is 4.525 μM. The probe was successfully used to visually detect H2O2 in living cells and zebrafish and was successfully used to monitor the changes in H2O2 levels in zebrafish due to APAP-induced liver injury.

The multi-step degradation process of PROteolysis TArgeting Chimeras (PROTACs) poses a challenge for their rational development, as the rate-limiting steps that determine PROTACs efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here, we developed cell-based assays using the NanoLuciferase and HaloTag that allow measuring PROTAC-induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for the degradation of WD40 repeat domain protein 5 (WDR5), the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC-induced degradation pathway.

Chemically manipulating bacterial surface structures, a cutting-edge research direction in the biomedical field, predominantly relies on metabolic labeling by now. However, this method may involve daunting precursor synthesis and only labels nascent surface structures. Here, we report a facile and rapid modification strategy based on a tyrosinase-catalyzed oxidative coupling reaction (TyOCR) for bacterial surface engineering. This strategy employs phenol-tagged small molecules and tyrosinase to initiate direct chemical modification of Gram-positive bacterial cell walls with high labeling efficiency, while Gram-negative bacteria are inert to this modification due to the hindrance of an outer membrane. By using the biotin‒avidin system, we further present the selective deposition of various materials, including photosensitizer, magnetic nanoparticle, and horseradish peroxidase, on Gram-positive bacterial surfaces, and realize the purification/isolation/enrichment and naked-eye detection of bacterial strains. This work demonstrates that TyOCR is a promising strategy for engineering live bacterial cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

For clinical research, the precise measurement of hydrogen peroxide (H2O2) and glucose (Glu) is of paramount importance, due to their imbalanced concentrations in blood glucose, and reactive oxygen species (ROS) play a huge role in COVID-19 viral disease. It is critical to construct and develop a simple, rapid, flexible, long-term, and sensitive detection of H2O2 and glucose. In this paper, we have developed a unique morphological structure of MOF(Cu) on a single-walled carbon nanotube-modified gold wire (swnt@gw). Highly designed frameworks with nanotube composites enhance electron rate-transfer behavior while extending conductance and electroactive surface area.The composite sensing system delivers wide linear-range concentrations, low detection limit, and interference-free performance in co-existence with other biomolecules and metal ions. Endogenous quantitative tracking of H2O2 was performed in macrophage live-cells with the help of a strong stimulator lipopolysaccharide.The composite device was effectively utilized for the measurement of H2O2 and glucose in turbid samples of whole blood and milk samples without a pretreatment process. The practical results of biofluids showed favorable voltammetric results and acceptance recovery percentage levels between 97.49 and 98.88%. Finally, a flexible MOF-based hybrid system may provide a suitable detection platform in the construction of electro-biosensors and hold potential promise for clinical-sensory applications.

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.