Toward quantification of hypoxia using fluorinated EuII/III-containing ratiometric probes.

Hypoxia is a prognostic biomarker of rapidly growing cancers, where the extent of hypoxia is an indication of tumor progression and prognosis; therefore, hypoxia is also used for staging while performing chemo- and radiotherapeutics for cancer. Contrast-enhanced MRI using EuII-based contrast agents is a noninvasive method that can be used to map hypoxic tumors, but quantification of hypoxia using these agents is challenging due to the dependence of signal on the concentration of both oxygen and EuII. Here, we report a ratiometric method to eliminate concentration dependence of contrast enhancement of hypoxia using fluorinated EuII/III-containing probes. We studied three different EuII/III couples of complexes containing 4, 12, or 24 fluorine atoms to balance fluorine signal-to-noise ratio with aqueous solubility. The ratio between the longitudinal relaxation time (T1) and 19F signal of solutions containing different ratios of EuII- and EuIII-containing complexes was plotted against the percentage of EuII-containing complexes in solution. We denote the slope of the resulting curves as hypoxia indices because they can be used to quantify signal enhancement from Eu, that is related to oxygen concentration, without knowledge of the absolute concentration of Eu. This mapping of hypoxia was demonstrated in vivo in an orthotopic syngeneic tumor model. Our studies significantly contribute toward improving the ability to radiographically map and quantify hypoxia in real time, which is critical to the study of cancer and a wide range of diseases.

Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

An aggregation-induced emission-based ratiometric fluorescent chemosensor for Hg(II) and its application in Caenorhabditis elegans imaging.

A chromone-based ratiometric fluorescent probe L2 was developed for the selective detection of Hg(II) in a semi-aqueous solution based on aggregation-induced emission (AIE) and chelation-enhanced fluorescence (CHEF) effect. The probe L2 fluoresced significantly at 498 nm in its aggregated state, and when chelated with Hg(II), the soluble state fluoresced 1-fold higher. In addition, Job's plot reveals that the probe forms a 1:1 stoichiometry complex with Hg(II) with an association constant of 9.10 × 103M-1 estimated by the BH plot. The probe L2 detects Hg(II) down to 22.47 nM without interference from other interfering ions. The FTIR, ESI mass, and DFT-based computational studies investigated the binding mechanism of probe L2 with Hg(II). Taking advantage of its AIE characteristics, the probe L2 was successfully applied for bio-capability analysis in Caenorhabditis elegans (a nematode worm) imaging of Hg(II) in a living model.

Fluorescence-based ratiometric sensors as emerging tools for CN- detection: Chemical structures, sensing mechanisms and applications.

Hazardous cyanide anions (CN-) are increasingly threatening the environment and human health due to their widespread use in industry and many other fields. Over the past three decades, a large number of probes have been reported to sensitively and selectively detect this toxic anion, while a rather limited number of ratiometric fluorescent probes have been developed. The ratiometric probes have significant potential in bio-imaging and biomedical applications because of the ability to detect CN- in a quick, convenient and affordable way. In this review, we introduce 42 ratiometric fluorescent probes reported in the past 6 years (2018-2023) for CN- detection. Our description includes the chemical structures, photo-physical properties, CN- sensing mechanisms, solution color changes, limits of detection (LODs) and/or various applications of these chemical probes. This review provides guidelines for design and development of a new ratiometric probe for effective CN- detection.

A ratiometric fluorescence probe for bisulfite detection in live cells and meat samples.

The development of reliable probe technology for the detection of bisulfite (HSO3-) in situ in food and biological samples is contributing significantly to food quality and safety assurance as well as community health. In this work, a responsive probe, EHDI, is developed for ratiometric fluorescence detection of HSO3- in aqueous solution, meat samples, and living cells. The probe is designed based on the HSO3- triggered 1,4-addition of electron deficit C = C bond of EHDI. As a result of this specific 1,4-addition, the π-conjugation system was destructed, resulting in blue shifts of the emission from 687 to 440 nm and absorption from 577 to 355 nm. The probe has good water solubility, high sensitivity and selectivity, allowing it to be used for imaging of HSO3- internalization and production endogenously. The capability of probe EHDI for HSO3- was then validated by traditional HPLC technology, enabling accurately detect HSO3- in beef samples. The successful development of this probe thus offers a new tool for investigating HSO3- in situ in food and biological conditions.

