For Fluorescence Research Articles

Imaging and Simulation of Ruthenium Derivative Coating Microbeads at the Opaque Electrode with Electrogenerated Chemiluminescence.

Electrogenerated chemiluminescence (ECL) imaging is gaining increasing attention in various fields because of its high sensitivity, low background, and good temporal and spatial resolution. However, ECL imaging of microsized objects at the opaque electrode via top-view configuration is challenged with the reactants' diffusion and light propagation. Here, we imaged and numerically simulated ruthenium derivative coating polystyrene microbeads (Ru1-PS@MB) at the glassy carbon electrode (GCE) via top-view configuration by ECL imaging. The ruthenium derivative (bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate), Ru1), a typical ECL reagent, was covalently linked onto the surface of aminated PS@MBs via the amide reaction. "Strong emission in edge and weak emission in center" phenomena for fluorescence (FL) and ECL emissions were obtained from Ru1-PS@MB on GCE. Z-Stack imaging of the microsized Ru1-PS@MB luminescence was performed on GCE in the presence of tri-n-propylamine (TPA). It is found that the clear luminescence range of Ru1-PS@MB perpendicular to the electrode surface in ECL image is slightly smaller than that in the FL image. The bigger was the diameter of the microbeads (from 5 to 18 μm), the larger was the ECL luminescence range of Ru1-PS@MB perpendicular to the electrode surface (from 5 to 7 μm). Our findings, which are also supported by numerical simulation, provide insights into the ECL imaging of microsized objects at the electrode surface, which will raise promising ECL applications in bioassays and cell imaging at the microscale level.

Evaluation of a photodynamic therapy agent using a canine prostate cancer model.

Male dogs can develop spontaneous prostate cancer, which is similar physiologically to human disease. Recently, Tweedle and coworkers have developed an orthotopic canine prostate model allowing implanted tumors and therapeutic agents to be tested in a more translational large animal model. We used the canine model to evaluate prostate-specific membrane antigen (PSMA)-targeted gold nanoparticles as a theranostic approach for fluorescence (FL) imaging and photodynamic therapy (PDT) of early stage prostate cancer. Dogs (four in total) were immunosuppressed with a cyclosporine-based immunosuppressant regimen and their prostate glands were injected with Ace-1-hPSMA cells using transabdominal ultrasound (US) guidance. Intraprostatic tumors grew in 4-5 weeks and were monitored by ultrasound (US). When tumors reached an appropriate size, dogs were injected intravenously (iv) with PSMA-targeted nano agents (AuNPs-Pc158) and underwent surgery 24 h later to expose the prostate tumors for FL imaging and PDT. Ex vivo FL imaging and histopathological studies were performed to confirm PDT efficacy. All dogs had tumor growth in the prostate gland as revealed by US. Twenty-four hours after injection of PSMA-targeted nano agents (AuNPs-Pc158), the tumors were imaged using a Curadel FL imaging device. While normal prostate tissue had minimal fluorescent signal, the prostate tumors had significantly increased FL. PDT wasactivated by irradiating specific fluorescent tumor areas with laser light (672 nm). PDT bleached the FL signal, while fluorescent signals from the other unexposed tumor tissues were unaffected. Histological analysis of tumors and adjacent prostate revealed that PDT damaged the irradiated areas to a depth of 1-2 mms with the presence of necrosis, hemorrhage, secondary inflammation, and occasional focal thrombosis. The nonirradiated areas showed no visible damages by PDT. We have successfully established a PSMA-expressing canine orthotopic prostate tumor model and used the model to evaluate the PSMA-targeted nano agents (AuNPs-Pc158) in the application of FL imaging and PDT. It was demonstrated that the nano agents allowed visualization of the cancer cells and enabled their destruction when they were irradiated with a specific wavelength of light.

Enzymatic Substrates and Fluorescence Imaging of Influenza Virus Sialidase.

Immunostaining with an antiviral antibody is usually performed to visualize virus-infected cells. In contrast, this study established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzymes "neuraminidase" or "hemagglutinin-neuraminidase," which show sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and applies to virus isolation and detection of an influenza virus resistant to sialidase inhibitors anti-influenza drugs.

Synthesis of Magnetic Ions-Doped QDs Synthesized Via a Facial Aqueous Solution Method for Optical/MR Dual-Modality Imaging Applications.

