[Determination of sodium cyclamate in Baijiu by pre-column derivatization-high performance liquid chromatography with fluorometric detection].

Sodium cyclamate in Baijiu is a key item in the China National Food Safety Supervision and Inspection Plan. A simple, economical, sensitive, and reliable method is urgently needed for routine analysis and internal quality control. A method based on high performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for the determination of sodium cyclamate in Baijiu by o-phthalaldehyde derivatization. First, the sodium cyclamate in the sample solution was converted into amino compounds using the desulfurization reaction under acidic conditions. Next, 400 g/L sodium hydroxide solution was added to the sample solution for neutralization. The amino compounds in the sample solution were then derivatized with o-phthalaldehyde to produce indole-substituted derivatives that are capable of producing fluorescence signals. Separation was carried out on a C18 column (250 mm×4.6 mm, 5 µm) in isocratic elution mode using a mobile phase consisting of acetonitrile and phosphate buffer. Finally, the eluate was monitored using a fluorescence detector, and an external standard method was used for quantification. A good linear relationship was obtained in the range of 0.1-2.0 mg/L, with correlation coefficients greater than 0.999. The average recoveries of sodium cyclamate spiked at levels of 0.1-1.0 mg/kg in Baijiu samples ranged from 90.7% to 100.9%, with relative standard deviations (RSDs) of 3.5%-5.6% (n=6). The limits of detection and quantification were 0.03 and 0.10 mg/kg, respectively. Nine Baijiu samples collected from the market were tested, and the results demonstrated that the contents of sodium cyclamate detected by the developed method were consistent with those obtained using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method described in GB 5009.97-2016 (the third method). The proposed method is economical, sensitive, specific, and accurate; thus, it provides a basic approach for the determination of sodium cyclamate in Baijiu samples and has great potential for routine analysis in foodstuffs.

Fluorometric Methods to Measure Bioavailable and Total Heme.

Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.

Fluorescence-Based Enzyme Activity Assay: Ascertaining the Activity and Inhibition of Endocannabinoid Hydrolytic Enzymes.

The endocannabinoid system, known for its regulatory role in various physiological processes, relies on the activities of several hydrolytic enzymes, such as fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), monoacylglycerol lipase (MAGL), and α/ß-hydrolase domains 6 (ABHD6) and 12 (ABHD12), to maintain homeostasis. Accurate measurement of these enzymes' activities is crucial for understanding their function and for the development of potential therapeutic agents. Fluorometric assays, which offer high sensitivity, specificity, and real-time monitoring capabilities, have become essential tools in enzymatic studies. This review provides a comprehensive overview of the principles behind these assays, the various substrates and fluorophores used, and advances in assay techniques used not only for the determination of the kinetic mechanisms of enzyme reactions but also for setting up kinetic assays for the high-throughput screening of each critical enzyme involved in endocannabinoid degradation. Through this comprehensive review, we aim to highlight the strengths and limitations of current fluorometric assays and suggest future directions for improving the measurement of enzyme activity in the endocannabinoid system.

High-Throughput Fluorometric Assay For Quantifying Polysorbate In Biopharmaceutical Products Using Micelle Activated Fluorescence Probe N-Phenyl-1-Naphthylamine.

PURPOSE: Polysorbates are among the most used surfactants in biopharmaceutical products containing proteins. Our work aims to develop a high-throughput fluorometric assay to further diversify the analytical toolbox for quantification of PSs. METHOD: The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference. RESULTS: All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75 mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer. CONCLUSION: A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.

An aptamer-based fluorometric method for the rapid berberine detection in Kampo medicines.