A Novel Ion Species- and Ion Concentration-Dependent Anti-Counterfeiting Based on Ratiometric Fluorescence Sensing of CDs@MOF-Nanofibrous Films.

Traditional fluorescent anti-counterfeiting labels based on "on-off" fluorescence can be easily cloned. It is important to explore advanced anti-counterfeiting fluorescent labels with high-level security. Here, a pioneering ion species- and ion concentration-dependent anti-counterfeiting technique is developed. By successive loading Cu2+ -sensitive yellow emitted carbon dots (Y-CDs) and Cu2+ non-sensitive blue emitted carbon dots (B-CDs) into metal-organic frameworks (MOFs) and followed by electrospinning, the B&Y-CDs@MOF-nanofibrous films are prepared. The results show that the use of MOF not only avoids the fluorescence quenching of CDs but also improves the fluorescence stability. The fluorescence Cu2+ -sensitivity of the CDs@MOF-nanofibrous films can be regulated by polymer coating or lamination. The fluorescent label consisting of different Cu2+ -sensitivity films will show Cu2+ concentration-dependent decryption information. Only at a specific ion species and concentration (Cu2+ solution of 40-90 µm), the true information can be read out. Less or more concentration (<40 or >90 µm) will lead to false information. The identification of the real information depends on both the species and the concentration. After Cu2+ treatment, the fluorescence of the label can be recovered by ethylenediaminetetraacetic acid disodium (EDTA-2Na) for further recycling. This work will open up a new door for designing high-level fluorescent anti-counterfeiting labels.

Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Naphthalimide-based, Single-Chromophore, Emission Ratiometric Fluorescent Sensor for Tracking Intracellular pH.

We report a novel, reversible, cell-permeable, pH-sensor, TRapH. TRapH afforded a pH-sensitive ratiometric emission response in the pH range ~3-6, enabling imaging and quantification of pH in living cells. The biological-applicability of TRapH was illustrated via live-tracking of intracellular pH dynamics in living mammalian cells induced by a synthetic H+-transporter.

A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology.

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self-sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red-light switch for tight regulation of metabolic pathways.

Design of an Acidic pH-Activated NIR Fluorescent Convertible Rhodamine-Hemicyanine Probe-Peptide Conjugate for Living Cancer Cell Active Targeted Selective Tracking of Lysosomes.

We have synthesized an acidic pH-activatable dual targeting ratiometric fluorescent probe-peptide conjugate using the SPPS protocol on resin. Living carcinoma cell specific active targeting, successive cell penetration, and selective staining of lysosomes are accomplished. Real-time monitoring of lysosomes, 3D, and multicolor cancer cell imaging are attained. The de novo design consists of the integration of multifunctionality into a single molecular scaffold, e.g., RGDS peptide to target cancer cell overexpressed receptor αVß3 integrin, live-cell penetrating  rhodamine-hemicyanine chromophore comprising a lysosome targeting morpholine group, and an acidic pH openable spiro-lactam ring for a visible-to-NIR switchable ratiometric response. Water-soluble probe-peptide conjugate exhibits intramolecular spirolactamization at basic pH through Arg amide N. The visible spirolactam state predominantly exists at physiological and basic pH and can be switched to the highly conjugated NIR open amide state (λem=735 nm) through spiro-lactam ring opening triggered by acidic pH with a huge bathochromic shift (Δλabs=336 nm, ΔλFL=265 nm). pH-sensitive ratiometric switching is achieved. This in situ acidic cancer cell lysosome activatable multifunctional fluorophore-peptide conjugate shows augmented molar absorptivity, enhanced quantum yield, and improved fluorescence lifetime at acidic lysosomal pH; negligible cytotoxicity; and dual targeted ratiometric imaging capability of living cancer cell selective lysosomes with pKa value of 5.1.

Tuning Tumor Targeting and Ratiometric Photoacoustic Imaging by Fine-Tuning Torsion Angle for Colorectal Liver Metastasis Diagnosis.

