Fluorescence Technique Research Articles

Enhancing Spectral Coverage, Efficiency, and Photometric Accuracy in Ultrafast Fluorescence Upconversion Spectroscopy

Time‐resolved fluorescence techniques are essential for studying the excited‐state dynamics of molecules and materials, as well as their interactions with the surrounding media. Fluorescence upconversion provides femtosecond time resolution, but its limited spectral coverage often restricts its application. Broadband variants of fluorescence upconversion spectroscopy (FLUPS) offer improved spectral coverage but require balancing spectral bandwidth against signal strength. In this paper, we introduce a three‐angle method (3‑AM) for FLUPS designed to overcome these limitations. This method enhances spectral coverage, photometric accuracy, and signal‐to‐noise ratio by recording and averaging spectrally resolved upconversion signals at three distinct crystal angles. Unlike other techniques that rely on spectral reconstruction or continuous crystal rotation during acquisition, the 3‑AM relies on fixed crystal positions, allowing for robust post‐acquisition corrections. We validate the performance and reproducibility of the 3‑AM by comparing it with the conventional fixed‐angle approach, as well as through blind testing in two independent laboratories. Despite variations in experimental configuration, the 3‑AM consistently produces reproducible spectral line shapes across the two setups. The enhanced performance and reliability demonstrate its practicality for laboratories with broadband FLUPS capabilities. Thus, the 3‑AM is a valuable tool for investigating ultrafast radiative processes in previously inaccessible molecular and material systems.

A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.

Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).

Integrated 3D geo-environmental assessment of acid-forming materials in historic coal waste piles for sustainable management.

Coal mining produces coal mine waste rock (CMWR), posing significant environmental risks, including acid mine drainage (AMD) if unmanaged. The Jerada Mine in eastern Morocco has accumulated CMWR since it began operations in 1936, with no rehabilitation efforts until 2001. This study assessed the stability of the T08 pile, which has been deposited over five decades across various oxidation zones. More than 400 samples from 13 drill holes were thoroughly analyzed, including particle size distribution, X-ray fluorescence (XRF), and other advanced techniques, culminating in a 3D model to identify potentially acid-forming (PAF) zones. Particle sizes (D30 and D90) ranged from 16.3 to 16.5μm in low-oxidation zones to 353.3-409μm in highly oxidized areas, respectively. Sulfur content varied from 0.32 to 2.05wt% for sulfide and from 0.0013 to 0.17wt% for sulfate, with an acidification potential ranging from 14.42 to 29.2kg CaCO₃/t and negative net neutralization potential (NNP) from -35.12 to -11.14kg CaCO₃/t. NAG tests revealed a low pH of approximately 4 and acidity levels exceeding safety thresholds, with low neutralizing minerals content. Pyrite was the dominant sulfide, alongside ankerite, hematite, and goethite. Highly oxidized zones exhibited larger particle size distributions, increasing porosity and airflow. Thereby, enhancing oxidation and converting iron into different oxidation states. This process affects sulfur speciation, leading to sulfate formation. The 3D model estimated 3.8Mt of PAF material in the upper pile, highlighting a heterogeneous distribution linked to porosity and oxidation levels, underscoring the necessity for further kinetic testing to evaluate long-term AMD risks.

A novel method of using indocyanine green fluorescence technique for nephron-sparing surgery

A novel method of using indocyanine green fluorescence technique for nephron-sparing surgery

Suppressing morphology-dependent resonances in laser-induced fluorescence signals of fuel droplets using a two-dye approach

The present study focuses on suppressing morphology-dependent resonances (MDRs) in laser-induced fluorescence signals of micrometric fuel droplets. The fluorescence signal is generated by doping the fuels with the fluorophore nile red and its excitation by a pulsed Nd:YAG laser at 532 nm. The fluorescence signals are collected by a spectrometer and an imaging setup. In general, the MDRs appear at longer wavelengths in the emission spectrum compared to the fluorescence maximum. These artefacts can lead to large measurement uncertainties, especially for ratio-based fluorescence techniques. An admixture of the second dye solvent blue 38 (S38), which shows an absorption band matching the MDR emission of nile red, enables a suppression of MDRs that are also shifted to larger wavelengths. The study investigates the influence of S38 at various concentrations to the fluorescence signal of nile red in micrometric spherical ethanol droplets between 40 µm and 80 µm. Furthermore, the investigations revealed that an increase in laser fluence inevitably requires a larger concentration of S38. A sufficient admixture of S38 for a certain concentration of nile red ensures a full suppression of the MDRs in nile red fluorescence signals and facilitates reliable measurement conditions for droplet studies.

