Chemiluminescence Detection Method Research Articles

Copper(II)−Alizarin Red S Complex as an Efficient Chemiluminescent Probe for the Detection of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry.

Development of a sensitive flow‐injection–chemiluminescence detection method for the determination of laevodopa

A simple chemiluminometric method using flow injection has been developed for the determination of levodopa, based on its sensitizing effect on the weak chemiluminescence (CL) reaction between Na(2)SO(3) and acidic KMnO4. Under optimum experimental conditions, the CL intensity was linearly related to the concentration of levodopa from 3.4 x 10(-8) to 2.4 x 10(-5) mol/L and the detection limit was 1.1 x 10(-8) mol/L (s:n = 3). The relative standard deviation (RSD) of the proposed method calculated from 20 replicate injection of 3 x 10(-7) mol/L levodopa was 3.3%. The correlation coefficient was 0.997. The method was successfully applied to the determination of levodopa in commercial pharmaceutical formulations and spiked urine samples.

Recent ozone investigations over Bulgaria by remote sensing: Ground-based and satellite data

This presentation deals with the recent investigations of the dynamics of the total ozone content (TOC) and of the lower troposphere ozone over Bulgaria, South-Eastern Europe. For analysis of the TOC we have used data obtained through measurements carried out by using a ground-based spectrophotometer Photon and data from satellite experiments TOMS-Nimbus 7 and TOMS-EP. The spectrophotometer Photon measures the direct solar ultraviolet (UV) radiation in the range 290–360 nm with 1 nm resolution, using which TOC is calculated. The ozone values received from the ground-based and satellite observations in the period 1999–2005 have been compared. In this period the ozone column did not show a statistically significant trend. The TOC data demonstrated typical for the middle latitudes seasonal variations. The monthly mean ozone values observed during the last 9 years have been examined also. It was found that the largest TOC fluctuations were detected in February and the smallest – in July. Ozone behaviour in the planetary boundary layer (PBL) has been analyzed by using data obtained with remote-sensing technique – ground-based aerosol LIDAR (LIght Detection And Ranging), which give information about the evolution of the PBL. The surface ozone concentrations have been measured with a fast and sensitive ozone monitor, based on chemiluminescent method for ozone detection. The investigation shows that the simultaneous observations of the surface ozone and of the boundary layer dynamics provide the opportunity accurately to establish relation between the BL structure and the features of ozone variations. In particular, the influence of the residual layer on the ozone values near the ground is demonstrated.

A Novel Probe Au(III) for Chemiluminescent Image Detection of Protein Blots on Nitrocellulose Membranes

A new method, based on the chloroauric acid-enhanced luminol chemiluminescence, is established for the chemiluminescent imaging detection of protein blots on nitrocellulose membranes. After transferring to the nitrocellulose (NC) membranes, various proteins in human serum can be easily detected using this method. Simplicity and wide applicability are achieved, without the need of expensive antibodies or tedious immunoassay procedures. Furthermore, neither noxious materials nor radioactive pollution is produced. The successful detection of proteins is due to the binding of Au(III) to the protein blots and the chemiluminescent character of the enhanced luminol signal. As a novel chemiluminescent detection method, it offers significant biological analytical potentials in biochemistry and in molecular biology.

Novel method for fast immunodetection using vacuum filtration

Western blotting is a well establish technique used for the routine detection of specific proteins from complex mixtures. The immunodetection step of western blotting is a long and tedious process, involving multiple changes of solutions, and is usually extended over several hours to overnight. This paper describes an innovative, vacuum‐based immunodetection approach, which allows the detection of the protein in less than 45 minutes. The system can be used for rapid antibody optimization studies, antibody screening, simultaneous detection of different proteins in multiples blots, or routine western blot analysis. Vacuum filtration enables efficient blocking and washing of the membrane, speeds up the overall process and make the system easy to use. Blocking of the membrane occurs rapidly in the presence of low concentration of non fat dry milk or other common blocking reagents. Since incubation steps are typically performed in ten minutes, users can sequentially process several blots in a day. The system in compatible with PVDF or nitrocellulose membranes and detection can be accomplished by any chemiluminescence or fluorescence detection method. The SNAP id™ system is a novel immunodetection system that uses the basic steps of the standard immunodetection protocol, is easy to use and offers a substantial reduction in processing time.This research has been totally funded by Millipore Corporation.