This research reports the preparation and examination of Cadmium Telluride (CdTe) Quantum Dots and doping CdTe QDs with Europium (Eu), Gadolinium (Gd), and Manganese (Mn) prepared in aqueous solution using TGA as a capping agent. Magnetic QDs (MQDs) are important agents for fluorescence (FL) /magnetic resonance (MR) dual-modal imaging due to their excellent optical and magnetic properties. Herein, the chemical bonds, structural, fluorescence, and magnetized properties of CdTe QDs and effect of Mn, Eu, and Gd ions doping on their properties were examined by X-ray powder diffraction (XRD), high-resolution transmission electron microscopy (HRTM), Energy-dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR), photoluminescence spectroscopy (PL), Ultraviolet-visible spectroscopy (UV-Vis), and vibrating sample magnetometer measurements (VSM). Almost similar X-Ray patterns with the absence/presence of ions for all samples with cubic crystal structures were obtained which indicated that the introduction of ions into CdTe QDs could not alter the cubic primary structure of CdTe. Monodisperse size distributed with seemingly-spherical shapes, and also, the estimated mean diameters about 3 and less than 3nm of QDs were obtained. The effect of X ions injection on the fluorescence and UV-Vis properties of the QDs were also investigated. Optical studies showed the decreases in bandgap as the heating time increases during synthesiswhile undergoing red-shift. The CdTe nanocrystals with high PL quantum yields were achieved in more than 6h of heating. Also, investigations have shown the quenching of fluorescence by the existence of ions in the CdTe QDs. Then, all the ions doped QDs exhibited ferromagnetic behavior at room temperature by a vibrating sample magnetometer which confirmed the success of the presentation of ions into CdTe cubic crystal structure. They can have been employed as a suitable contrast agent successfully for biological applications such as FL/MR dual-modal imaging.

Light‐Triggered Transformable Ferrous Ion Delivery System for Photothermal Primed Chemodynamic Therapy

Chemodynamic therapy (CDT) involves the catalytic generation of highly toxic hydroxyl radicals (. OH) from hydrogen peroxide (H2 O2 ) through metal-ion-mediated Fenton or Fenton-like reactions. Fe2+ is a classical catalyst ion, however, it suffers easy oxidation and systemic side-effects. Therefore, the development of a controllable Fe2+ delivery system is a challenge to maintain its valence state, reduce toxicity, and improve therapeutic efficacy. Reported here is a near-infrared (NIR) light-triggered Fe2+ delivery agent (LET-6) for fluorescence (FL) and photoacoustic (PA) dual-modality imaging guided, photothermal primed CDT. Thermal expansion caused by 808 nm laser irradiation triggers the transformation of LET-6 to expose Fe2+ from its hydrophobic layer, which primes the catalytic breakdown of endogenous H2 O2 within the tumor microenvironment, thus generating . OH for enhanced CDT. LET-6 shows remarkable therapeutic effects, both in vitro and in vivo, achieving 100 % tumor elimination after just one treatment. This high-performance Fe2+ delivery system provides a sound basis for future synergistic metal-ion-mediated cancer therapy.

Light‐Triggered Transformable Ferrous Ion Delivery System for Photothermal Primed Chemodynamic Therapy

AbstractChemodynamic therapy (CDT) involves the catalytic generation of highly toxic hydroxyl radicals (.OH) from hydrogen peroxide (H2O2) through metal‐ion‐mediated Fenton or Fenton‐like reactions. Fe2+ is a classical catalyst ion, however, it suffers easy oxidation and systemic side‐effects. Therefore, the development of a controllable Fe2+ delivery system is a challenge to maintain its valence state, reduce toxicity, and improve therapeutic efficacy. Reported here is a near‐infrared (NIR) light‐triggered Fe2+ delivery agent (LET‐6) for fluorescence (FL) and photoacoustic (PA) dual‐modality imaging guided, photothermal primed CDT. Thermal expansion caused by 808 nm laser irradiation triggers the transformation of LET‐6 to expose Fe2+ from its hydrophobic layer, which primes the catalytic breakdown of endogenous H2O2 within the tumor microenvironment, thus generating .OH for enhanced CDT. LET‐6 shows remarkable therapeutic effects, both in vitro and in vivo, achieving 100 % tumor elimination after just one treatment. This high‐performance Fe2+ delivery system provides a sound basis for future synergistic metal‐ion‐mediated cancer therapy.

Live Imaging of Virus-Infected Cells by Using a Sialidase Fluorogenic Probe.