Scaffolds fabricated via two-photon polymerisation for regulating cell morphology and differentiation

ABSTRACT Three-dimensional (3D) cell culture scaffolds play a key role in guiding cell fate. The fine-tunability of these scaffolds is essential for accurately replicating in vivo conditions and revealing cellular behaviours. In this study, two-photon polymerisation (TPP) technology was employed to create 3D scaffolds with woodpile and honeycomb architectures. The influence of scaffold geometry and pore dimensions on the proliferation, morphology, orientation, and differentiation of human mesenchymal stem cells (hMSCs) was investigated through multi-staining fluorescence and 3D confocal imaging technique. It was revealed that hMSCs cultured within these 3D scaffolds demonstrated higher density up to 15.04 ± 1.46 cells/100 × 100 μm2 after a 6-day culture compared to their 2D counterparts (7.47 ± 1.42 cells/100 × 100 μm2). Furthermore, hMSCs grown on the woodpile scaffolds displayed elongated morphology, with approximately 50% aligning along the columns. In contrast, hMSCs cultivated on honeycomb structures exhibited triangular and crescent cellular shapes, with a random orientation. Notably, compared to the control group, the cells on the scaffolds exhibited smaller nucleus areas, lower circularity, and aspect ratios, but these values increased as pore size increased. Furthermore, ALP staining showed a greater tendency for osteogenic differentiation with larger pore sizes. These TPP-fabricated 3D scaffolds hold immense promise for advancing understanding of cellular behaviour.

Perspective: fluorescence lifetime imaging and single-molecule spectroscopy for studying biological condensates

Abstract Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These are critical for cellular processes and implicated in diseases like cancer and neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM), FLIM-FRET, and fluorescence correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, and their responses to environmental stimuli in live cells. This perspective highlights the utility of time-resolved fluorescence and single-molecule spectroscopy techniques for shedding light on condensate functions, properties, and interactions with membranes, offering insights into cellular physiology and pathology.

A Redox-Active Copper Complex for Orthogonal Detection of Homocysteine Involving Fluorescence and Electrochemical Techniques.

The present work reports the synthesis, characterization, and excited state photo-physical studies of two copper(II) compounds, 1 & 2, which show interference-free emission with homocysteine (Hcy). Cu(II) complexes offer an orthogonal detection strategy involving fluorescence and electrochemical methods, paving the way for improved point-of-care diagnostics and early cardiovascular diseases intervention. The reduction-induced emission enhancement (RIEE) of Cu complexes facilitates the fluorescence measurement of Hcy at physiological pH. The fluorogenic redox-active 1 and 2 are deposited onto gold electrode surfaces to construct the electrochemical sensors 1@Au and 2@Au, respectively. Under specific alkaline conditions, a distinct and selective redox peak at 0.6V (vs Ag/AgCl) emerges for 1@Au upon interaction with homocysteine. Further, square wave voltammetry confirms the non-interference of its congener (cysteine) even at high concentrations (200µM) while detecting Hcy (5-100µM), demonstrating its potential for real-world applications. The fabricated 1@Au exhibits excellent sensitivity of 31.88µA/µM, with an impressive detection limit of 2.26nM, and a limit of quantification of 6.85nM toward Hcy. The analytical applicability of the 1@Au is validated by quantifying Hcy levels in human blood plasma samples. The results highlighted the feasibility of the proposed technique as a rapid and portable monitoring of Hcy in diagnosing cardiovascular diseases (CVDs).