Analysis of Monosaccharides by Capillary Electrophoresis with Electrochemiluminescence Detection

A capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II)-based electrogenerated chemiluminescence detection method was investigated for the determination of monosaccharides using 2-diethylaminoethanethiol as a derivatizing reagent. Xylose, rhamnose, glucose and glucosamine were selected for the demonstration of the method. Under optimal conditions, the calibration curve of glucose was linear over the range of 1.0 x 10(-4) to 1.0 x 10(-7) mol/L (R = 0.999) and the detection limit was 6.0 x 10(-8) mol/L (S/N = 3). The method was successfully applied for the assay of glucose in angelica.

Liquid Chromatographic Arsenic Speciation with Gas-Phase Chemiluminescence Detection

We present a newly developed gas-phase chemiluminescence (CL) detection method for the separation and quantification of inorganic and organic arsenic species. Arsenite, arsenate, dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) were separated by anion exchange using carbonate-bicarbonate and NaOH eluents with step-gradient elution. The separated species were passed through a UV photooxidation reactor which decomposed the organic species and converted them to inorganic As(V). Subsequent on-line hydride generation with acid and sodium borohydride produces AsH3 and H2, which are separated from the liquid in a gas-liquid separator. The produced AsH3, driven by H2, reacts with ozone in a small reflective cell located atop a photomultiplier tube, resulting in intense CL. In the present form, the limits of detection (LODs, signal-to-noise = 3), based on peak height, for arsenite, arsenate, MMA, and DMA are 0.4, 0.2, 0.5, and 0.3 microg/L, respectively, for a 100 microL injected sample. This analyzer demonstrates the robustness of the CL detection system for arsenic and provides an affordable alternative to atomic spectrometry for use as a detector after chromatographic speciation. We found no significant practical interferences.

Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic beta-cells (isoform 1) and hepatocytes (isoforms 2 and 3). Although certain cellular proteins are known to either stimulate or inhibit its activity, little is known about post-translational modifications of this enzyme and their possible regulatory functions. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin-conjugating enzyme system of the rabbit reticulocyte lysate. Both isoforms were polyubiquitinated on at least two lysine residues, and mutation analysis indicated that multiple lysine residues functioned as redundant acceptor sites. Deletion of its C-terminal alpha-helix, as part of a ubiquitin-interacting motif, affected the polyubiquitination at one of the sites and resulted in a completely inactive enzyme. Evidence is presented that poly/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, the recombinant hGK was found to interact with and to be allosterically activated up to approximately 1.4-fold by purified free pentaubiquitin chains at approximately 100 nm (with an apparent EC(50) of 93 nm), and possibly also by unidentified polyubiquitinated proteins assigned to their equilibrium binding to the ubiquitin-interacting motif site. The affinity of pentaubiquitin binding to hGK is regulated by the ligand (d-glucose)-dependent conformational state of the site. Both ubiquitination of hGK and its activation by polyubiquitin chains potentially represent physiological regulatory mechanisms for glucokinase-dependent insulin secretion in pancreatic beta-cells.

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1–20 μg mL −1 for MTU and 0.1–10 μg mL −1 for PTU, the detection limits were 0.03 μg mL −1 for PTU, 0.03 μg mL −1 for MTU and the quantification limits were 0.1 μg mL −1 for PTU, 0.1 μg mL −1 for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.

