Chemiluminescent Solution Research Articles

Miniaturizable Chemiluminescence System for ATP Detection in Water.

We present the design, fabrication, and testing of a low-cost, miniaturized detection system that utilizes chemiluminescence to measure the presence of adenosine triphosphate (ATP), the energy unit in biological systems, in water samples. The ATP-luciferin chemiluminescent solution was faced to a silicon photomultiplier (SiPM) for highly sensitive real-time detection. This system can detect ATP concentrations as low as 0.2 nM, with a sensitivity of 79.5 A/M. Additionally, it offers rapid response times and can measure the characteristic time required for reactant diffusion and mixing within the reaction volume, determined to be 0.3 ± 0.1 s. This corresponds to a diffusion velocity of approximately 44 ± 14 mm2/s.

Magnetic Field Effects in Triplet-Triplet Annihilation Upconversion: Revisiting Atkins and Evans' Theory.

In their seminal description of magnetic field effects on chemiluminescent fluid solutions, Atkins and Evans considered the spin-dependent interactions between two triplets, incorporating the effects of the diffusion of the molecules in the liquid phase. Their results, crucial for the advancement of photochemical upconversion, have received renewed attention due to the increasing interest in triplet-triplet annihilation for photovoltaic and optoelectronic applications. Here we revisit their approach, using a modern formulation of open quantum system dynamics and extend their results. We provide corrections to the theory of the magnetic field response of the fluorescent triplet pair state with singlet multiplicity. These corrections are timely, as improvements in the precision and range of available experimental methods are supported by the determination of quantitatively accurate rotational and interaction model parameters. We then extend Atkins and Evans' theory to obtain the magnetic field response of triplet pair states with triplet and quintet multiplicity. Although these states are not optically active, transitions between them are becoming imperative to study the working mechanism of spin-mediated upconversion and downconversion processes, thanks to advances in electron spin resonance and time-resolved transient absorption spectroscopy.

Chemiluminescence imaging immunoassay for multiple aminoglycoside antibiotics in cow milk

SummaryA simple and rapid multiplexed direct competitive chemiluminescent (CL) imaging immunoassay had been developed for the simultaneous detection of kanamycin (KAN) and streptomycin (STR), which were widely used against bacterial infections in animals. To achieve the multiplexed detection of the two targets, a microarray format based on nitrocellulose membranes (NCMs) has been designed. Then, the peroxidase activity of the captured HRP‐labelled immunocomplex in each channel was measured by an enhanced luminol‐based chemiluminescent solution using a cooled low‐light CCD with high resolution. For qualitative analysis, the limit of detection (LOD) was 0.5 ng mL−1 for KAN and 3.13 ng mL−1 for STR by the naked eye. Through the CL data collected, KAN and STR could be detected quantitatively at the LOD of 0.03 and 0.33 ng mL−1, respectively. This method can be utilised as a screening test to evaluate the presence of antibiotics in milk samples.

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.

ABSTRACTThe in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.

Molecular identification of tick-borne pathogens infecting cattle in Mymensingh district of Bangladesh reveals emerging species of Anaplasma and Babesia.

Tick-borne diseases are considered a major hindrance to the health and productive performance of cattle in Bangladesh. To elucidate the epidemiology of tick-borne pathogens (TBPs) in local cattle, a cross-sectional study was performed in the 12 subdistricts (Upazilas) of Mymensingh district in Bangladesh. Blood samples and ticks were collected from 384 clinically healthy cattle kept by 135 farmers from 96 randomly selected villages. DNA extracted from the blood samples was subsequently screened by polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay using an in-house prepared chemiluminescence solution for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria spp. A total of 2,287 ticks were collected from 232 infested cattle (60.4%, 232/384) and identified morphologically as Rhipicephalus (Boophilus) microplus (n=1,432, 62.6%) and Haemaphysalis bispinosa (n=855; 37.4%). The RLB results demonstrated that the majority of the cattle (62.2%) were infected with at least one TBP. Theileria orientalis infections were most common (212/384, 55.2%) followed by infections with Anaplasma bovis (137/384, 35.67%), Anaplasma marginale (16/384, 4.17%), Babesia bigemina (4/384, 1.04%) and Babesia bovis (2/384, 0.52%). A previously uncharacterized Anaplasma sp. (Anaplasma sp. Mymensingh) and Babesia sp. (Babesia sp. Mymensingh), which are genetically closely related to Anaplasma platys and B.bigemina, were detected in 50 of 384 (13.0%) and 1 of 384 (0.3%) of the blood samples, respectively. Key risk factors for the occurrence of T.orientalis, A.marginale and Anaplasma sp. Mymensingh were identified. In conclusion, this study revealed that cattle in Mymensingh district are mainly infested with R.microplus and H.bispinosa ticks and may carry multiple TBPs. In addition, two previously uncharacterized pathogens were detected in the bovine blood samples. The pathogenicity of these species remains to be determined.