Exploring Microbial Signatures in Endometrial Tissues with Endometriosis.

The endometrial microbiota exerts a crucial role in maintaining the health of the female reproductive system. As such, in this study, we have examined the composition of the microbiota in endometrium tissues with and without endometriosis, with the objective of identifying key species that may potentially contribute to the progression of endometriosis. We obtained endometrial tissues from 43 women diagnosed as either having endometriosis or not. Subsequently, we utilized a diverse array of techniques, including fluorescence in situ hybridization (FISH), immunohistochemistry (utilizing anti-LPS and anti-LTA antibodies), transmission electron microscopy (TEM), and 16S rRNA sequencing, to undertake a comprehensive examination of the presence of microorganisms in the endometrium and their potential role in endometriosis. Our findings consistently indicated the existence of bacteria in both normal endometrium tissues and those affected by endometriosis. By employing the fluorescent co-staining technique, we observed the colocalization of macrophages and bacteria in both tissue types. Notably, we discovered a significant increase in microbial diversity in endometrial tissue from women with endometriosis compared to normal endometrial tissues. Additionally, we identified 13 species that were more abundant in the normal group, such as Acinetobacter guillouiae. In contrast, seven species were prominent in the endometriosis group, with Faecalibacterium prausnitzii being a notable one. Finally, our results suggest that Faecalibacterium prausnitzii may play an essential role in the progression of endometriosis. We carried out a comprehensive analysis of the endometrial microbial landscape in endometriosis tissue and revealed that Faecalibacterium prausnitzii is a pivotal species that may potentially play a crucial role in the pathogenesis of endometriosis.

Rapid identification of marine microplastics by laser-induced fluorescence technique based on PCA combined with SVM and KNN algorithm.

The laser-induced fluorescence technique has the advantage of fast and non-destructive detection and can be used to classify types of marine microplastics. However, spectral overlap poses a challenge for qualitative and quantitative analysis by conventional fluorescence spectroscopy. In this paper, a 405 nm excitation laser source was used to irradiate 4 types of microplastic samples with different concentrations, and a total of 1600 sets of fluorescence spectral data were obtained. The 726 data points contained in each sample spectrum were first analyzed by PCA, and the 4 microplastics were differentiated by their position in the PCA score plot. The classification and identification are then performed by SVM, KNN, PCA-SVM and PCA-KNN algorithms respectively. The classification accuracy of microplastics in seawater using SVM and KNN algorithms is higher than 86%. The classification accuracy can be increased to 100% by PCA combined with SVM and KNN algorithm. Concentration inversion was conducted by SVM and KNN algorithms after classification. The correlation coefficients between the predicted values and the actual values were higher than 0.8, and the RMSE was less than 0.47%, which indicated that both algorithms had good prediction results. These machine learning methods provide accurate and reliable identification results in the rapid identification of microplastic types and their concentrations without complex spectral data preprocessing and fluorescence background removal algorithms.

The Application of X-Ray Fluorescence to Assess Proportions of Fresh Concrete

Any transportation infrastructure system is concerned with durability and performance issues. The proportioning and uniformity control of concrete mixtures are critical factors that directly affect the longevity and performance of concrete pavements. Currently, the only means available to monitor mix proportions of any batch are to track batch tickets created at the batch plant. This does not take into account potential errors in loading materials into storage silos, calibration errors, and addition of water after dispatch. Therefore, there is a need for a rapid, cost-effective, and reliable field test that estimates the proportions of as delivered concrete mixtures. In addition, performance based specifications will be more easily implemented if there were a way to readily demonstrate whether any given batch is similar to the proportions already accepted based on laboratory performance testing. This paper describes a preliminary investigation into the potential use of a portable x-ray fluorescence (XRF) technique to assess the proportions of concrete mixtures as they are delivered. Tests were conducted on the raw materials, paste and mortar samples using a portable XRF device. There is a reasonable correlation between the actual and calculated mix proportions of the paste samples, but data on mortar samples was less reliable.

