Light-emitting Diode-induced Fluorescence Detection Research Articles

A Miniaturized and Highly Sensitive "Windmill" Three-Channel Fluorescence Detector for Simultaneous Detection of Various Mycotoxins.

A novel "windmill" three-channel light-emitting diode induced fluorescence detector (LED-IF) was proposed to maximize the excitation efficiency and fluorescence collection efficiency. Compared with the typical collinear arrangement, the fluorescence intensity of the three channels was increased by 7.85, 3.88, and 2.94 times, respectively. The compact shaping optical path was designed to obtain higher excitation efficiency and a lower background stray light effect caused by high divergence angle high-power ultraviolet (UV)-LEDs simultaneously, which increased the sensitivity of three channels by 4.6 to 5.7 times. It was found that using a photodiode (PD) with a flat window and a larger photosensitive surface can collect the Lambertian emission fluorescence in the flow cell more efficiently, increasing the signal-to-noise ratio of each channel 1.3 to 1.8 times. The limits of detection (LODs, 3 times peak-peak noise) of aflatoxin B2 (AFB2), ochratoxin (OTA), and zearalenone (ZEN) were 0.33, 1.80, and 28.2 ng/L, respectively. Finally, six mycotoxins were analyzed simultaneously by the detector coupling with HPLC. The results showed that the sensitivity of the detector was at the best level to date, which was better than that of the top commercial fluorescence detectors (FLDs). The developed detector has the advantages of having small volume, low cost, and long lifetime and being robust, which has wide application and market prospects.

Capillary Zone Electrophoresis with Light-Emitting Diode-Induced Fluorescence Detection for the Analysis of Monoclonal Antibodies: Detector Optimization through Design of Experiments and Comparison to UV Detection

Capillary zone electrophoresis (CZE) is an important technique for the analysis of monoclonal antibodies (mAbs). A recently released light-emitting diode (LED)-induced fluorescence (LEDIF) detector equipped with a 275 nm LED for the detection of proteins through their native fluorescence was used in this study and compared to results obtained using the predominant detection mode, the measurement of the absorption of ultraviolet light (UV detection). This was accomplished using an established CZE method for the analysis of three mAbs: NISTmAb, matuzumab, and Intact Mass Check Standard (Waters). For this purpose, the detector’s settings were first optimized using a design of experiments approach. Three factors, rise time, photomultiplier high voltage supply, and acquisition frequency, were optimized by means of a D-optimal design. The optimal settings were then used for the investigation of signal-to-noise ratios (S/Ns), linearity, and precision. LEDIF detection offered a similar separation quality, up to 12 times higher S/Ns, and lower limits of detection compared to UV detection. Repeatability was excellent, with relative standard deviations (RSDs) of approximately 1% for percentage areas. For intermediate precision, RSDs of <2% (n = 3 × 10) were typically achieved. Overall, LEDIF detection was found to be an excellent and easily optimizable alternative to UV detection.

Analysis of Sugars in Honey Samples by Capillary Zone Electrophoresis Using Fluorescence Detection

The applicability of capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF) for the separation of sugars in honey samples was studied. An amount of 25 mM ammonium acetate (pH 4.5) with 0.3% polyethylene oxide (PEO) was found to be optimal for the efficient separation of carbohydrates. 8-aminopyrene-1,3,6-trisulfonic acid (APTS) was used for the labeling of the carbohydrate standards and honey sugars for fluorescence detection. The optimized method was applied in the quantitative analysis of fructose and glucose by direct injection of honey samples. Apart from the labeling reaction, no other sample preparation was performed. The mean values of the fructose/glucose ratio for phacelia honey, acacia honey and honeydew honey were 0.86, 1.61 and 1.42, respectively. The proposed method provides high separation efficiency and sensitive detection within a short analysis time. Apart from the labeling reaction, it enables the injection of honeys without sample pretreatment. This is the first time that fluorescence detection has been applied for the CE analysis of sugars in honeys.

Taylor dispersion analysis discloses the impairment of Aβ peptide aggregation by the presence of a fluorescent tag.