Ultrasensitive electrogenerated chemiluminescence detection of DNA hybridization using carbon-nanotubes loaded with tris(2,2′-bipyridyl) ruthenium derivative tags

An ultrasensitive electrogenerated chemiluminescence (ECL) detection method of DNA hybridization based on single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium complex tags was developed. The probe single strand DNA (ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe. When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode, and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate, a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4 × 10 −14 to 1.7 × 10 −12 M with a detection limit of 9.0 × l0 −15 M. The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA. This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization.

食物アレルゲンタンパク質の近赤外蛍光標識プローブによる検出

We aimed to detect the allergen proteins in food materials using recently developed near-infrared fluorescent probes. Sensitivities of this method were comparable to chemiluminescence detection methods, which are known to be sensitive. In addition, the sensitivities of this near-infrared fluorescent method were at least 10-50-times higher than those of the conventional visible fluorescent methods using Cy3 and Cy5 dyes. This method was effectively applicable to immunoblotting, dot-blotting and plate-assay (direct FLISA : fluorescence-linked immunosorbent assay) with ELISA plate. Allergen levels of the food sample were quantified by standard curves using standard allergen protein using the dot-blotting technique. This highly sensitive detection system also provided multiple detections of different allergens for different antibodies and dyes with distinct properties of wavelength. This enables high-throughput screening of characteristic allergen contents of target food materials, or cultivars. Generally, allergen proteins are recognized by patient's serum IgE. Therefore, we tried to detect patient's IgE-binding proteins, the putative allergens in foodstuffs. In this detection system, it was possible to detect IgE-binding proteins with sensitivity almost equivalent to a chemiluminescent detection system. Taken together, it was shown that this novel detection system was an effective technique for the sensitive detection and screening of food allergens.

Nicorandil Elevates Tissue cGMP Levels in a Nitric-Oxide-Independent Manner

The K+ channel opener nicorandil is a hybrid compound that contains nitrate in its structure. It has been reported that nicorandil can relax vascular tissue in vitro via a mechanism that involves activation of KATP channels and stimulation of soluble guanylyl cyclase. However, it is not known whether the increase of cGMP levels occurs through an elevation of nitric oxide (NO). The aim of the present study was to determine whether NO release was a direct effect of nicorandil. We reported here that nicorandil did not generate NO using ozone chemiluminescence detection methods in human or rat liver microsomes (P450-rich fractions) with addition of NADPH. However, nicorandil elevated cGMP levels in rat liver, aorta, and human coronary smooth muscle cells in vitro. The elevation was not inhibited by the NO trapping agent carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO). These results suggest that nicorandil elevates cGMP without NO generation.

Chemiluminescent detection of DNA hybridization using gold nanoparticles as labels

A chemiluminescent (CL) detection method has been developed for DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which gold nanoparticles modified with alkylthiol-capped oligonucleotide strands are used as probes to monitor the presence of the specific target DNA. The AuCl(4)(-), which is the dissolving product of the gold nanoparticles anchored on the DNA hybrids, serves as an analyte in the H(2)O(2)-luminol- AuCl(4)(-) CL reaction for the indirect measurement of the target DNA. The combination of the remarkable sensitivity of the CL analysis with the large number of AuCl(4)(-) released from each DNA hybrid allows a detection limit at levels as low as 0.1 pM of the target DNA. Moreover, with a further silver amplification step, the detection limit will be pushed down to the femtomolar domain.