Photoacoustic (PA) tomography is an emerging biomedical imaging technology for precision cancer medicine. Conventional small-molecule PA probes usually exhibit a single PA signal and poor tumor targeting that lack the imaging reliability. Here, we introduce a series of cyanine/hemicyanine interconversion dyes (denoted Cy-HCy) for PA/fluorescent dual-mode probe development that features optimized ratiometric PA imaging and tunable tumor-targeting ability for precise diagnosis and resection of colorectal cancer (CRC). Importantly, Cy-HCy can be presented in cyanine (inherent tumor targeting and long NIR PA wavelength) and hemicyanine (poor tumor targeting and short NIR PA wavelength) by fine-tuning torsion angle and the ingenious transformation between cyanine and hemicyanine through regulation optically tunable group endows the NIR ratiometric PA and tunable tumor-targeting properties. To demonstrate the applicability of Cy-HCy dyes, we designed the first small-molecule tumor-targeting and NIR ratiometric PA probe Cy-HCy-H2S for precise CRC liver metastasis diagnosis, activated by H2S (a CRC biomarker). Using this probe, we not only visualized the subcutaneous tumor and liver metastatic cancers in CRC mouse models but also realized PA and fluorescence image-guided tumor excision. We expect that Cy-HCy will be generalized for creating a wide variety of inherently tumor-targeting NIR ratiometric PA probes in oncological research and practice.

A Pilot Study of Ratiometric Creatine CEST MRI Assessment of Rabbit Skeletal Muscle Energy Metabolism at 3 T.

BACKGROUND: pH MRI may provide useful information to evaluate metabolic disruption following ischemia. Radiofrequency amplitude-based creatine chemical exchange saturation transfer (CrCEST) ratiometric MRI is pH-sensitive, which could but has not been explored to examine muscle ischemia. PURPOSE: To investigate skeletal muscle energy metabolism alterations with CrCEST ratiometric MRI. STUDY TYPE: Prospective. ANIMAL MODEL: Seven adult New Zealand rabbits with ipsilateral hindlimb muscle ischemia. FIELD STRENGTH/SEQUENCE: 3 T/two MRI scans, including MRA and CEST imaging, were performed under two B1 amplitudes of 0.5 and 1.25 µT after 2 hours of hindlimb muscle ischemia and 1 hour of reperfusion recovery, respectively. ASSESSMENT: CEST effects of two energy metabolites of creatine and phosphocreatine (PCrCEST) were resolved with the multipool Lorentzian fitting approach. The pixel-wise CrCEST ratio was quantified by calculating the ratio of the resolved CrCEST peaks under a B1 amplitude of 1.25 µT to those under 0.5 µT in the entire muscle. STATISTICAL TESTS: One-way ANOVA and Pearson's correlation. P < 0.05 was considered statistically significant. RESULTS: MRA images confirmed the blood flow loss and restoration in the ischemic hindlimb at the ischemia and recovery phases, respectively. Ischemic muscles exhibited a significant decrease of PCr at the ischemia (under both B1 amplitudes) and recovery phases (under B1 amplitude of 0.5 µT) and significantly increased CrCEST from normal tissues at both phases (under both B1 levels). Specifically, CrCEST decreased, and PCrCEST increased with the CrCEST ratio. Significantly strong correlations were observed among the CrCEST ratio, and CrCEST and PCrCEST under both B1 levels (r > 0.80). DATA CONCLUSION: The CrCEST ratio altered substantially with muscle pathological states and was closely related to CEST effects of energy metabolites of Cr and PCr, suggesting that the pH-sensitive CrCEST ratiometric MRI is feasible to evaluate muscle injuries at the metabolic level. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY STAGE: 1.

Remote temperature sensing in microelectronics: optical thermometry using dual-center phosphors.

Remote thermal sensing has emerged as a temperature detection technique for tasks in which standard contact thermometers cannot be used due to environment or dimension limitations. One of such challenging tasks is the measurement of temperature in microelectronics. Here, optical thermometry using co-doped and mixed dual-center Gd2O3:Tb3+/Eu3+samples were realized. Ratiometric approach based on monitoring emission intensities of Tb3+(5D4-7F5) and Eu3+(5D0-7F2) transition provided sensing in the range of 30 °C-80 °C. Dispersion system type only slightly affected relative sensitivity, accuracy and precision. The applicability of phosphors synthesized to be utilized as remote optical thermometers for microelectronics has been proved with an example on a surface mount resistor and microcontroller.