BACKGROUND: Berberine (BBR), a key component in Kampo medicine, is a cationic benzylisoquinoline alkaloid whose detection plays a critical role in the quality control of these traditional remedies. Traditional methods for detecting BBR often involve complex procedures, which can be time-consuming and costly. To address this challenge, our study focuses on developing a simpler, faster, and more efficient detection method for BBR in Kampo medicine formulations. RESULTS: We successfully developed a rapid fluorometric detection method for BBR using colloidal gold nanoparticle-based systematic evolution of ligands by exponential enrichment (GOLD-SELEX). Initially, specific single-stranded DNA (ssDNA) sequences were selected for their ability to enhance BBR's fluorescence intensity. The optimal ssDNA sequence, identified as BBR38, was further truncated to produce BBR38S, a stem-loop ssDNA that improved fluorescence upon interaction with BBR. To further enhance the fluorescence, the BBR38S aptamer underwent additional modifications, including stem truncation and nucleotide mutations, resulting in the higher fluorescence variant BBR38S-3 A10C. The final product, TetBBR38S, a tetramer version of BBR38S-3 A10C, exhibited a linear detection range of 0.780-50.0 µg mL-1 and a limit of detection of 0.369 µg mL-1. The assay demonstrated sufficient selectivity and was successfully applied to analyze 128 different Kampo medicine formulations, accurately detecting BBR content with high precision. SIGNIFICANCE: This study represents an advancement in Kampo medicine research, marking the first successful application of an aptamer-based approach for BBR detection in complex matrices. The developed method is not only simple and rapid (with a detection time of 5 min) but also cost-effective, which is crucial for widespread application.

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements.

Herein, a new, direct paper-based fluorimetric method is described for the quantitative determination of glutathione (GSH) molecules in nutritional supplements. Briefly, the proposed analytical method is based on the fluorescence emission resulting from the direct and selective chemical reaction of GSH molecules with the derivatization reagent that is o-phthalaldehyde (OPA) in acidic conditions at room temperature. The intensity of the emitted fluorescence on the surface of the analytical paper devices after irradiation with a lamp at 365 nm is proportional to the concentration of GSH and is measured using a smartphone as the detector. This methodology, which is suitable for measurements in laboratories with limited resources, does not require specialized instrumentation or trained personnel. The protocol governing the proposed method is simple and easily applicable. Essentially, the chemical analyst should adjust the value of pH on the surface of the paper by adding a minimal amount of buffer solution; then, after adding a few microliters of the derivatization reagent, wait for the surface of the paper to dry and, finally, add the analyte. Subsequently, the irradiation of the sensor and the measurement of the emitted fluorescence can be recorded with a mobile phone. In the present study, several parameters affecting the chemical reaction and the emitted fluorescence were optimized, the effect of interfering compounds that may be present in dietary supplements was examined, and the stability of these paper sensors under different storage conditions was evaluated. Additionally, the chemical stability of these paper devices in various maintenance conditions was studied, with satisfactory results. The detection limit calculated as 3.3 S/N was 20.5 µmol L-1, while the precision of the method was satisfactory, ranging from 3.1% (intra-day) to 7.3% (inter-day). Finally, the method was successfully applied to three different samples of dietary supplements.

Voltage Clamp Fluorometry: Illuminating the Dynamics of Ion Channels.

Ion channels comprise one of the largest targets for drug development and treatment and have been a subject of enduring fascination since first discovered in the 1950s. Over the past decades, thousands of publications have explored the cellular biology and molecular physiology of these proteins, and many channel structures have been determined since the late 1990s. Trying to connect the dots between ion channel function and structure, voltage clamp fluorometry (VCF) emerges as a powerful tool because it allows monitoring of the conformational rearrangements underlying the different functional states of the channel. This technique represents an elegant harmonization of molecular biology, electrophysiology, and fluorescence. In the following chapter, we will provide a concise guide to performing VCF on Xenopus laevis oocytes using the two-electrode voltage clamp (TEVC) modality. This is the most widely used configuration on Xenopus oocytes for its relative simplicity and demonstrated success in a number of different ion channels utilizing a variety of attached labels.

Synthesis of biscarbazole derivative, detection of the "on-off" sensor property of Cu2+ by fluorimetry, and anti-cancer evaluation.