Visualization of virus-infected cells is usually performed by immunostaining with an antiviral antibody. On the other hand, we established an easy method for fluorescence (FL) imaging of cells infected with influenza A and B viruses and some paramyxoviruses without the need for cell fixation and an antiviral antibody. These viruses and the cells they have infected express the viral surface enzyme "neuraminidase" or "hemagglutinin-neuraminidase" that shows sialidase activity. Sialidase activity is fluorescently visualized by using a sialidase fluorogenic probe developed in our previous study. The probe enables histochemical FL imaging of the virus-infected cells and is applicable to virus isolation and detection of an influenza virus resistant to antiinfluenza drugs of sialidase inhibitors.

Gadolinium-doped carbon dots with high-performance in dual-modal molecular imaging.

Carbon dots (CDs), possessing unexpected advantages of photostability, biocompatibility and low toxicity, are regarded as novel nanomaterials in fluorescence (FL) imaging. Doping Gd element in CDs makes them have the ability to be used for magnetic resonance (MR) and FL imaging simultaneously. However, CDs reported before exhibit obvious defects like low photoluminescence (PL) quantum yield (QY) or biotoxicity. In this work, we use gadolinium meglumine, a material with relatively low biotoxicity, along with citric acid and diethylenetriamine to synthesize Gd-doped CDs (Gd-CDs) by a one-step hydrothermal method. The prepared Gd-CDs exhibit excitation-independent emission with a PL QY of 78.05% and a longitudinal relaxivity of 7.37 mM-1 S-1, which endows the composite with high-performance in MR/FL imaging. Meanwhile, the FL intensity of Gd-CDs remains stable in the presence of multiple amino acids, which indicates that the FL imaging effect should not be impacted significantly in microenvironments in vivo. In addition to the inconspicuous cytotoxicity, Gd-CDs could be used efficiently for dual-modal molecular imaging to detect diseases such as tumors in the early stages.

Tumor Microenvironment Stimuli‐Responsive Fluorescence Imaging and Synergistic Cancer Therapy by Carbon‐Dot–Cu2+ Nanoassemblies

AbstractA method is developed to fabricate tumor microenvironment (TME) stimuli‐responsive nanoplatform for fluorescence (FL) imaging and synergistic cancer therapy via assembling photosensitizer (chlorine e6, Ce6) modified carbon dots (CDs‐Ce6) and Cu2+. The as‐obtained nanoassemblies (named Cu/CC nanoparticles, NPs) exhibit quenched FL and photosensitization due to the aggregation of CDs‐Ce6. Their FL imaging and photodynamic therapy (PDT) functions are recovered efficiently once they entering tumor sites by the stimulation of TME. Introducing of Cu2+ not only provides extra chemodynamic therapy (CDT) function through reaction with hydrogen peroxide (H2O2), but also depletes GSH in tumors by a redox reaction, thus amplifying the intracellular oxidative stress and enhancing the efficacy of reactive oxygen species (ROS) based therapy. Cu/CC NPs can act as a FL imaging guided trimodal synergistic cancer treatment agent by photothermal therapy (PTT), PDT, and thermally amplified CDT.

Tumor Microenvironment Stimuli-Responsive Fluorescence Imaging and Synergistic Cancer Therapy by Carbon-Dot-Cu2+ Nanoassemblies.

A method is developed to fabricate tumor microenvironment (TME) stimuli-responsive nanoplatform for fluorescence (FL) imaging and synergistic cancer therapy via assembling photosensitizer (chlorine e6, Ce6) modified carbon dots (CDs-Ce6) and Cu2+ . The as-obtained nanoassemblies (named Cu/CC nanoparticles, NPs) exhibit quenched FL and photosensitization due to the aggregation of CDs-Ce6. Their FL imaging and photodynamic therapy (PDT) functions are recovered efficiently once they entering tumor sites by the stimulation of TME. Introducing of Cu2+ not only provides extra chemodynamic therapy (CDT) function through reaction with hydrogen peroxide (H2 O2 ), but also depletes GSH in tumors by a redox reaction, thus amplifying the intracellular oxidative stress and enhancing the efficacy of reactive oxygen species (ROS) based therapy. Cu/CC NPs can act as a FL imaging guided trimodal synergistic cancer treatment agent by photothermal therapy (PTT), PDT, and thermally amplified CDT.

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators.

SummaryFunctional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs.

