Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging between the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest to the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared to POT1 alone by bridging single- and double-stranded telomeric tracts.

Cotranscriptional folding of a 5́ stem-loop in the Escherichia coli tbpA riboswitch at single-nucleotide resolution.

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5́ stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5́ stem-loop along the elongation process, consistent with RNAP interacting with the 5́ stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation.

DNA Origami Vesicle Sensors with Triggered Single-Molecule Cargo Transfer.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and controllable stoichiometric loading of vesicles with single-molecule control.

A homo-FRET assay for patatin-specific proteolytic activity.

The potato protein patatin embeds bioactive peptides that require targeted hydrolysis to be released as promising food additives. This study presents a patatin-specific protease assay for assessing a wide range of protease activities in high-throughput format. Conjugating patatin to the amine reactive fluorogenic BODIPY FL dye provided a stable protease substrate with efficient homo-FRET quenching at a low degree (7-8) of labeling. Compared to commercial BODIPY-casein, BODIPY-patatin provided higher fluorescence enhancement (by de-quenching) at high protease concentrations, while the sensitivity was generally comparable for both highly specific (e.g. Trypsin) and industrial relevant proteases (e.g. Alcalase and Neutrase) at low doses. For Chymotrypsin, BODIPY-patatin provided a 39 % response improvement at 5 ng dose. A peptide-centric analysis of mass spectrometry-based bottom-up proteomics data identified several BODIPY-labeling sites with varying occupancies in patatin, indicating heterogenous labeling under the applied conjugation conditions. BODIPY-labeled patatin complements commercial BODIPY-labeled casein as a globular, plant-based alternative for screening of proteolytic activity.

Elucidation of PGPR-responsive OsNAM2 regulates salt tolerance in Arabidopsis by AFP2 and SUS protein interaction.

This study investigates the molecular mechanisms underlying salt stress responses in plants, focusing on the regulatory roles of OsNAM2, a gene influenced by the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens (SN13). The study examines how SN13-modulated OsNAM2 enhances salt tolerance in Arabidopsis through physiological, biochemical, and molecular analyses. Overexpression of OsNAM2, especially with SN13 inoculation, improves germination, seedling growth, root length, and biomass under high NaCl concentrations compared to wild-type plants, indicating a synergistic effect. OsNAM2 overexpression enhances relative water content, reduces electrolyte leakage and malondialdehyde accumulation, and increases proline content, suggesting better membrane integrity and stress endurance. Furthermore, SN13 and OsNAM2 overexpression modulates essential metabolic genes involved in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, facilitating metabolic adjustments crucial for salt stress adaptation. The interaction of OsNAM2 with SUS, facilitated by SN13, suggests enhanced sucrose metabolism efficiency, providing substrates for protective responses. Additionally, OsNAM2 plays a regulatory role in the ABA signaling pathway through significant protein-protein interactions like with AFP2. This study highlights the intricate interplay between SN13-responsive OsNAM2 and key signaling pathways, suggesting strategies for enhancing crop salt tolerance through targeted genetic and microbial interventions.

Methods for Studying Fusion of Bacterial Extracellular Vesicles with Intact Bacteria and Host Cells.

Bacterial extracellular vesicles (BEVs) are nano- or micrometer-sized membrane-bound lipid vesicles released from both Gram-negative and Gram-positive bacteria. Cellular transport, communication, pathogenesis, and host-pathogen interactions are some of the major biological processes impacted by BEVs. Among these, host-pathogen interactions and bacterial pathogenesis are emerging as highly important targetable avenues underlined by the issues of antimicrobial resistance, thus demanding novel targets and approaches to treat bacterial infections. In this aspect, the study of the interaction of BEVs with bacteria and/or host cells becomes imperative and brings the membrane fusion process to the forefront. Furthermore, membrane fusion also underscores the performance of BEVs as nano-therapeutic delivery platforms. Here, we report methods to study fusion kinetics between mycobacteria-derived extracellular vesicles, which we refer to as MEVs, and intact mycobacteria or MEVs themselves. We also discuss the isolation of MEVs and their characterization. We outline critical factors that affect fusion kinetics by MEVs. The same principle can be extended for studying fusion between BEVs and mammalian host cells important for understanding how BEVs influence host-pathogen crosstalk.