A photochemical method for in vitro evaluation of fluid flow in human dentine

ObjectiveTo evaluate the flow dynamics of dentine fluid using a chemiluminescence method in vitro. Materials and methodsHorizontally sliced coronal dentine specimens with thicknesses of 1.4, 1.6, 1.8, and 2.0mm (n=10 each) were prepared from extracted human third molars. After cleaning with EDTA, a mounted specimen was clamped between 2 acrylic chambers attached to both the occlusal and pulpal sides. The occlusal chamber, which was closed with a glass coverslip, was filled with a chemiluminescent solution (0.02% luminol and 1% sodium hydroxide in water). A trigger solution of 1% hydrogen peroxide and 1% potassium ferricyanide was injected into the pulpal chamber at a constant pressure of 2.5kPa, and allowed to immediately flow into the patent dentinal tubules. Four consecutive measurements (T1–T4) were performed on each sample by recording the emission of chemiluminescence with a photodetector. The relationship between the crossing time of the liquid through the slice and dentine thickness was examined. ResultsAn apparent time delay was detected between the starting points of the trigger solution run and photochemical emission at T1. Dt (Dt, s) values of each thickness group were 13.6±4.25 for 1.4mm, 18.1±2.38 for 1.6mm, 28.0±2.46 for 1.8mm, and 39.2±8.61 for 2.0mm, respectively. Dt significantly decreased as dentine became thinner towards the pulp chamber (P<0.001). ConclusionsThe velocity of fluid flow increased both with increasing dentine depth or reduction of remaining dentine thickness.

Sensitive and selective capillary electrophoretic analysis of proteins by zirconia nanoparticle-enhanced copper (II)-catalyzed luminol–hydrogen peroxide chemiluminescence

We report herein a sensitive, selective, convenient CE determination of heme proteins in complex matrices by a sodium-dodecyl-sulfate-assisted, zirconia nanoparticle-enhanced copper (II)-catalyzed luminol–hydrogen peroxide chemiluminescence (CCLHPCL). Introducing a segment of sodium dodecyl sulfate to the capillary after sample injection not only rendered selective detection by quenching the luminescence signals from the non-heme proteins but also owning to the suppressed protein adsorption, led to significant improvement in separation efficiency and detection sensitivity. The signals were further improved by addition of ZrO2 nanoparticles to the chemiluminescence solution. Compared with the conventional CCLHPCL, the detection limits (S/N=3) were improved by 10.2–22.0 folds, with 7.8×10−9, 3.3×10−9 and 1.5×10−9M for three model proteins, viz, myoglobin, hemoglobin and cytochrome C, respectively. Because the method did not require sophisticated pretreatment, it was convenient to analyze heme proteins in complex matrices, as demonstrated, hemoglobin in human blood and spiked human urine samples.

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.