Polymer-based gadolinium oxide nanocomposites for FL/MR/PA imaging guided and photothermal/photodynamic combined anti-tumor therapy

Recently, ultrasmall gadolinium oxide (Gd2O3) nanoparticles with high longitudinal relaxation rate have received enormous attention. However, it can't be concentrated in tumor site through intravenous administration due to its ultrasmall size. In this project, we coated ultrasmall Gd2O3 nanoparticles with a near-infrared (NIR) light-absorbing polymer polypyrrole (PPy), modifying with hyaluronic acid (HA) and loaded aluminum phthalocyanine (AlPc), the Gd2O3@PPy/AlPc-HA nanoparticles could be used for fluorescence (FL)/magnetic resonance (MR)/photoacoustic (PA) imaging guided as well as remotely controlled PTT/PDT combined anti-tumor therapy. Polymerized PPy with high photothermal conversion efficiency was introduced to assemble the ultrasmall Gd2O3 nanoparticles which have high longitudinal relaxation rate and signal-to-noise ratio, thus obtaining Gd2O3@PPy nanoparticles which possess a larger particle size and can be more suitable for tumor targeting based on the EPR effect. HA and AlPc were adsorbed on PPy for HA-mediated tumor targeting and photodynamic therapy respectively. The in vivo triple-modal imaging revealed that Gd2O3@PPy/AlPc-HA nanoparticles possess enhanced tumor uptake effect after intravenous injection. More importantly, the nanoparticles exhibited an obvious photothermal effect, which can trigger the release and de-quench of AlPc. The anti-tumor efficiency further corroborated that the combined therapy achieved an excellent tumor inhibition therapeutic effect which was much better than any other mono-therapy. Consequently, our work encouraged further exploration of polymer-based multifunctional theranostic nanoparticles for cancer combination therapy under remote near-infrared (NIR) light controls.

Yellow-Emissive Carbon Dot-Based Optical Sensing Platforms: Cell Imaging and Analytical Applications for Biocatalytic Reactions.

Carbon dots (CDs) have attracted increasing interest in bioimaging and sensing recently. Herein, we present a simple synthetic strategy to prepare yellow-emissive CDs (λem = 535 nm) by one-pot hydrothermal treatment of p-phenylenediamine and aspartic acid. The as-prepared CDs possess outstanding optical features, excellent biocompatibility, and low cytotoxicity, especially for fluorescence (FL) cellular imaging. Interestingly, by combining the quenching and recognition ability of silver nanoparticles (AgNPs) with the optical capacity of CDs, a label-free strategy for specifically monitoring H2O2-generated biocatalytic processes was proposed, such as glucose oxidase-induced conversion of glucose, cholesterol oxidase-catalyzed oxidization of cholesterol, and bienzyme of acetylcholinesterase and choline oxidase-mediated reaction of acetylcholine. In this process, AgNPs act as a "nanoquencher" to decrease the FL intensity of CDs via surface plasmon-enhanced energy-transfer mechanism. The enzymatic oxidation product (H2O2) subsequently etches the AgNPs to silver ions, thus recovering the FL of CDs, which enabled this proposed nanosensor to sensitively detect H2O2-generated biocatalytic processes. The above results pave the way to implement CDs as FL labels for biosensors and biological imaging.

Indocyanine Green-holo-Transferrin Nanoassemblies for Tumor-Targeted Dual-Modal Imaging and Photothermal Therapy of Glioma.

Active-targeted cancer imaging and therapy of glioma has attracted much attention in theranostic nanomedicine. As a promising tumor-targeting ligand, holo-transferrin (holo-Tf) has been applied for enhancing delivery of nanotheranostics. However, holo-Tf-based nanoassemblies for active targeting mediated multimodal imaging and therapeutics have not been previously reported. Here, we develop a one-step method for the preparation of holo-Tf-indocyanine green (holo-Tf-ICG) nanoassemblies for fluorescence (FL) and photoacoustic (PA) dual-modal imaging and photothermal therapy (PTT) of glioma. The nanoassemblies are formed by hydrophobic interaction and hydrogen bonds between holo-Tf and ICG, which exhibit excellent active tumor-targeting and high biocompability. The brain tumor with highly expressed Tf receptor can be clearly observed with holo-Tf-ICG nanoassemblies base on FL and PA dual-modal imaging in subcutaneous and orthotopic glioma models. Under the near-infrared laser irradiation, the holo-Tf-ICG nanoassemblies accumulated in tumor regions can efficiently convert laser energy into hyperthermia for tumor ablation. The novel theranostic nanoplatform holds great promise for precision diagnosis and treatment of glioma.