The use of fluorescently tagged amyloid peptides, implicated in Alzheimer's disease, to study their aggregation at low concentrations is a common method; however, the fluorescent tag should not introduce a bias in the aggregation process. In this work, native amyloid peptides Aβ(1-40) and Aβ(1-42) and fluorescein-5-isothiocyanate (FITC), tagged ones, were studied using Taylor dispersion analysis coupled with a simultaneous UV and light-emitting diode-induced fluorescence detection, to unravel the effect of FITC on the aggregation process. For that, a total concentration of 100µM of peptides consisting of a mixture of native and tagged ones (up to 10% in moles) was applied. Results demonstrated that FITC had a strong inhibition effect upon the aggregation behaviour of Aβ(1-42), whereas for Aβ(1-40), only a retardation in kinetics was observed. It was also shown that when mixed solutions of Aβ(1-40) and Aβ(1-42) are used, the Aβ(1-42) alloform was the leading peptide in the aggregation process, and when the latter was tagged, the aggregation kinetics decreased but the lifetime of potentially toxic oligomers was drastically increased. These results confirmed that the hydrophilicity of the N-terminus part of the peptide plays a major role in the aggregation process.

Assessment of bioaccumulation of glyphosate and aminomethylphosphonic acid in marine mussels using capillary electrophoresis with light‐emitting diode‐induced fluorescence detection

Glyphosate or N-(phosphonomethyl)glycine, widely used as herbicide in agriculture to control weeds and to facilitate harvesting, has been included in Group 2A pollutants (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC). In intensive agricultural areas, runoff and soil leaching are likely to drive glyphosate to surface waters, where the compound is often detected together with its main microbial metabolite, aminomethylphosphonic acid (AMPA).In the present study a method based on capillary electrophoresis coupled with light-emitting diode-induced fluorescence detection has been developed and validated for the determination of the two compounds in whole soft mass of marine mussels (Mytilus galloprovincialis). The method is based on the acidic hydrolysis of lyophilized tissue using 6 M HCl (oven at 110 °C for 22 h) to release the target analytes; their subsequent derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole, was found to be suitable for the sensitive fluorescence detection. To achieve optimum separation of the analytes from the matrix and degradation reagent interferences, the background electrolyte constituted by borate buffer (pH 9.2, 30 mM) was supplemented with 10 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin. The method was validated for linearity, precision, accuracy, robustness and sensitivity showing LOQ of 0.2 and 1.0 µg/g in fresh tissues, for AMPA and glyphosate, respectively; the recovery values ranged within 88.5 – 94.6% for glyphosate and 70.4 – 76.6% for AMPA. Experimental samples of Mediterranean mussels M. galloprovincialis treated with 100 µg/L or 500 µg/L of both glyphosate and AMPA, showed a dose dependent bioaccumulation of the compounds reaching maximum level of 77.0 µg/g and 11.3 µg/g of AMPA and glyphosate, respectively. The study demonstrates for the first time M. galloprovincialis as potential sentinel organisms for the environmental occurrence of these small amphoteric pollutants.

Determination of three antiepileptic drugs in pharmaceutical formulations using microfluidic chips coupled with light-emitting diode induced fluorescence detection.

In this study, a facile, sensitive, and precise lab-on-a-chip electrophoretic method coupled with light-emitting diode induced fluorescence (LED-IF) detection was developed to assay three antiepileptic drugs, namely, vigabatrin, pregabalin, and gabapentin, in pharmaceutical formulations. The analytes were derivatised offline for the first time with fluorescine-5-isothiocyanate (FITC) to yield highly fluorescent derivatives with λex/em of 490/520nm. The FITC-labelled analytes were injected, separated, and quantitated by a microfluidic electrophoresis device using fluorescence detection. The labelled analytes were monitored using a blue LED-IF system. The separation conditions were significantly optimised adding specific concentrations of heptakis-(2,6-di-O-methyl)-β-cyclodextrin (HDM-β-CD) and methylcellulose to the buffer solution (40mM borate buffer). HDM-β-CD acted as a selective host for the studied antiepileptic drugs, rendering a high separation efficiency. Methylcellulose was used as an efficient dynamic coating polymer to prevent the labelled drugs from being adsorbed on the inner surfaces of the poly (methylmethacrylate) microchannels. A laboratory-prepared ternary mixture of the three antiepileptic drugs was separated within 100s with acceptable resolution between all the three analytes (Rs>3) and a high number of theoretical plates (N) for each analyte (N≈106 plates/m). The sensitivity of the method was enhanced approximately 80-fold by stacking to yield a detection limit below 0.6ngmL-1 in the concentration range of 2.0-200.0ngmL-1. The method was successfully validated for analysing the studied drugs in their pharmaceutical formulations.