Ratiometric fluorescence detection of dopamine based on copper nanoclusters and carbon dots.

Nanoclusters for fluorescence detection are generally comprised of rare and expensive noble metals, and the nanoclusters based on more affordable transition metal have attracted increasing attention. This study designed a ratiometric fluorescent probe to detect dopamine (DA), an important neurotransmitter. With carbon dots encapsulated within silica (CDs@SiO2) as the reference, the emitted reference signal was almost unchanged due to the protection of inert silicon shell. Meanwhile, copper nanoclusters modified with 3-aminophenyl boronic acid (APBA-GSH-CuNCs) provided the sensing signal, in which the phenylboric acid could specifically recognize the cis-diol structure of DA, and caused the fluorescence quenching by photoinduced electron transfer. This dual emission ratiometric fluorescent probe exhibited high sensitivity and anti-interference, and was able to selectively responded to DA with a linear range of 0-1.4 mM, the detection limit of 5.6 nM, and the sensitivity of 815 mM-1. Furthermore, the probe successfully detected DA in human serum samples, yielding recoveries ranging from 92.5% to 102.7%. Overall, this study highlights the promising potential of this ratiometric probe for detecting DA.

Hemicyanine-Phenothiazine Based Highly Selective Ratiometric Fluorescent Probes for Detecting Hypochlorite Ion in Fruits, Vegetables and Beverages.

Hypochloric acid (HClO) is a reactive oxygen species (ROS) that functions as a bacteriostatic and disinfectant in food production. Excessive levels of ClO-, however, have been linked to various health issues, including cardiovascular diseases (Halliwell and Gutteridge in Oxford University press, USA, 2015), arthritis, and neurodegenerative diseases (Heinzelmann and Bauer in Biol Chem. 391(6):675-693, 2010). Therefore, synthesizing highly selective and sensitive probes for rapidly detecting endogenous ClO- in daily foods is currently a popular research topic (Kalyanaraman et al. in Redox Biol. 15:347-362, 2018; Winterbourn in Nat Chem Biol. 4(5):278-286, 2008; Turrens in J Physiol. 552(2):335-344, 2003). Thus, we have developed two highly selective ratiometric fluorescent probes (Probe1 and Probe2) based on indole-phenothiazine to detect ClO- in common vegetables, fruits and beverages qualitatively and quantitatively. Moreover, Both Probe1 and Probe2 have shown good specificity and stability, with high fluorescence intensity and long duration (Feng et al. in Adv Sci. 5:1800397, 2018; Wei et al. in Angew Chem. 131(14):4595-4599, 2019; Baruah et al. in J Mater Chem B, 2022).

Smartphone-Aided Fluorescence Detection of Cardiac Biomarker Myoglobin by a Ratiometric Fluorescent AuNCs-QDs Nanohybrids Probe with High Sensitivity.

Simple and sensitive detection of cardiac biomarkers is of great significance for early diagnosis and prevention of acute myocardial infarction (AMI). Here, a ratiometric fluorescent nanohybrids probe (AuNCs-QDs) was synthesized through the coupling of bovine serum albumin-functionalized gold nanoclusters (AuNCs) with CdSe/ZnS quantum dots (QDs) to realize simple and sensitive detection of cardiac biomarker myoglobin (Mb). The AuNCs-QDs probe shows pink fluorescence under UV light, with two emission peaks at 468 nm and 630 nm belonging to QDs and AuNCs, respectively. Importantly, the presence of Mb caused fluorescence quenching of the blue-emitting QDs, thereby inhibiting the fluorescence resonance energy transfer (FRET) process between QDs and AuNCs, and reducing the fluorescence intensity ratio (F468/F630) of AuNCs-QDs probe effectively. As the concentration of Mb increases, the ratiometric fluorescent probe also exhibits a visible fluorescence color change. The detection limit was as low as 4.99 µg/mL, and the response of the probe to Mb showed a good linear relationship up to 0.52 mg/mL. Moreover, the probe has excellent specificity for Mb. Besides, the AuNCs-QDs has been applied to detect Mb of urine samples. More importantly, we also developed an AuNCs-QDs probe modified smartphone-aided paper-based strip for on-site monitoring of Mb. As far as we know, this is the first report of a smartphone-aided paper-based strip for on-site quick monitoring of Mb, which provides a useful approach for AMI biomarker monitoring and may can be extended to other medical diagnostics.

