Investigating ligand-protein complexes is essential in the areas of chemical biology and drug discovery. However, detailed information on key reagents such as fluorescent tracers and associated data for the development of widely used bioluminescence resonance energy transfer (BRET) assays including NanoBRET, time-resolved Förster resonance energy transfer (TR-FRET) and fluorescence polarization (FP) assays are not easily accessible to the research community. We created tracerDB, a curated database of validated tracers. This resource provides an open access knowledge base and a unified system for tracer and assay validation. The database is freely available at https://www.tracerdb.org/ .

The reliable application of field deployable fluorescent dissolved organic matter (fDOM) probes is hindered by several influencing factors which need to be compensated. This manuscript describes the corrections of temperature, pH, turbidity and inner filter effect on fluorescence signal of a commercial fDOM probe (fDOMs). For this, Australian waters with wide ranging qualities were selected, e.g. dissolved organic carbon (DOC) ranging from ∼1 to ∼30 mg/L, specific UV absorbance at 254 nm from ∼1 to ∼6 L/m/mg and turbidity from ∼1 to ∼ 350 FNU. Laboratory-based model calibration experiments (MCEs) were performed. A model template was developed and used for the development of the correction models. For each factor, data generated through MCEs were used to determine model coefficient (α) values by fitting the generated model to the experimental data. Four discrete factor models were generated by determination of a factor-specific α value. The α values derived for each water of the MCEs subset were consistent for each factor model. This indicated generic nature of the four α values across wide-ranging water qualities. High correlation between fDOMs and DOC were achieved after applying the four-factor compensation models to new data (r, 0.96, p < 0.05). Also, average biases (and %) between DOC predicted through fDOMs and actual DOC were decreased by applying the four-factor compensation model (from 3.54 (60.9%) to 1.28 (16.7%) mg/L DOC). These correction models were incorporated into a Microsoft EXCEL-based software termed EXOf-Correct for ready-to-use applications.

BACKGROUND: Glutathione (GSH), a highly abundant thiol compound within cells, plays a critical role in physiological processes and exhibits close correlation with cancer. Among molecular imaging technologies, most probes have relatively short emission wavelengths and lack photoacoustic imaging (PA) capability, resulting in the inability to obtain tissue images with high penetration depth. The presence of GSH in the tumor microenvironment neutralizes ROS, diminishing the therapeutic effect of PDT, thus resulting in often unsatisfactory therapeutic efficacy. Therefore, it is imperative to develop a dual-modal probe for the detection of GSH and the diagnosis and treatment of cancer.
RESULTS: In this study, we synthesized a novel dual-modal probe, Cy-Bio-GSH, utilizing near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging techniques for GSH detection. The probe integrates cyanine dye as the fluorophore, nitroazobenzene as the recognition moiety, and biotin as the tumor-targeting moiety. Upon reacting with GSH, the probe emits NIR fluorescence at 820 nm and generates a PA signal. Significantly, this reaction activates the photodynamic and photothermal properties of the probe. By depleting GSH and employing a synergistic photothermal therapy (PTT) treatment, the therapeutic efficacy of photodynamic therapy (PDT) is remarkably enhanced. In-vivo experiments confirm the capability of the probe to detect GSH via NIRF and PA imaging. Notably, the combined tumor-targeting ability and PDT/PTT synergistic therapy enhance therapeutic outcomes for tumors and facilitate their ablation.
CONCLUSIONS: A novel tumor-targeting and dual-modal imaging probe (Cy-Bio-GSH) is synthesized, exhibiting remarkable sensitivity and selectivity to GSH, enabling the visualization of GSH in cells and the differentiation between normal and cancer cells. Cy-Bio-GSH enhances PDT/PTT with effective killing of cancer cells and makes the ablation of tumors in mice. This work represents the first tumor-targeting probe for GSH detection, and provides crucial tool for cancer diagnosis and treatment by dual-modal imaging with improved PDT/PTT synergistic therapy.