Quantifying Sample Collection and Processing Impacts on Fiber-Based Tear Fluid Chemical Analysis.

PurposeNoninvasive analyses of tear fluid from humans and animal models in clinical and research settings most commonly use absorbent material for collection and processing. Still, the impact of these analytical techniques on tear chemical analyses remains largely unknown. The purpose of this study was to quantify the impacts of phenol red thread fiber-based tear sample collection and processing on the primary amine content.MethodsHuman tears were collected by placing the folded end of phenol red thread on the palpebral conjunctiva of the right eye for 20 seconds. The wetted thread was then processed using elution or extraction, and capillary electrophoresis with light-emitting diode-induced fluorescence detection was used for analysis and quantitation.ResultsDistinct processing methods impacted tear analysis differently. Primary amines adsorbed onto the thread partitioned in a chromatographic manner and thus any single portion of the wetted thread might not be representative of the whole sample. Quantitative assessment of five small molecule standards after on-thread processing showed significant overestimation of the actual concentration, with increased accuracy for larger volume samples. Yet collection of larger tear volumes introduced error in volume determination owing to evaporation and reduced small molecule separation resolution.ConclusionsThese results indicated that absorption-based tear fluid collection and processing significantly alter chemical content analysis, suggesting that the impacts of methods used should be regularly evaluated to standardize results drawn from different studies.Translational RelevanceThis study identifies potential inconsistencies and inaccuracies in tear analyses that are widespread across the published literature and clinical care.

Enhanced light-emitting diode induced fluorescence detection system with capillary electrophoresis

An enhanced fluorescence detection system of capillary electrophoresis (CE) was equipped with a concave silver mirror, by which the detection sensitivity of light-emitting diode induced fluorescence (LEDIF) can be increased greatly. The silver concave mirror and the cathode window in photomultiplier tube (PMT) were accurately set face to face at the same axis. When the two labeled tumor markers exactly moved to the center of detection window, the emission from analytes are excitated by LED source. Currently, the analytes may be regarded as a luminescent source point. When the source point exactly moves to the focus of the concave mirror, the emission of the labeled sample was collected effectively, enhanced by convergence and reflected by the concave mirror. Then it was sensitively detected by the PMT. The optical mechanism of enhancing detection sensitivity was explored. A simple comparative test on sensitivity was carried out, which aimed to compare sensitivity of the new detection system with concave mirror to that without concave mirror but the other conditions were kept the same. Two tumor markers labeled with FITC were selected for the test, using the simple LEDIF detect system. The results (LOD, 150 nM for L-Leu and L-Val) showed that the detection sensitivity matched with concave mirror reached more 16 times than the detection method without concave mirror.

Thread-based assay for quantitative small molecule analysis of mice tear fluid by capillary electrophoresis

Tear fluid plays blood-like roles in the cornea, and changes in its chemical composition may be reflective of ocular surface disease pathogenesis. Studies of mice tears are limited by the small volume available for collection and difficulty in obtaining representative samples. Here, we establish a non-invasive assay for small volume analysis of small molecules in mice tears that requires no pre-treatment of mice. To the best of our knowledge, this is the first small molecule analysis of mice tears. Nanoliters of mice tears (70 ± 25nL) was collected via a single insertion of phenol red thread in the corner of the eye without anesthesia to prevent any tear production alteration. The processing and elution of tear samples were optimized for minimal sample handling and dilution while maintaining high separation resolution. A capillary electrophoresis separation with light-emitting diode-induced fluorescence detection was developed for the analysis of primary amine-containing small molecules. The levels of arginine, alanine, aspartate, and glutamate after elution were in the micromolar range as seen in human tears. However, taurine and histamine levels were decreased and increased, respectively, compared to human tears, which may be indicative of restraint-induced emotional stress. No significant differences were seen for any of the small molecules between 20-week-old ND4 Swiss Webster females and 12-week-old CD-1 males (N = 3). The developed assay represents a means to assess the chemical composition of tear fluid in mouse models of human disease, which could significantly improve our understanding of ocular surface diseases. Graphical abstract ᅟ.