Advances in Fluorescence Techniques for the Detection of Hydroxyl Radicals near DNA and Within Organelles and Membranes.

Hydroxyl radicals (•OH), the most potent oxidants among reactive oxygen species (ROS), are a major contributor to oxidative damage of biomacromolecules, including DNA, lipids, and proteins. The overproduction of •OH is implicated in the pathogenesis of numerous diseases such as cancer, neurodegenerative disorders, and some cardiovascular pathologies. Given the localized nature of •OH-induced damage, detecting •OH, specifically near DNA and within organelles, is crucial for understanding their pathological roles. The major challenge of •OH detection results from their short half-life, high reactivity, and low concentrations within biological systems. As a result, there is a growing need for the development of highly sensitive and selective probes that can detect •OH in specific cellular regions. This review focuses on the advances in fluorescence probes designed to detect •OH near DNA and within cellular organelles and membranes. The key designs of the probes are highlighted, with emphasis on their strengths, applications, and limitations. Recommendations for future research directions are given to further enhance probe development and characterization.

Association Between 5-HTRs Gene Polymorphism and Alcohol Use Disorder in Han Males from Yunnan, China.

Alcohol use disorder (AUD) has become a very serious medical and social problem. It is found that genetic polymorphisms of the 5-hydroxytryptamine receptors (5-HTRs) genes were associated with the risk of AUD. However, the results are controversial among different ethnic groups. At present, the correlation between 5-HTRs gene polymorphism and AUD in Han population from Yunnan Province remains unclear. In this study, 13 single nucleotide polymorphisms (SNPs) of HTR1B, HTR2A, HTR3A, HTR3B and HTR7 were detected by universal fluorescent probe technique. The CT genotype frequency of HTR3A rs1062613 was significantly higher in AUD case group than that in control group (P = 0.037, OR = 2.193, 95% CI: 1.048 ∼ 4.366). The study indicated that the genetic polymorphisms of 5-HTRs were significantly associated with risk of AUD in Han male from Yunnan,China. In addition, this study further demonstrated the impact of alcohol on human health, especially liver damage, by analyzing the blood biochemical indicators of patients with AUD and combining them with their medical history.

ICT-based fluorescent nanoparticles for selective cyanide ion detection and quantification in apple seeds.

In this report, we successfully engineered a novel probe based on an acceptor-donor-acceptor (A-D-A) architecture featuring dicyanovinyl-substituted thieno[3,2-b]thiophene, termed DCVTT. The designed probe self-assembles into luminous nanoparticles (DCVTT NPs) upon introducing mixed aqueous solutions. These fluorescent nanostructures served as a ratiometric probe for detecting cyanide (CN-) ions in aqueous-based environments, owing to the robust Intramolecular Charge Transfer (ICT) characteristics of DCVTT. The A-D-A substituents in DCVTT significantly enhanced ICT behavior by promoting more efficient electron transfer between the donor and acceptor groups. This improved electron transfer process leads to heightened sensitivity in detection applications. In the case of cyanide (CN) sensing, this enhanced ICT behavior manifests as a strong colorimetric response, allowing for a visible color change before and after interaction with cyanide. Speculation regarding the interaction mechanism between DCVTT and CN- is proposed based on the findings of various experimental analyses. The detection limit (LOD) for DCVTT in identifying CN- is 0.83 nM, significantly lower than the CN- concentration thresholds deemed safe by the World Health Organization (WHO) and the United States Environmental Protection Agency (EPA). Time-Dependent Density Functional Theory (TD-DFT) has been utilized to theoretically analyze the optical properties of DCVTT both before and after the introduction of the CN- ions. A paper-based test strip was developed to demonstrate its practical application to enable efficient qualitative CN- detection by visual inspection. Furthermore, this sensing platform demonstrates highly accurate quantitative detection of CN- in apple seeds. No prior reports have utilized fluorescence techniques to estimate apple seeds' CN levels.