Biscarbazole derivative probe (6) (Z)-2-(3-(((9-heptyl-9H-carbazol-3-yl)methylene)amino)-9H-carbazol-9-yl)ethan-1-ol containing an imine group, which is a sensitive and selective fluorescence chemosensor, was designed and synthesized for the effective evaluation of Cu2+ metal ion levels. The synthesized compounds were characterized using 1H NMR, 13C NMR, FT-IR, and MALDI-TOF MS (for compound 6) spectroscopic data. The interaction model between probe 6 and Cu2+ was determined by combining fluorescence methods, 1H NMR titration, Job's plot, and theoretical calculations. For probe 6, the fluorogenic recognition of Cu2+ was investigated by fluorescence spectroscopy, and the optical changes caused by Cu2+ ions were carried out in ACN/H2O (50:50) solution at pH 7.0. Fluorescence probe 6 was found to "turn-off" its fluorescence in the presence of paramagnetic Cu2+ ions. Probe 6 was determined to have a rapid response within 40s and showed a fluorescence response to Cu2+ with a low detection limit of 0.16 µM. Additionally, in vitro anticancer activity and cell imaging studies of probe 6 against the prostate cell line (PC-3) were performed.

A dual-signals fluorometric and colorimetric peptide-based probe for Cu(II) and glyphosate detection and its application for bioimaging and water testing.

A novel dual-signal fluorometric and colorimetric probe FMDH (5-FAM-Met-Asp-His-NH2), incorporating a tripeptide (Met-Asp-His-NH2) linked to 5-carboxyfluorescein (5-FAM), was firstly synthesised. FMDH demonstrated exceptional selectivity and sensitivity, rapid response, wide pH response range and robust anti-interference capabilities for monitoring Cu2+. This was achieved through a distinctive naked-eye colorimetric and fluorescent quenching behaviour. A good linearity within the range of 0-3 µM (R2 = 0.9914) was attained, and the limit of detection (LOD) for Cu2+ was 47.4 nM. Furthermore, the FMDH-Cu2+ ensemble responded to glyphosate with notable selectivity and sensitivity. A good linear correlation (R2 = 0.9926) was observed at the lower concentration range (2.4-7.8 µM) and achieving a detection limit as low as 29.9 nM. The response time of FMDH with Cu2+ and glyphosate were less than 20 s, and the pH range of 7-11 that was suitable for practical application under physiological pH conditions. MTT assays confirmed that FMDH offers good permeability and low toxicity, facilitating successful application in imaging analysis of Cu2+ and glyphosate in living cells and zebrafish. In addition, FMDH was employed in the detection of these analytes in real water samples. Cost-effective, highly sensitive and easily prepared FMDH-impregnated test strips were developed for the efficient visual detection of Cu2+ and glyphosate under 365 nm UV light. Increasing concentrations of Cu2+ and glyphosate resulted in notable colour changes under 365 nm UV light, enabling visual semi-quantitative analysis via a smartphone colour-analysis App.

Mapping the molecular motions of 5-HT3 serotonin-gated channel by voltage-clamp fluorometry.

The serotonin-gated ion channel (5-HT3R) mediates excitatory neuronal communication in the gut and the brain. It is the target for setrons, a class of competitive antagonists widely used as antiemetics, and is involved in several neurological diseases. Cryo-electron microscopy (cryo-EM) of the 5-HT3R in complex with serotonin or setrons revealed that the protein has access to a wide conformational landscape. However, assigning known high-resolution structures to actual states contributing to the physiological response remains a challenge. In the present study, we used voltage-clamp fluorometry (VCF) to measure simultaneously, for 5-HT3R expressed at a cell membrane, conformational changes by fluorescence and channel opening by electrophysiology. Four positions identified by mutational screening report motions around and outside the serotonin-binding site through incorporation of cysteine-tethered rhodamine dyes with or without a nearby quenching tryptophan. VCF recordings show that the 5-HT3R has access to four families of conformations endowed with distinct fluorescence signatures: 'resting-like' without ligand, 'inhibited-like' with setrons, 'pre-active-like' with partial agonists, and 'active-like' (open channel) with partial and strong agonists. Data are remarkably consistent with cryo-EM structures, the fluorescence partners matching respectively apo, setron-bound, 5-HT bound-closed, and 5-HT-bound-open conformations. Data show that strong agonists promote a concerted motion of all fluorescently labeled sensors during activation, while partial agonists, especially when loss-of-function mutations are engineered, stabilize both active and pre-active conformations. In conclusion, VCF, though the monitoring of electrophysiologically silent conformational changes, illuminates allosteric mechanisms contributing to signal transduction and their differential regulation by important classes of physiological and clinical effectors.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe.