Study of metabolic activity of human embryos focused on amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Assisted reproduction is a quickly developing field of reproductive medicine whose importance is growing every year due to the increasing number of patients suffering from infertility. As a result, there is a need for the continuous development and/or improvement of assisted reproductive technologies. This paper presents a new method for the in vitro measurement of the amino acid turnover of developing embryos based on capillary electrophoresis with light-emitting diode-induced fluorescence detection. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde/NaCN, and the resulting fluorescent derivatives were baseline resolved within 25min in a background electrolyte comprised of 50mM sodium tetraborate, 73mM sodium dodecyl sulphate, 5mM sodium deoxycholate and 2.5mM (2-hydroxypropyl)-β-cyclodextrin (pH≈9.3). The migration time and the peak area repeatability (n=10) were below 0.5 and 4.3%, respectively. The limits of detection ranged from 12.6nM (histidine) to 39.3nM (taurine). The developed method, which only requires 2μL of raw sample, was successfully applied for determining the metabolic activity of human embryos exposed to different environmental stress conditions.

Sensitive determination of l-hydroxyproline in dairy products by capillary electrophoresis with in-capillary optical fiber light-emitting diode-induced fluorescence detection

A sensitive capillary electrophoretic method with in-capillary optical fiber light-emitting diode-induced fluorescence detection (ICOF-LED-IFD-CE) was developed for determination of l-Hyp and further for identification of hydrolyzed leather proteins in dairy products.

Sensitive determination of sulfonamides in environmental water by capillary electrophoresis coupled with both silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector.

A new detector, silvering detection window and in-capillary optical fiber light-emitting diode-induced fluorescence detector (SDW-ICOF-LED-IFD), is introduced for capillary electrophoresis (CE). The strategy of the work was that half surface of the detection window was coated with silver mirror, which could reflect the undetected fluorescence to the photomultiplier tube to be detected, consequently enhancing the detection sensitivity. Sulfonamides (SAs) are important antibiotics that achieved great applications in many fields. However, they pose a serious threat on the environment and human health when they enter into the environment. The SDW-ICOF-LED-IFD-CE system was used to determine fluorescein isothiocyanate (FITC)-labeled sulfadoxine (SDM), sulfaguanidine (SGD) and sulfamonomethoxine sodium (SMM-Na) in environmental water. The detection results obtained by the SDW-ICOF-LED-IFD-CE system were compared to those acquired by the CE with in-capillary optical fiber light-emitting diode-induced fluorescence detection (ICOF-LED-IFD-CE). The limits of detection (LODs) of SDW-ICOF-LED-IFD-CE and ICOF-LED-IFD-CE were 1.0-2.0 nM and 2.5-7.7 nM (S/N = 3), respectively. The intraday (n = 6) and interday (n = 6) precision of migration time and corresponding peak area for both types of CE were all less than 0.86% and 3.68%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 92.5-102.9%. The results indicated that the sensitivity of the SDW-ICOF-LED-IFD-CE system was improved, and that its reproducibility and accuracy were satisfactory. It was successfully applied to analyze SAs in environmental water.