Development of a time-resolved fluorescence resonance energy transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Evaluation of High-Affinity Monoclonal Antibodies and Antibody-Drug Conjugates by Homogenous Time-Resolved FRET.

The rapid growth of therapeutic monoclonal antibodies demands greater accessibility to scalable methods of evaluating antigen binding. Homogenous TR-FRET is ideal for preliminary screening but has not been reported to assay these interactions due to their high-affinity and complex solution-phase kinetics. Here we report the development of a competition assay to rank-order the relative affinities of these drugs for a common antigen. The assay is compatible with automation, requires no modification of the analytes, and measures affinities as low as single-digit picomolar. We further demonstrate applications to inform the development of antibody-drug conjugates. The assay may aid discovery and manufacturing of therapeutic antibodies as a low-cost, high-throughput alternative to existing technologies.

A universal optical aptasensor for antibiotics determination based on a new high-efficiency Förster resonance energy transfer pair.

A novel "turn-on" aptasensor for kanamycin (Kana) detection based on a new Förster resonance energy transfer (FRET) pair is reported. A new organic small molecule was employed as a high-efficiency quencher for fluorophore. Based on specific interactions between ssDNA and the quencher, an ingenious and amplified strategy was designed. In the absence of the target, the fluorescence of the fluorophore labeled at the end of the aptamer was quenched. After the binding of the aptamer to the target, the fluorescence was recovered and amplified. The proposed aptasensor showed high specificity, selectivity, and stability in complicated systems. With the P3-based strategy, the limit of detection for Kana is estimated to be 10 nM, which is much lower than the maximum allowable concentration in milk. The recoveries of spiked Kana in milk were in the range 99.8 ~ 105.3% (n = 3). Fortunately, this novel method can be easily extended to other antibiotics such as tobramycin by simply replacing the aptamer, showing great potential as a universal platform for selective, sensitive, and rapid detection of hazardous analytes in food samples.

Construction of CDs@ß-CD@CCM ratiometric fluorescence probe for FRET-based ClO--sensing.

ß-Cyclodextrin (ß-CD)-functionalized carbon quantum dots (CDs) loaded with curcumin (CCM) were used for ClO-sensing with high sensitivity and selectivity. This fluorescence resonance energy transfer (FRET)-based sensor was created through attaching CCM to the CDs via ß-CD linker. CCM could get into the interior of ß-CD triggering the FRET from CDs to CCM, providing an 'off' state of the CDs. However, the effect of FRET was weakened by the ClO-, because the o-methoxyphenol structure from CCM was oxidized to be benzoquinone. The fluorescence intensity of CDs@ß-CD@CCM at 440 nm can be heightened and 520 nm from CCM can decrease along with the increased ClO-. Therefore, a ratiometric fluorescence probe for ClO-sensing is successfully constructed. It conforms to a polynomial curve equation which is I440/I520= -0.0268 + 0.0315 CClO-+ 0.0055[CClO-]2(R2= 0.9958) between 0 and 18.4µM ClO-. Furthermore, we also obtain excellent results using this spectrophotometric method for ClO--sensing in pure water and commercial disinfectants, which afford potential in the environment monitoring area. We expect this sensing platform could be helpful in other analogous probes in relevant fields.

Pilot-Scale Screening of Clinically Approved Drugs to Identify Uridine Insertion/Deletion RNA Editing Inhibitors in Trypanosoma brucei.

RNA editing pathway is a validated target in kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) that cause severe diseases in humans and livestock. An essential large protein complex, the editosome, mediates uridine insertion and deletion in RNA editing through a stepwise process. This study details the discovery of editosome inhibitors by screening a library of widely used human drugs using our previously developed in vitro biochemical Ribozyme Insertion Deletion Editing (RIDE) assay. Subsequent studies on the mode of action of the identified hits and hit expansion efforts unveiled compounds that interfere with RNA-editosome interactions and novel ligase inhibitors with IC50 values in the low micromolar range. Docking studies on the ligase demonstrated similar binding characteristics for ATP and our novel epigallocatechin gallate inhibitor. The inhibitors demonstrated potent trypanocidal activity and are promising candidates for drug repurposing due to their lack of cytotoxic effects. Further studies are necessary to validate these targets using more definitive gene-editing techniques and to enhance the safety profile.