Evaluation of Brilliance and Visibility of Fluorescence and Chemiluminescence Solution for Training of Preparing Injections

Personnel who prepare and administer chemotherapeutic agents have been reported to develop untoward effects. The use of appropriate techniques for preparing these agents is encouraged, and educational training systems that involve the use of a fluorescent or chemiluminescence reagent as placebos have been established to minimize potential exposure to these agents. However, the optimum conditions for the use and visibility of these placebos remain obscure. In this study, our results indicated that the fluorescence intensity of fluorescent reagent decreased when it was used at a concentration greater than 0.01%. Because drops created due to splashes and leaks are extremely small and easily evaporate, it is possible that the fluorescence resulting from such drops readily disappears despite using an anti-evaporation reagent. We also developed a method to evaluate the visibility of the small drop; using this method, we determined the distance at which the drop present on the pin could be seen by the observer. The distance at which the drop was clearly recognized as a pinpoint by using the fluorescence method was almost comparable to that for the chemiluminescence method. In the chemiluminescence method, the drop on the pin was faintly visible as a slightly bright area because of low background when observed at a certain distance that was much greater than that at which the drop was clearly visible; however, such an area was not observed in the fluorescence method. The results of our study will help in the selection of a training method depending on the situation.

Chemiluminescence detection in liquid-core microstructured optical fibers

We reported the first measurement of chemiluminescence photon emission in microstructured optical fibers (MOFs). The hollow-core of a polymer MOF was infiltrated with fluid to support an index-guiding mechanism. The selective filling method involved sealing the cladding air-holes during polishing, leaving the hollow core available for the chemiluminescence solution. Experimental results, compared with a solid-core index-guiding fiber, indicated that the luminescent intensity collected was highly improved by the hollow-core structure. Due to the robustness and flexibility of optical fibers, the MOF can be coiled, making it suitable for microchip integration. Moreover, this novel technique of luminescence emission inside a channel will eliminate the difficulty in coupling alignment of optical waveguide to light sources.

Ligand Regulation of the Quaternary Organization of Cell Surface M3 Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET

Flp-In(TM) T-REx(TM) 293 cells expressing a wild type human M(3) muscarinic acetylcholine receptor construct constitutively and able to express a receptor activated solely by synthetic ligand (RASSL) form of this receptor on demand maintained response to the muscarinic agonist carbachol but developed response to clozapine N-oxide only upon induction of the RASSL. The two constructs co-localized at the plasma membrane and generated strong ratiometric fluorescence resonance energy transfer (FRET) signals consistent with direct physical interactions. Increasing levels of induction of the FRET donor RASSL did not alter wild type receptor FRET-acceptor levels substantially. However, ratiometric FRET was modulated in a bell-shaped fashion with maximal levels of the donor resulting in decreased FRET. Carbachol, but not the antagonist atropine, significantly reduced the FRET signal. Cell surface homogeneous time-resolved FRET, based on SNAP-tag technology and employing wild type and RASSL forms of the human M(3) receptor expressed stably in Flp-In(TM) TREx(TM) 293 cells, also identified cell surface dimeric/oligomeric complexes. Now, however, signals were enhanced by appropriate selective agonists. At the wild type receptor, large increases in FRET signal to carbachol and acetylcholine were concentration-dependent with EC(50) values consistent with the relative affinities of the two ligands. These studies confirm the capacity of the human M(3) muscarinic acetylcholine receptor to exist as dimeric/oligomeric complexes at the surface of cells and demonstrate that the organization of such complexes can be modified by ligand binding. However, conclusions as to the effect of ligands on such complexes may depend on the approach used.

Modulation of the Tumor Suppressor Protein α-Catenin by Ischemic Microenvironment

Dysregulation or mislocalization of cell adhesion molecules and their regulators, such as E-cadherin, beta-catenin, and alpha-catenin, usually correlates with loss of polarity, dedifferentiation, invasive tumor growth, and metastasis. A subpopulation of alpha-catenin-negative cells within the DLD-1 colorectal carcinoma cell line causes it to display a heterogeneous morphological makeup, thus providing an excellent model system in which to investigate the role of alpha-catenin in tumorigenesis. We re-established expression of alpha-catenin protein in an alpha-catenin-deficient subpopulation of the DLD-1 cell line and used it to demonstrate that loss of alpha-catenin resulted in increased in vitro tumorigenic characteristics (increased soft agarose colony formation, clonogenic survival after suspension, and survival in suspension). When the cells were used to form tumor xenografts, those lacking alpha-catenin showed faster growth rates because of increased cellular cycling but not increased tumor microvascular recruitment. alpha-Catenin-expressing cells were preferentially located in well perfused areas of xenografts when tumors were formed from mixed alpha-catenin-positive and -negative cells. We therefore evaluated the role of the ischemic tumor microenvironment on alpha-catenin expression and demonstrated that cells lose expression of alpha-catenin after prolonged exposure in vitro to hypoglycemic conditions. Our findings illustrate that the tumor microenvironment is a potent modulator of tumor suppressor expression, which has implications for localized nutrient deficiency and ischemia-induced cancer progression.