Facile synthesis of CdTe@GdS fluorescent-magnetic nanoparticles for tumor-targeted dual-modal imaging

Multimodal imaging has made great contribution for diagnosis and therapy of disease since it can provide more effective and complementary information in comparison to any single imaging modality. The design and fabrication of fluorescent-magnetic nanoparticles for multimodal imaging has rapidly developed over the years. Herein, we demonstrate the facile synthesis of GdS coated CdTe nanoparticles (CdTe@GdS NPs) as multimodal agents for fluorescence (FL) and T1-weighted magnetic resonance (MR) imaging. These nanoparticles obtain both prominent fluorescent and paramagnetic properties by coating the GdS shell on the surface of CdTe core via a simple room-temperature route in aqueous solution directly. It is shown that the as-prepared CdTe@GdS NPs have high quantum yield (QY) value of 12% and outstanding longitudinal relaxation rate (r1) of 11.25mMs−1, which allow them to be employed as FL/MR dual-modal imaging contrast agents. They also exhibit small particle size of 5nm, excellent colloidal stability and low cellular toxicity for concentrations up to 750μgmL−1. In addition, with the conjugation of folic acid, the nanoparticles were successfully used for tumor-targeted FL/MR dual-modal imaging in vitro and in vivo.

Subnanomolar fluorescent-molecule sensing by guided resonances on nanoimprinted silicon-on-insulator substrates

We have produced nanoimprinted silicon-on-insulator (SOI) substrates with high precision and applied them for fluorescence (FL) enhancement of typical dye molecules of Rhodamine series. We experimentally found that the FL signals on the nanoimprinted SOI substrates are enhanced by more than 200 fold as compared with the signals on Si wafers. The FL enhancement mechanism was investigated using spectroscopic and theoretical approaches. The results indicated that guided resonances with large density of states significantly contribute to the FL enhancement. We also substantiated that the FL-enhancing substrates well serve as rapid-sensing platforms for a very dilute FL-molecule solution with subnanomolar concentration. The nanoimprinted SOI substrates were repeatedly reused without degradation.

Facile synthesis of functional gadolinium-doped CdTe quantum dots for tumor-targeted fluorescence and magnetic resonance dual-modality imaging.

Magnetic quantum dots (MQDs) are an important class of agents for fluorescence (FL)/magnetic resonance (MR) dual-modal imaging due to their excellent optical and magnetic properties. However, functional MQDs prepared by a simple room-temperature route as FL/MR dual-modal imaging probes are lacking. Herein, we report the fabrication of Gd-doped CdTe quantum dots (Gd:CdTe QDs) as an agent for FL/MR dual-modality imaging. The as-designed QDs with an ultrasmall particle size are synthesized by a facile one-pot aqueous synthesis approach at room temperature. They emit strong fluorescence at 640 nm with a quantum yield of 37% in water, and they have a high longitudinal relaxation rate (r1) value of 3.27 mM-1 s-1. With the further conjugation of folic acid, the Gd:CdTe QDs can successfully label live HepG2 cells for targeted cellular imaging and present no evidence of cellular toxicity up to the concentration of 0.5 mg mL-1. They have been employed as a suitable contrast agent successfully for tumor-targeted FL/MR dual-modal imaging in a mouse model.

Simultaneous determination of 5-hydroxyindoles and catechols from urine using polymer monolith microextraction coupled to high-performance liquid chromatography with fluorescence detection

To make analytes amenable for fluorescence (FL) detection, polymer monolith microextraction (PMME) coupled to high-performance liquid chromatography with FL detection was developed for the simultaneous determination of catechols and 5-hydroxyindoleamines (5-HIAs) from urine samples. In this method, a two-step pre-column derivatization method was employed to derivatize the analytes and a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was used as the extraction medium for PMME. The conditions for the derivatization and subsequent extraction of 5-HIAs and catechols derivatives were optimized. Using our optimum conditions, the detection limit of the target analytes were 0.11–21 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations less than 12% and a recovery of higher than 82%. In this study, we show how our proposed method can be used as a rapid sensitive technique for the determination of catechols and 5-HIAs from urine samples.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

30-day oral toxicity study of domoic acid in Cynomolgus monkeys: Lack of overt toxicity at doses approaching the acute toxic dose

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.