Ratiometric Sensing of Azithromycin and Sulfide Using Dual Emissive Carbon Dots: A Turn On-Off-On Approach.

A novel ratiometric fluorescence probe was developed for the determination of azithromycin (AZM) and sulfide ions based on the differential modulation of red emissive carbon dots (R-N@CDs) and blue emissive carbon dots (B-NS@CDs). The addition of sulfide anion selectively quenched the red emission of R-N@CDs while the blue emission of B-NS@CDs unaffected. Upon subsequent introduction of AZM to this R-N@CDs@sulfide system, the quenched red fluorescence was restored. Comprehensive characterization of the CDs was performed using UV-Vis, fluorescence, FTIR spectroscopy, XPS, and TEM. The proposed method exhibited excellent sensitivity and selectivity, with limits of detection of 0.33 µM for AZM and 0.21 µM for sulfide. Notably, this approach enabled direct detection of sulfide without requiring prior modulation of the CDs with metal ions, as is common in other reported methods. The ratiometric probe was successfully applied for the determination of AZM in biological fluids and sulfide in environmental water samples with high selectivity. This work presents the first fluorometric method for the detection of AZM in biological fluids.

A Reaction-based Ratiometric Fluorescent Probe with Large STOKES Shift for Cu2+ in Neat Aqueous Solution.

A novel reaction-based ratiometric fluorescent probe 1 for Cu2+ using picolinate as the reaction site and hemicyanine as the fluorophore was developed. 1 displayed maximum absorption peak at 355 nm and fluorescence emission peak at 500 nm, with large Stokes shift of 145 nm. Upon reaction with Cu2+, the maximum absorption and fluorescence emission peaks red-shifted to 390 nm and 570 nm respectively, owing to Cu2+-induced hydrolysis of the picolinate moiety in 1. Meanwhile, the solution of 1 turned from green to orange under a 365 nm UV lamp. 1 not only could detect Cu2+ ratiometrically by the ratios of both absorbance (A390 nm/A355 nm) and fluorescence intensity (F570 nm/F500 nm), but also displayed large Stokes shift, fast response, high sensitivity and excellent selectivity over other metal ions in neat aqueous solution.

Development of a Pyrone-Fused Tricyclic Scaffold-based Ratiometric Fluorescent Probe for Al3+ Detection.

Aluminum (Al3+) is environmentally abundant and can harm living organisms in various ways, such as by inhibiting root growth, damaging faunal nervous systems, and promoting tumor cell proliferation. However, the dynamics of Al3+ in living organisms are largely unknown; thus, detecting Al3+ in the environment and organisms is crucial. Fluorescent probes are useful tools for the selective detection of metal ions. In particular, ratiometric fluorescent probes exhibit a detection response at two different maximum fluorescence emission wavelengths; which is advantageous for avoiding the influence of background fluorescence. A novel pyrone-fused tricyclic scaffold-based ratiometric fluorescent probe for detecting Al3+, ethyl 11-imino-1-oxo-3-phenyl-1H,11H-pyrano[4,3-b] quinolizine-5-carboxylate (PQ), was developed in this study. The PQ fluorescence blue shifted from 505 to 457 nm upon the addition of Al3+. The blue shift was accompanied by a change in the fluorescence color of the PQ solution from green to blue. Fluorescence titration experiments demonstrated that the fluorescence intensity ratio at the two peaks of interest (457/505 nm) increased in a concentration-dependent manner upon the addition of Al3+. Moreover, this study demonstrated that a PQ-soaked paper displays a visible color change under ultraviolet light upon exposure to Al3+. The above results suggest that PQ is an effective ratiometric probe for the detection of Al3+ in the environment. Future studies will be conducted to introduce various substituents and develop fluorescent probes by leveraging the fluorescence property of a pyrone-fused tricyclic scaffolds.