Physicochemical Characteristics of Gum Arabic From Acacia mearnsii Grown in Alahan Panjang, West Sumatera

Acacia gum produced from Acacia mearnsii is a natural material with great potential for various industrial applications due to its unique physicochemical properties. This study aimed to characterize gum acacia from Alahan Panjang, West Sumatra. Characterizatio was done using Fourier Transform Infrared Spectroscopy (FTIR), X-ray fluorescence (XRF), and X-ray diffraction (XRD) techniques. FTIR results revealed the presence of hydroxyl (-OH), carbonyl (-C=O), and glycosidic (C-O-C) groups, indicating the complex polysaccharide structure of the gum. XRF analysis showed the dominance of calcium oxide (CaO) with a concentration of 81.652%, followed by potassium oxide (K₂O) at 10.052% and phosphate (P₂O₅) at 6.341%. Meanwhile, the XRD diffraction pattern identified a near amorphous diffraction with some weak crystalline peaks. This combination of results suggests that gum acacia has chemical and physical properties that support its use as an emulsifier, thickener, and filler in various industries, such as food, pharmaceutical, and cosmetics.

Prospective Investigation of Nanoplastic Accumulation in Healthy Subjects, Autoimmune Diseases, Hematological Malignancies, Lung Cancer, and Murine Models

Nanoplastics (NPs) and microplastics (MPs) are an emerging threat to global health. They negatively impact ecosystems and many physiological processes, causing alterations in xenobiotic metabolism, nutrient uptake, energy metabolism, or cytotoxicity. In humans, we are beginning to analyze these plastics for the mechanisms by which they enter the organism, accumulate, and diffuse as well as for their pathogenic potential. NP accumulation has been demonstrated in human tissues, such as blood or placenta, while in others it remains largely unstudied. In this work, we detected NP accumulation in bronchoalveolar lavage fluids (BALFs), cerebrospinal fluids (CSFs), lymph nodes (LNs), urine, pleural fluids (PFs), ascitic fluids (AFs), and peripheral blood (PB) by combining fluorescence and nanocytometry techniques. NP analysis was compared with two strains of mice, and the results support that inhalation is the main route of NP accumulation.

Extrachromosomal DNA in Breast Cancer Cell Lines: Detection and Characterization.

This study delves into the intriguing world of extrachromosomal DNA (ecDNA) in breast cancer, uncovering its pivotal role in cancer's aggressiveness and genetic variability. ecDNA, a form of circular DNA found outside chromosomes, is known to play a significant role in cancer progression by increasing oncogene expression. Focusing on two contrasting cell lines, MDA-MB-231 (triple-negative) and MCF-7 (Luminal-A), we utilized advanced microscopy and fluorescence techniques to detect and characterize ecDNA. Our findings reveal a stark difference: MDA-MB-231 cells, known for their high metastatic potential, exhibit a striking abundance of ecDNA, manifested as double minutes and single form with intense fluorescence signals. In contrast, the less aggressive MCF-7 cells harbor significantly fewer ecDNA. This disparity highlights the potential of ecDNA as a key player in cancer progression and a promising target for novel therapies. This research sheds light on the unseen genetic forces driving breast cancer and opens the door to new strategies in cancer treatment. Further research is necessary to understand the mechanisms of ecDNA formation and its role in different breast cancer subtypes.

Construction of colorimetric-fluorescent dual-signal aptamer-based assay using COF-Au nanozyme and magnetic nanoparticle-based CdTe quantum dots for sensitive zearalenone determination.

Adual-signal aptamer-based assay utilizing colorimetric and fluorescence techniques was developed for the determination of zearalenone (ZEN). The CdTe quantum dots, serving as the fluorescent signal source, were surface-modified onto Fe3O4@SiO2 and subsequently functionalized with the aptamer. The COF-Au was modified with complementary chain, which possessed peroxide (POD)-like enzyme properties, and could catalyze the peroxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to ox TMB, resulting in the generation of colorimetric signals. The two parts were merged based on the principle of base complementary pairing, resulting in an assembled structure exhibiting a diminished fluorescence signal due to the Förster resonance energy transfer (FRET) effect. Due to the higher affinity of the aptamer towards the target, the presence of ZEN resulted in the detachment of COF-Au, leading to an increase in supernatant concentration of COF-Au proportional to ZEN concentration. Consequently, this enhanced thecatalytic ability and amplified thecolorimetric signal. The fluorescence of precipitation increased simultaneously with the reduction of FRET, enabling linear detection of colorimetry in the range 0.5 ~ 10,000μg·kg-1 and fluorescence in the range 0.1 ~ 10,000μg·kg-1, with respective detection limits of 0.36μg·kg-1 and 0.09μg·kg-1. The spike recovery in wheat flour and corn ranged from 93.4 to122.0%. This technology was simple to operate and had low cost and good application prospects.