Subtype-specific conformational landscape of NMDA receptor gating.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.

Demystifying Trion Emission in CdSe Nanoplatelets.

At cryogenic temperatures, the photoluminescence spectrum of CdSe nanoplatelets (NPLs) usually consists of multiple emission lines, the origin of which is still under debate. While there seems to be consensus that both neutral excitons and trions contribute to the NPL emission, the prominent role of trions is rather puzzling. In this work, we demonstrate that Förster resonant energy transfer in stacks of NPLs combined with hole trap states in specific NPLs within the stack trigger trion formation, while single NPL spectra are dominated by neutral excitonic emission. This interpretation is verified by implementing copper (Cu+) dopants into the lattice as intentional hole traps. Trion emission gets strongly enhanced, and due to the large amount of hole trapping Cu+ states in each single NPL, trion formation does not necessarily require stacking of NPLs. Thus, the ratio between trion and neutral exciton emission can be controlled by either changing the amount of stacked NPLs during sample preparation or implementing copper dopants into the lattice which act as additional hole traps.

Mutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics.

BACKGROUND: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. METHODS: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. RESULTS: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. CONCLUSIONS: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.

Freezing Conformers for Gas-Phase Förster Resonance Energy Transfer.

Various techniques are available to illuminate geometric structures of molecular ions in gas phase, such as Förster Resonance Energy Transfer (FRET) informing on distances between two dyes covalently attached to a molecule. Typically, cationic rhodamines, which absorb and emit visible light, are used for labeling. Extensive work has revealed that the transition energy of a rhodamine is intricately linked to its nearby microenvironment, with nearby charges causing Stark-shifted emission. This occurs because the inter-dye Coulomb interaction is weaker in the excited state (S1) than in the ground state (S0) due to the increase in polarizability upon excitation. Therefore, absorption and emission spectra, along with FRET efficiencies, provide insights into structural motifs. At room temperature, multiple conformers often co-exist, leading to overlapping absorption bands among different conformers and broad spectra. To study specific conformers, it is necessary to isolate them, for example, using ion-mobility spectrometry. Another approach is to reduce temperature, which results in spectral narrowing and distinct absorption bands, allowing for the selection of specific conformers through selective excitation. Here, we describe the instrumentation used for cryogenically cold FRET experiments and discuss recent results for small model systems, as well as future directions for a technique still in its infancy.

Development of new NGLY1 assay systems - toward developing an early screening method for NGLY1 deficiency.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1  -/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

Multimodal integrated and broadband light-driven antibacterial cellulose fabric based on π-π coupling enhanced intermolecular FRET.

Fabrication of antimicrobial photodynamic therapy (aPDT) materials based on organic photosensitizers has garnered considerable attention within functional textiles. However, the UV- or narrow-band absorption range of the photosensitizers results in poor photon utilization of the fabrics, limiting the photodynamic efficiency and wasting solar energy. In this study, a broadband light-driven antibacterial cellulose fabric (CF-ZnPc/NAD) was developed by loading carboxyl-modified zinc(II) phthalocyanine photosensitizer (CAZnPc) and cationic 1,8-naphthalimide fluorescent molecule (NAD) on the fabric via covalent binding and electrostatic adsorption assembly, facilitating the intermolecular π-π coupling and fluorescence resonance energy transfer (FRET) process. There is a 2.54-fold increase in photo-induced ROS generation capacity of CF-ZnPc/NAD via the FRET process compared to that of CF-ZnPc, and it also exhibited a strong photothermal effect (PTT), wherein the temperature of the fabric increased from 24.5 to 53.5 °C within 80 s of illumination (λ > 400 nm, 75 mW/cm2). CF-ZnPc/NAD exhibited strong light-harvesting capacity and a combination of aPDT and PTT, achieving excellent antibacterial performance against Staphylococcus aureus (Gram-positive, S. aureus) and Escherichia coli (Gram-negative, E.coli) with 99.99 % bacterial reduction under 90 min of illumination (λ > 400 nm, 10 ± 1 mW/cm2). This study demonstrates a novel and facile strategy for successfully fabricating high-performance antibacterial cellulose fabrics with potential biomedical prospects.