BACKGROUND: As promising biomarkers of diabetes, α-glucosidase (α-Glu) and β-glucosidase (β-Glu) play a crucial role in the diagnosis and management of diseases. However, there is a scarcity of techniques available for simultaneously and sensitively detecting both enzymes. What\'s more, most of the approaches for detecting α-Glu and β-Glu rely on a single-mode readout, which can be affected by multiple factors leading to inaccurate results. Hence, the simultaneous detection of the activity levels of both enzymes in a single sample utilizing multiple-readout sensing approaches is highly attractive.
RESULTS: In this work, we constructed a facile sensing platform for the simultaneous determination of α-Glu and β-Glu by utilizing a luminescent covalent organic framework (COF) as a fluorescent indicator. The enzymatic hydrolysis product common to both enzymes, p-nitrophenol (PNP), was found to affect the fluorometric signal through an inner filter effect on COF, enhance the colorimetric response by intensifying the absorption peak at 400 nm, and induce changes in RGB values when analyzed using a smartphone-based color recognition application. By combining fluorometric/colorimetric measurements with smartphone-assisted RGB mode, we achieved sensitive and accurate quantification of α-Glu and β-Glu. The limits of detection for α-Glu were determined to be 0.8, 1.22, and 1.85 U/L, respectively. Similarly, the limits of detection for β-Glu were 0.16, 0.42, and 0.53 U/L, respectively.
CONCLUSIONS: Application of the proposed sensing platform to clinical serum samples revealed significant differences in the two enzymes between healthy people and diabetic patients. Additionally, the proposed sensing method was successfully applied for the screening of α-Glu inhibitors and β-Glu inhibitors, demonstrating its viability and prospective applications in the clinical management of diabetes as well as the discovery of antidiabetic medications.

BACKGROUND: Fluoroquinolones (FQs) are widely used in livestock and poultry industry because of their satisfactory effects in preventing and treating bacterial infection. However, due to irrational use and poor biodegradability, FQs can easily remain in food animals and further enter the human body through the food chain. Therefore, accurate and sensitive detection of FQs residues in animal-origin food is significant. The traditional methods commonly used for FQs detection have some limitations. Ratiometric fluorescence detection technology has the advantages of fast, sensitive, self-correcting, and easy visualization. However, the reports on the use of ratiometric fluorescence probes for FQs detection are limited.
RESULTS: In this work, a novel probe was proposed for ratiometric fluorescent analysis of FQs. In this probe, the fluorescence of dithioerythritol stabilized copper nanoclusters (DTE-Cu NCs) was significantly enhanced due to the Tb3+ triggered aggregation-induced emission effect. FQs bound Tb3+ in Tb3+/DTE-Cu NCs through carboxyl and carbonyl groups, so that Tb3+ was effectively sensitized to emit green fluorescence. However, the red fluorescence of DTE-Cu NCs was not interfered. The fluorescence of the probe transformed from red to green with the increase of FQs concentration. Using norfloxacin (NOR), difloxacin (DIF), and enrofloxacin (ENR) as FQs simulants, this probe showed a sensitive linear response ranged from 0.025 to 22.5 μM, with the limits of detection of 9.6 nM, 9.3 nM, and 7.7 nM. The application potential for FQs detection was verified via a standard addition assay of egg samples with the recovery rate of 90.4 %-114.7 %.
CONCLUSIONS: The fluorescence probe based on Tb3+/DTE-Cu NCs is expected to realize the ratiometric fluorescence sensitive detection of FQs. The establishment of this simple, effective, and rapid detection platform opens up a new way for the detection of FQs residues in animal-origin foods, and also provides a new idea for the design of rapid detection platforms for other hazard factors.

BACKGROUND: As is well documented, prostate cancer (PCa) being the second most prevalent cancer in men worldwide, emphasizing the importance of early diagnosis for prognosis. However, conventional prostate-specific antigen (PSA) testing lacks sufficient diagnostic efficiency due to its relatively low sensitivity and limited detection range. Mounting evidence suggests that matrix metalloproteinase 9 (MMP-9) expression increases with the aggressive behavior of PCa, highlighting the significance of detecting the serum level of MMP-9 in patients. Developing a non-immune rapid, portable MMP-9 detection strategy and investigating its representativeness of PCa serum markers hold considerable implications.
RESULTS: Herein, our study developed a simple, homogeneous dual fluorescence and smartphone-assisted red-green-blue (RGB) visualization peptide sensor of MMP-9, utilizing cadmium telluride quantum dots (CdTe QDs) and calcein as signal reporters. The essence of our approach revolves around the proteolytic ability of MMP-9, exploiting the selective recognition of molecule-Cu2+ complexes with different molecular weights by CdTe QDs and calcein. Under optimized conditions, the limits of detection (LODs) for MMP-9 were 0.5 pg/mL and 6 pg/mL using fluorescence and RGB values readouts, respectively. Indeed, this strategy exhibited robust specificity and anti-interference ability. MMP-9 was quantified in 42 clinical serum samples via dual-fluorescence analysis, with 12 samples being visually identified with a smartphone. According to receiver operating characteristic curve (ROC) analysis, its sensitivity and specificity were 90 % and 100 %, respectively, with an area under curve (AUC) value of 0.903.
UNASSIGNED: Of note, the results of the aforementioned analysis were highly consistent with the serum level of PSA, clinical color Doppler flow imaging (CDFI), and histopathological results. Therefore, this simple, rapid, homogeneous fluorescence and visualization strategy can reliably measure MMP-9 levels and exhibit promising potential in point-of-care testing (POCT) applications for PCa patients.