Fixed-angle observation of surface plasmon coupled chemiluminescence from palladium thin films

In this letter, the fixed-angle observation of surface plasmon coupled chemiluminescence (SPCC) from palladium thin films is reported. Fresnel calculations predict that light 492–549 nm spectral range can efficiently induce surface plasmon modes in 15 nm palladium thin films. Free-space emission from blue, green, and chartreuse chemiluminescent solutions on palladium thin films was isotropic, while the SPCC emission was highly directional and predominantly p-polarized in accordance with the predictions of the Fresnel calculations. In addition, the decay rates of the SPCC and free-space emission were similar, which suggests that palladium thin films have no catalytic effect on chemiluminescence emission.

Surface plasmon coupled chemiluminescence from iron thin films: Directional and approaching fixed angle observation

We report the observation of surface plasmon coupled chemiluminescence (SPCC) from iron thin films. Theoretical Fresnel calculations were employed to determine the optimum thickness of iron thin films to be 15 nm, which is assessed by the value of minimum reflectivity curves for incident light upon the thin film. An overlayer 10 nm thick SiO2 was used to protect the iron thin films from oxidation by air or physical changes induced by chemiluminescent solutions. SPCC emission from blue, green, and chartreuse chemiluminescent solutions on iron films can be observed at a fixed angle of 60° for all the chemiluminescent solutions, while free-space emission was isotropic. Iron thin films do not have a catalytic effect on chemiluminescence emission.

Directional surface plasmon coupled chemiluminescence from nickel thin films: Fixed angle observation

Directional surface plasmon coupled chemiluminescence (SPCC) from nickel thin films is demonstrated. Free-space and angular-dependent SPCC emission from blue, green and turquoise chemiluminescent solutions placed onto nickel thin films attached to a hemispherical prism were measured. SPCC emission was found to be highly directional and preferentially p-polarized, in contrast to the unpolarized and isotropic chemiluminescence emission. The largest SPCC emission for all chemiluminescence solutions was observed at a fixed observation angle of 60°, which was also predicted by theoretical Fresnel calculations. It was found that nickel thin films did not have a catalytic effect on chemiluminescence emission.

Surface plasmon coupled chemiluminescence from zinc substrates: Directional chemiluminescence

In this letter, the observation of surface plasmon coupled directional chemiluminescence (SPCC) from zinc thin films is reported. Fresnel calculations predict that light in the ultraviolet and visible spectral range can induce surface plasmon modes in 30 nm zinc thin films. Free-space emission from blue and green chemiluminescent solutions on zinc thin films was isotropic, and the SPCC emission was highly directional and p-polarized in accordance with the Fresnel calculations. In addition, the decay rates of the free-space and SPCC emission were similar, which suggests that zinc thin films have no catalytic effect on chemiluminescence.

Constitutive Activity of the Cannabinoid CB1 Receptor Regulates the Function of Co-expressed Mu Opioid Receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

Evaluation of a Chemiluminescent Assay for Analysis of Choline in Human Plasma and Whole Blood

The chemiluminescent indicator solution consisting of a 4 µM solution of 9-[[(3-carboxypropyl)[(4-methylphenyl)sulfonyl] amino]-carbonyl]-10-(3-sulfopropyl)acridinium inner salt in a phosphate buffer (pH 8.0, 0.2 M, 1% sodium cholate) was supplied by Abbott (Chicago). All other reagents and solutions (including 10 U/mL choline oxidase solution) were prepared as described by Adamczyk. 6

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.