Label-Free and Ultra-Sensitive Detection of Dexamethasone Using a FRET Aptasensor Utilizing Cationic Conjugated Polymers.

Dexamethasone (Dex) is a widely used glucocorticoid in medical practice, with applications ranging from allergies and inflammation to cerebral edema and shock. Despite its therapeutic benefits, Dex is classified as a prohibited substance for athletes due to its potential performance-enhancing effects. Consequently, there is a critical need for a convenient and rapid detection platform to enable prompt and accurate testing of this drug. In this study, we propose a label-free Förster Resonance Energy Transfer (FRET) aptasensor platform for Dex detection utilizing conjugated polymers (CPs), cationic conjugated polymers (CCPs), and gene finder probes (GFs). The system operates by exploiting the electrostatic interactions between positively charged CCPs and negatively charged DNA, facilitating sensitive and specific Dex detection. The label-free FRET aptasensor platform demonstrated robust performance in detecting Dex, exhibiting high selectivity and sensitivity. The system effectively distinguished Dex from interfering molecules and achieved stable detection across a range of concentrations in a commonly used sports drink matrix. Overall, the label-free FRET Dex detection system offers a simple, cost-effective, and highly sensitive approach for detecting Dex in diverse sample matrices. Its simplicity and effectiveness make it a promising tool for anti-doping efforts and other applications requiring rapid and accurate Dex detection.

Stabilization of interdomain closure by a G protein inhibitor.

Inhibitors of heterotrimeric G proteins are being developed as therapeutic agents. Epitomizing this approach are YM-254890 (YM) and FR900359 (FR), which are efficacious in models of thrombosis, hypertension, obesity, asthma, uveal melanoma, and pain, and under investigation as an FR-antibody conjugate in uveal melanoma clinical trials. YM/FR inhibits the Gq/11/14 subfamily by interfering with GDP (guanosine diphosphate) release, but by an unknown biophysical mechanism. Here, we show that YM inhibits GDP release by stabilizing closure between the Ras-like and α-helical domains of a Gα subunit. Nucleotide-free Gα adopts an ensemble of open and closed configurations, as indicated by single-molecule Förster resonance energy transfer and molecular dynamics simulations, whereas GDP and GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) stabilize distinct closed configurations. YM stabilizes closure in the presence or absence of GDP without requiring an intact interdomain interface. All three classes of mammalian Gα subunits that are insensitive to YM/FR possess homologous but degenerate YM/FR binding sites, yet can be inhibited upon transplantation of the YM/FR binding site of Gq. Novel YM/FR analogs tailored to each class of G protein will provide powerful new tools for therapeutic investigation.

Unlocking multi-mode sensing potential: Phosphorus-doped graphitic carbon nitride quantum dots for Ag+, ciprofloxacin, and riboflavin analysis in environment and food matrices.

The simultaneous detection of multiple analytes through a single fluorescence sensor is highly attractive. In this study, phosphorus-doped graphitic carbon nitride quantum dots (P-CNQDs) were developed, achieving multi-mode sensing through three distinct response mechanisms. The preparation involved using melamine as the carbon and nitrogen source and ammonium dihydrogen phosphate as the phosphorus source. Uniform and narrowly distributed P-CNQDs were successfully synthesized through chemical oxidation and hydrothermal methods, with an average size of 2.4 nm. These unique P-CNQDs exhibited fluorescence quenching through photo-induced electron transfer (PET) in response to Ag+. Additionally, the formation of hydrogen bonds and coordination interactions between P-CNQDs-Ag+ and ciprofloxacin (CIP) led to a pronounced fluorescence response to CIP by the chelation enhanced fluorescence (CHEF) mechanism. Furthermore, leveraging the principle of fluorescence resonance energy transfer (FRET), P-CNQDs-CIP served as a ratio fluorescence sensor for riboflavin (RF), enabling ultra-sensitive detection of RF. The combination of PET, CHEF, and FRET response mechanisms successfully facilitated multi-mode sensing for Ag+, CIP, and RF. The detection ranges were 0.05-100 µM, 0.002-2 µM, and 0.05-60 µM, with corresponding lowest detection limits of 17.1 nM, 1.1 nM, and 29.2 nM, respectively. This versatile sensor has been effectively applied to real samples, including the detection of river water and vitamin B2 tablets.