BACKGROUND: Cirrhosis represents the terminal stage of liver disease progression and timely intervention in a diseased liver can enhance the likelihood of recovery. Viscosity, a crucial parameter of the cellular microenvironment, is intricately linked to the advancement of cirrhosis. However, viscosity monitoring still faces significant challenges in achieving non-invasive and rapid early diagnosis of cirrhosis. Near-infrared (NIR) fluorescence imaging has the advantages of high sensitivity, non-destructive detection, and ignoring background fluorescence interference, plays an important role in diagnosing and treating various biological diseases. Hence, monitoring cellular viscosity changes with NIR fluorescence probe holds great significance in the early diagnosis of cirrhosis.
RESULTS: In this study, the NIR fluorescence probe based on the intramolecular charge transfer (TICT) mechanism was developed for imaging applications in mouse model of liver cirrhosis. A molecular rotor-type viscosity-responsive probe was synthesized by linking dioxanthracene groups via carbon-carbon double bonds. The probe demonstrated remarkable sensitivity, high selectivity and photostability, with its responsiveness to viscosity largely unaffected by factors such as polarity, pH, and interfering ions. The probe could effectively detect various drug-induced changes in cellular viscosity, enabling the differentiation between normal cells and cancerous cells. Furthermore, the enhanced tissue penetration capabilities of probe facilitated its successful application in mouse model of liver cirrhosis, allowing for the assessment of liver disease severity based on fluorescence intensity and providing a powerful tool for early diagnosis of cirrhosis.
CONCLUSIONS: A NIR viscosity-sensitive fluorescent probe was specifically designed to effectively monitor alterations in cellular and organ viscosity, which could advance the understanding of the biological characteristics of cancer and provide theoretical support for the early diagnosis of cirrhosis. Overall, this probe held immense potential in monitoring viscosity-related conditions, expanding the range of biomedical tools available.

Fluorescence analysis has been regarded as one of the commonly used analytical methods because of its advantages of simple operation, fast response, low cost and high sensitivity. So far, various fluorescent probes, with noble metal nanoclusters, quantum dots, organic dyes and metal organic frameworks as representatives, have been widely reported. However, single fluorescent probe often suffers from some deficiencies, such as low quantum yield, poor chemical stability, low water solubility and toxicity. To overcome these disadvantages, the introduction of cyclodextrins into fluorescent probes has become a fascinating approach. This review (with 218 references) systematically covers the research progress of fluorescent composites based on cyclodextrins in recent years. Preparation strategies, fluorescence properties, response mechanisms and applications in sensing (ions, organic pollutants, bio-related molecules, temperature, pH) and bioimaging of fluorescent composites based on cyclodextrins are summarized in detail. Finally, the current challenges and future perspectives of these composites in relative research fields are discussed.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Iron is an essential element for life owing to its ability to participate in a diverse array of oxidation-reduction reactions. However, misregulation of iron-dependent redox cycling can also produce oxidative stress, contributing to cell growth, proliferation, and death pathways underlying aging, cancer, neurodegeneration, and metabolic diseases. Fluorescent probes that selectively monitor loosely bound Fe(II) ions, termed the labile iron pool, are potentially powerful tools for studies of this metal nutrient; however, the dynamic spatiotemporal nature and potent fluorescence quenching capacity of these bioavailable metal stores pose challenges for their detection. Here, we report a tandem activity-based sensing and labeling strategy that enables imaging of labile iron pools in live cells through enhancement in cellular retention. Iron green-1 fluoromethyl (IG1-FM) reacts selectively with Fe(II) using an endoperoxide trigger to release a quinone methide dye for subsequent attachment to proximal biological nucleophiles, providing a permanent fluorescent stain at sites of elevated labile iron. IG1-FM imaging reveals that degradation of the major iron storage protein ferritin through ferritinophagy expands the labile iron pool, while activation of nuclear factor-erythroid 2-related factor 2 (NRF2) antioxidant response elements (AREs) depletes it. We further show that lung cancer cells with heightened NRF2 activation, and thus lower basal labile iron, have reduced viability when treated with an iron chelator. By connecting labile iron pools and NRF2-ARE activity to a druggable metal-dependent vulnerability in cancer, this work provides a starting point for broader investigations into the roles of transition metal and antioxidant signaling pathways in health and disease.