Chemiluminescent Method for Detection of Eutrophication Sources by Estimation of Organic Amino Nitrogen and Ammonium in Water

An automatic method has been developed for the estimation of organic amino nitrogen (CH2-NH) and ammonium in water samples. We propose a continuous flow system in which nitrogen compounds react with hypochlorite reagent to produce chloramines. Subsequently, the mixture is mixed with luminol, generating a chemiluminescence signal. The signal emission at 425 nm, registered as a function of time, decreases as nitrogen concentration increases, due to the decrease on hypochlorite concentration. A large number of nitrogen compounds have been assayed and their sensitivities compared, in milligrams per liter nitrogen. The ammonium calibration graph, expressed as N, can be used for most of the assayed compounds. The linear interval was 0.24-4 mg L(-1) N, with the detection limit 0.07 mg L(-1) N. The chemiluminescence method was applied to the analysis of several kinds of real water samples, natural, lake, irrigation ditch, fountain, residual, and seawater in order to detect possible sources of eutrophication. The accuracy (% relative error) and precision were satisfactory, with mean values of 5 +/- 4 and 3 +/- 2, respectively. This procedure has been used to estimate nitrogen content in samples before and after Kjeldahl treatment. In the same samples, the N found for the untreated samples provided a good estimation of the N Kjeldahl. Sixty samples per hour can be analyzed, and the procedure can also be used for in situ monitoring.

Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods

Although the formation of reactive oxygen species (ROS) in the skin induced by the ultraviolet (UV) light has been shown to lead to many cutaneous disorders, skin cancer and photoageing, the mechanism and distribution of ROS generation has not yet been definitively determined. In the present study, we examined the distribution of UVA-induced ROS in the skin of live hairless mice, using our proposed in vivo imaging chemiluminescent (CL) method to detect ROS combined with a CL probe (cypridina hilgendorfii luciferin analogue; CLA) and tape stripping (TS) technique. The CL intensities in the skin of live hairless mice were confirmed to significantly increase by UVA exposure. When TS was conducted five times in a maximum level after CL measurement following UVA exposure and subsequent CLA application, CL intensities due to UVA-induced ROS generation in the residual skin decreased to 10% of the original levels; and those in the stripped skin on each tape decreased in the stripped order such as 52%, 16%, 11%, 6% and 5%. Next, CLA was applied and then CL intensities were measured in the residual skin after advance 1, 3 and 5 tape strippings, and CL intensities due to ROS were detected primarily in the outer layer of the skin. On the basis of these results, we concluded that ROS induced by UVA exposure occurs and distributes in the outermost layer of the stratum corneum.

Reactive oxygen species and their chemiluminescence-detection methods

This review extensively covers information on chemiluminescence-detection systems for five reactive oxygen species, including superoxide radical, singlet oxygen, hydroxyl radical, hydrogen peroxide and peroxynitrite. We evaluate in detail the current state and the limitations of different chemiluminescence systems. We give some pointers to the likely directions of future developments and prospects.

Simultaneous determination of benserazide and levodopa by capillary electrophoresis–chemiluminescence using an improved interface

A capillary electrophoresis coupling with indirect chemiluminescence detection method for the simultaneous determination of benserazide and levodopa has been developed. The detection interface was improved to simplify the capillary electrophoresis–chemiluminescence (CE–CL) system and the features of this improved interface were illustrated in this paper. The CE–CL conditions for the simultaneous determination of benserazide and levodopa were optimized. Under the optimal conditions, the CL intensity was linear with concentrations of levodopa in the range of 1.0 to 100.0 μg ml −1, and benserazide in the range of 10.0 to 1000 μg ml −1, respectively. The detection limits (S/N = 3) in turn were 1.85 μg ml −1 for BS and 0.12 μg ml −1 for L-dopa with relative standard deviations of less than 3%. The proposed method has been successfully applied to the determination of benserazide and levodopa in medopar tablets and spiked urine samples, demonstrating the feasibility and reliability of the proposed method.