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

Catalysis-Based Fluorometric Method for Semiquantifying Trace Palladium in Sulfur-Containing Compounds and Ibuprofen.

Trace palladium in synthetic materials can be rapidly and inexpensively semiquantified by a catalysis-based fluorometric method that converts resorufin allyl ether to resorufin. However, whether sulfur compounds would interfere with this method has not been systematically studied. Herein, we show that although thiourea in solution interferes with quantification, sulfide, thiol, and thiocarbamate do not. The fluorometric method can also detect palladium bound to sulfur-based scavenger resin and outperform inductively coupled plasma mass spectrometry for detecting trace palladium in ibuprofen.

DSFworld: A flexible and precise tool to analyze differential scanning fluorimetry data.

Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature-dependent signal decay. When tested these models against DSFbase, an open-source database of 6235 raw, real-life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open-source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.

Cellulose-based adsorbent using in mercury detection and removal from water via an efficient grafting strategy of fluorometric sensors by click reaction.

Mercury pollution in waters attracts lots of attention due to its serious toxicity and high bioenrichment and many efforts have been devoted in the development of adsorbents for mercury detection and removal. Herein, a cellulose-based adsorbent Cell-TriA-HQ is functionalized with quinoline fluorophore by covalent immobilization through "Click reaction" with high yield. In addition to the admirable adsorptive performance, the prepared adsorbent exhibits excellent selectivity and sensitivity towards Hg (II) in water that the detection limit for Hg (II) is determined to be as low as 1.92 × 10-7 M. The sensitive fluorescence enhancement response is considered to be resulted from the inhibition of photo-induced electron transfer between triazole and quinoline groups and the reinforcement of structural rigidity. The easy manipulation along with excellent performance of adsorption capacity, detective ability and reusability for the multifunctional adsorbent makes it potential in mercury monitoring and removal from aqueous solutions in the field of water treatment.

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates. The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Simultaneous quantitative respirometry and fluorometric assays in dissected hippocampal tissue from mice.

Respirometry is a technique for studying mitochondrial function that has proven compatibility with ≥0.5 mg of brain tissue. Here, we present a protocol for assessing oxygen consumption and H2O2 production rates in hippocampal tissue using the Oroboros O2k system. We describe steps for brain harvesting, tissue preparation, hippocampal microdissection, and respirometry assays. This approach has been valuable to study the metabolism of dentate granule cells of the hippocampus and could be applicable to other brain subregions. For complete details on the use and execution of this protocol, please refer to Rose et al.1.

A dual-mode fluorometric/colorimetric sensor for sulfadimethoxine detection based on Prussian blue nanoparticles and carbon dots.

A dual-mode (colorimetric/fluorescence) nanoenzyme-linked immunosorbent assay (NLISA) was developed based on Au-Cu nanocubes generating Prussian blue nanoparticles (PBNPs). It is expected that this method can be used to detect the residues of sulfonamides in the field, and solve the problem of long analysis time and high cost of the traditional method. Sulfadimethoxine (SDM) was selected as the proof-of-concept target analyte. The Au-Cu nanocubes were linked to the aptamer by amide interaction, and the Au-Cu nanocubes, SDM and antibody were immobilized on a 96-well plate using the sandwich method. The assay generates PBNPs by oxidising the Cu shells on the Au-Cu nanocubes in the presence of hydrochloric acid, Fe3+ and K3[Fe (CN)6]. In this process, the copper shell undergoes oxidation to Cu2+ and subsequently Cu2 + further quenches the fluorescence of the carbon point. PBNPs exhibit peroxidase-like activity, oxidising 3,3',5,5'-tetramethylbenzidine (TMB) to OX-TMB in the presence of H2O2, which alters the colorimetric signal. The dual-mode signals are directly proportional to the sulfadimethoxine concentration within the range 10- 3~10- 7 mg/mL. The limit of detection (LOD) of the assay is 0.023 ng/mL and 0.071 ng/mL for the fluorescent signal and the colorimetric signal, respectively. Moreover, the assay was successfully applied to determine sulfadimethoxine in silver carp, shrimp, and lamb samples with satisfactory results.