Validation of Wavelength-Dispersive X-Ray Fluorescence Technique for Rapid Screening Iron and Zinc Concentration in Rice Seed

This study focuses on the validation of a wavelength dispersive X-ray fluorescence (WD-XRF) spectrometer for the rapid determination of iron (Fe) and zinc (Zn) concentrations in certified reference materials (CRMs). Five CRMs were used to establish calibration curves. The results were analyzed in terms of accuracy, precision, repeatability and linearity to assess the performance of the WD-XRF method. Accuracy was evaluated by comparing measured values to certified values, with relative accuracy calculated for each sample. Precision was determined through repeatability, where samples were measured in at least 7 replicates across different days, and relative standard deviation (RSD) was calculated. Linearity was assessed by plotting measured intensities and concentrations against certified concentrations, with the correlation coefficient (R2) values close to 1, indicating a strong linear relationship. Additionally, the WD-XRF method demonstrated high repeatability, with low RSD values across multiple measurements. The study concluded that WD-XRF is a reliable method for the rapid analysis of elemental composition, providing strong accuracy, precision and linearity for Fe and Zn determination in CRMs and can be applied as a valuable method for rapid and non-destructive analysis in various agricultural and food-related applications. HIGHLIGHTS The effectiveness of wavelength dispersive X-ray fluorescence (WD-XRF) method was evaluated to confirm its reliability for determining the elemental composition, specifically iron (Fe) and zinc (Zn), in plant-based certified reference materials (CRMs). Five CRMs were used for calibration and 2 known samples were used as the test samples to determine the analytical performance of WD-XRF. The linearity and correlation with the certified values of WD-XRF results revealed strong correlation with R-square being greater than 0.95 and the recovery percentage of 2 known samples were range from 98.75 - 109.66 for Fe and Zn concentration. WD-XRF spectrometer can be used as a rapid method for determination of Fe and Zn concentrations in rice seeds. GRAPHICAL ABSTRACT

Multisubstrate-based system: a kinetic mechanism study of catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT, EC2.1.1.6) can transfer the methyl group from S-adenosyl-l-methionine (SAM) to one of the hydroxyl groups of a catechol substrate in the presence of Mg2+. However, there is no consensus view of the kinetic mechanism of COMT involving multiple substrates. Further progress requires the development of methods for determining enzyme kinetic behavior and the binding mode of ligands to the protein. Here, we establish a multisubstrate kinetic system covering the fluorescence and mass spectrometry techniques to quantify the products in a COMT-catalyzed reaction. The catechol substrate, 3-BTD, can be methylated by COMT to form a single product, 3-BTMD, with a sensitive fluorescence response and the conversion of SAM to S-adenosyl-l-homocysteine (SAH) was monitored by LC-MS/MS. The kinetic assays suggested that the reaction occurred via an ordered sequential mechanism, in which SAM first bound to COMT, followed by the addition of Mg2+ and 3-BTD. The chemical step involved a quaternary complex of COMT-SAM-Mg2+-3-BTD, followed by the ordered dissociation of 3-BTMD, Mg2+, and SAH. In cooperation with molecular dynamics simulation, the binding of COMT to Mg2+ induced a shape change in the catechol-binding site, which accommodated 3-BTD binding and facilitated catalysis. These findings provide new insights into the kinetic mechanism of COMT, contributing to the development of previously undescribed functional COMT ligands.