Effect of Exercise on the Expression of HSP20 in the Hypertensive Rat Heart

Previous observations show that levels of Hsp20, a small heat shock protein, are elevated in the rat left ventricle by exercise training. PURPOSE: To determine whether exercise training would induce an increase in the levels of Hsp20 in hearts of spontaneously hypertensive rats (SHR). METHODS: SHR were randomly assigned to a sedentary (SHR-SED) or trained (SHR-TRN) group. The trained group was exercised on a treadmill at 0% grade for eight weeks, using a high intensity interval training protocol. After 3 weeks of introductory running, 2.5 min of sprint running at 29–45 m/min was interspersed with steady running at 24 m/min for five minutes for a total duration of 37 minutes. The number of sprints was increased progressively so that the duration was approximately 60 minutes after eight weeks. During the last week of the study, sedentary rats were habituated to the treadmill for 5 days for 10 min each at 15 m/min. On the last day of the study, sedentary and exercise-trained rats were run for 10 minutes at 15 or 25 m/min respectively, and hearts were harvested at various times (t=0,10,30,60,180 min) after the last bout of exercise. From groups of SHR-SED and SHR-TRN rats, heart left (LV) and right ventricle (RV) free walls and septum (SEP) were used to measure the levels of Hsp20. Western blotting was performed using 12% gels, PVDF membranes, and an enhanced chemiluminescent detection method. A rabbit polyclonal anti- HSP20 antibody was used to detect the protein. Levels of Hsp20 were normalized to those of glyceraldehyde-3-phosphate dyhydrogenase (GAPDH). RESULTS: The level of HSP20 expression was significantly increased in response to exercise training in the soluble fraction (+61%) and whole homogenate (+70%) of the RV. In the SEP, the increase in HSP20 expression level was observed only in the soluble fraction (+54%) while the expression level was not statistically different in the whole homogenate. Exercise training did not trigger increased expression of HSP20 in the hypertensive LV free wall. In the RV and LV free walls, the level of HSP20 was higher in the soluble fraction than in the whole homogenate, and the ratio of the two measurements was unchanged by exercise-training. On the other hand, the level of HSP20 in the SEP was greater in the whole homogenate, and the soluble-to-whole homogenate ratio was increased as a result of exercise training (p < 0.05). CONCLUSION: Exercise training triggers increased expression of HSP20 in the unaffected RV free wall of hypertensive rats, but not in the pressure-overloaded LV free wall. Supported by the American Heart Association Midwest Affiliate and the Undergraduate Research Opportunity Program at the University of Michigan

Chemiluminescent detection of clonal immunoglobulin and T cell receptor gene rearrangements in Tunisian lymphoid malignancies, leukemias and lymphomas

Clonal rearrangement of antigen receptor genes is commonly used to characterize the lymphoproliferative diseases. In order to perform molecular characterization in the diagnostics and monitoring of lymphoid malignancies, leukemias and lymphomas in Tunisia, we have introduced the use of chemiluminescent probes for immunoglobulin (IG) and T cell receptor (TR) gene rearrangement detection employing the Southern blot method. The chemiluminescent and radioactive detection methods tested with alkaline phosphatase and 32P labelled probes, respectively, were used for the IG and TR gene rearrangement characterization. Our results show the same pattern of rearrangement. Moreover, the chemiluminescent signal is detected faster and it is as sensitive as the radioactive one. We report the optimized conditions for using IGH, IGK, IGL, TRB and TRG probes in non radioactive detection. We have applied the chemiluminescent Southern blot method to analyze examples of Tunisian leukemias and lymphomas. The results allowed the assessment of clonality and the T or B cell lineage of these cases. The use of non radioactive probes makes chemiluminescent Southern blot detection reliable, safe and sensitive. As the use of radioactivity is not common in our laboratories and the licensing requirements needed for its use prohibitive, the chemiluminescent technique will be of great help for detection and characterization of molecular markers in lymphoid malignancies in Tunisia.

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (−)-epicatechin and (−)-epigallocatechin gallate solutions, were linear over the range of 0.1–50 μM. The detection limits (signal-to-noise ratio = 3) were 0.8 pmol for (−)-epigallocatechin gallate and 1.2 pmol for (−)-epicatechin. The high-performance liquid chromatography–chemiluminescence (HPLC–CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.