Analysis of difference of serum proteins between patients with cirrhosis and normal people by capillary electrophoresis with light-emitting diode induced fluorescence detection

An experimental method of protein separation by capillary electrophoresis coupled with light-emitting diode induced fluorescence detection (CE-LED-IF) was established. The fluorescein isothiocyanate (FITC) was used as fluorescent dye in this method. An optical fiber was inserted into the separation capillary and was suited right at the detection window which was used to transmit the excitation light of LED. The efficiency of the light of LED and the detection limits of FITC was improved. The difference of serum proteins between the patients with cirrhosis and normal people was detected by this method. A series of effects on CE were studied, such as the wavelength of the light source and filter, the concentration and pH of the running buffer and the separation voltage. Then the optimized method was established for the determination of the serum proteins. The repeatability of CE-LED-IF was tested and the RSDs of migration time and peak area were 1. 14% and 3. 23% , respectively. The results showed that there were two specific peaks in cirrhosis serum. The differences between the patients with cirrhosis and normal people having statistical significance indicated that these proteins might be the potential biomarkers for cirrhosis diagnosis. The developed CE-LED-IF method proved suitable for the separation of serum proteins with good repeatability and supportable for the clinical diagnosis of cirrhosis.

LED induced fluorescence detector integrated in microfluidic cell chip

A light-emitting diode (LED) induced fluorescence detector for the microfluidic cell chip has been proposed in this contribution. Compared with the other competitor, the optical path does not require re-calibration after the microfluidic chip has been reloaded and relocated. Last but not the least, the system has a compact size and is more energy efficient. The model selection of the excitation light module, filter sets, aperture and photoelectric detector were analysed. The angle between the exciting light path and fluorescence optical path was optimised to be 135 degrees by experiment. The optimal value of photomultiplier (PMT) working voltage and LED working current were experimentally optimised to be 3.5 V and 200 mA. HepG2 hepatoma carcinoma cells were used to validate the cell counting function of the fluorescence detector. The experimental results showed that the output was peak signals that can be obviously distinguished from background noise; there were eight peak signals with an average value of 0.7 V within 250 s, which agreed with that of fluorescence microscope imaging results. It provides a new technological approach for novel micro total cell analysis system.

Immuno-capture and in situ detection of Salmonella typhimurium on a novel microfluidic chip

The new method presented in this article achieved the goal of capturing Salmonella typhimurium via immunoreaction and rapid in situ detection of the CdSe/ZnS quantum dots (QDs) labeled S. typhimurium by self-assembly light-emitting diode-induced fluorescence detection (LIF) microsystem on a specially designed multichannel microfluidic chip. CdSe/ZnS QDs were used as fluorescent markers improving detection sensitivity. The microfluidic chip developed in this study was composed of 12 sample channels, 3 mixing zones, and 6 immune reaction zones, which also acted as fluorescence detection zones. QDs–IgG–primary antibody complexes were generated by mixing CdSe/ZnS QDs conjugated secondary antibody (QDs–IgG) and S. typhimurium antibody (primary antibody) in mixing zones. Then, the complexes went into immune reaction zones to label previously captured S. typhimurium in the sandwich mode. The capture rate of S. typhimurium in each detection zone was up to 70%. The enriched QDs-labeled S. typhimurium was detected using a self-assembly LIF microsystem. A good linear relationship was obtained in the range from 3.7×10 to 3.7×105cfumL−1 using the equation I=0.1739 log (C)−0.1889 with R2=0.9907, and the detection limit was down to 37cfumL−1. The proposed method of online immunolabeling with QDs for in situ fluorescence detection on the designed multichannel microfluidic chip had been successfully used to detect S. typhimurium in pork sample, and it has shown potential advantages in practice.

Highly sensitive analysis of flavonoids by zwitterionic microemulsion electrokinetic chromatography coupled with light-emitting diode-induced fluorescence detection

A rapid zwitterionic microemulsion electrokinetic chromatography (ZI-MEEKC) approach coupled with light-emitting-diode-induced fluorescence (LED-IF, 480nm) detection was proposed for the analysis of flavonoids. In the optimization process, we systematically investigated the separation conditions, including the surfactants, cosurfactants, pH, buffers and fluorescence parameters. It was found that the baseline separation of the seven flavonoids was obtained in less than 5min with a running buffer consisting of 92.9% (v/v) 5mM sodium borate, 0.6% (w/v) ZI surfactant, 0.5% (w/v) ethyl acetate and 6.0% (w/v) 1-butanol. High sensitivity was obtained by the application of LED-IF detection. The limits of detection for seven flavonoids were in the range of 3.30×10−8 to 2.15×10−6molL−1 without derivatization. Ultimately, the detection method was successfully applied to the analysis of flavonoids in hawthorn plant and food products with satisfactory results.

Design and Implementation of Light-Emitting Diode Induced Fluorescence Detection System

Design and Implementation of Light-Emitting Diode Induced Fluorescence Detection System

Determination of doxorubicin in plasma by using CE coupled with in‐column tapered optic‐fiber light‐emitting diode induced fluorescence detection

This paper proposes a novel strategy to enhance detection of doxorubicin in human plasma, using homemade CE combined with normal stacking mode (NSM). The detection system of CE named as in-column tapered optic-fiber light-emitting diode induced fluorescence detection system is economic and more sensitive that has been demonstrated in our previous work. The influence of sample matrix, BGE, applied voltage, and injection time on the efficiency of NSM were systematically investigated. The clean extracts were subjected to CE separation with optimal experimental conditions: Ethanol-water (1:1, v/v) was used as sample matrix, pH 4.12 15 mM sodium phosphate buffer solution containing 70% v/v ACN, applied voltage 23 kV and 45 s hydrodynamic injection at a height of 20 cm. The detection system displayed linear dynamic range from 6.4 to 1.13 × 10(3) ng/mL with a correlation coefficient of 0.9990 and LOD 2.2 ng/mL for doxorubicin (DOX). The proposed CE method has been successfully applied to determine DOX in human plasma which the recoveries of standard DOX added to human plasma were found to been the range of 93.8-104.6%. The results obtained demonstrate that our detection system combined with NSM is a good idea to enhance sensitivity in CE for routine determination of DOX in some biological specimens.

Determination of Sulfonamides in Pharmaceuticals and Rabbit Plasma by Microchip Electrophoresis with LED-IF Detection

In this paper, we describe a compact and low-cost light-emitting diode-induced fluorescence (LED-IF) detection coupled to microchip electrophoresis for the determination of sulfonamides in pharmaceutical formulations and rabbit plasma. Three fluorescein isothiocyanate-labeled sulfonamides in rabbit plasma were separated in the running buffer of 40 mM phosphate buffer (pH 7.0) at the separation voltage of 2.0 kV, and detected by LED-IF detector in which the high-power blue LED was driven at the constant current of 150 mA and the emitted fluorescence over 510 nm was collected by a planar photodiode. The linear concentration ranged from 2.0 to 125.0 μg mL−1, both for sulfadiazine and sulfamethazine with the correlation coefficients (r2) of 0.995 and 0.997, respectively, and from 2.0 to 100.0 μg mL−1 with the correlation coefficients (r2) of 0.997 for sulfaguanidine. The limits of detection for the three sulfonamides were 0.36–0.50 μg mL−1 (S/N = 3). Intra-day and inter-day precision of migration time and peak area for the determination of sulfonamides were <4.5 %. This method has been successfully applied to the analysis of sulfonamides in pharmaceuticals, and could be used to study the pharmacokinetics of sulfonamides in rabbit.

Rapid Determination of Creatinine in Human Urine by Microchip Electrophoresis with LED Induced Fluorescence Detection

In clinical medicine, urine creatinine concentration is an important marker in the evaluation of renal function and muscular dysfunctions. Herein, we reported a novel method for rapid determination of creatinine in urine by microchip electrophoresis with light-emitting diode induced fluorescence detection. Creatinine was derivatized by fluorescein isothiocyanate, and then quantitatively detected by the developed microchip LED induced fluorescence detection system. The excitation and emission wavelengths were 490 and 523 nm, respectively. The urine samples were analyzed after centrifuge and filtration. A baseline separation was obtained in <30 s using 10 mM borate buffer (pH 9.0, containing 45 mM sodium dodecylsulfate), with separation voltage of 1.5 kV. Good linearity was obtained (r2 = 0.9978) in the concentration range of 10.0–2.00 × 103 μM, and the limit of detection was 2.87 μM (S/N = 3). The recovery was 96.0–107 %, and the interday precision was <4.5 % (n = 6). To validate assay results, we compared the present method with the Jaffe’s colorimetric assay by measuring real urine samples. The method was reliable, sensitive, high-speed, low-cost and suitable for the routine analysis of creatinine in biofluids.