Effect of surfactants on the chemiluminescence of acridinium dimethylphenyl ester labels and their conjugates

Chemiluminescent acridinium dimethylphenyl esters, containing two methyl groups flanking the phenolic ester bond, display excellent chemiluminescence stability and are used as labels in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of the cationic surfactant cetyltrimethylammonium chloride. Under these conditions, light emission is rapid and is complete in <5 s. In the present study we examined the effect of various surfactants on light emission from acridinium dimethylphenyl ester labels and their conjugates containing hydrophilic linkers derived either from hexa(ethylene)glycol or a sulfobetaine zwitterion. Sulfobetaine zwitterions are very polar and incorporation of these functional groups in acridinium dimethyphenyl esters and their conjugates represents a new approach to improving the aqueous solubility of these chemiluminescent labels. Our results indicate that in general, surfactants affect light emission from these labels and their conjugates by two discrete mechanisms. Cationic surfactants, but not anionic or non-ionic surfactants, accelerate overall light emission kinetics and a more modest effect is observed with zwitterionic surfactants. Surfactants also enhance total light output and the magnitude of this enhancement is maximal for cationic surfactants and a sulfobetaine zwitterionic surfactant. These observations are the first to clearly delineate the role of the surfactant on the chemiluminescence reaction pathway of acridinium esters and can be rationalized based on known effects of surfactant aggregates on bimolecular and unimolecular reactions.     

A green synthesis of chemiluminescent N-sulfopropyl acridinium esters in ionic liquids without using the carcinogen 1,3-propane sultone

Chemiluminescent acridinium dimethylphenyl esters containing hydrophilic N-sulfopropyl groups in the acridinium ring are used as labels in automated immunoassays for clinical diagnostics. Introduction of the N-sulfopropyl group in these labels is normally accomplished by N-alkylation of the corresponding, nonchemiluminescent acridine ester precursors with the toxic carcinogen 1,3-propane sultone. In the current study, we report that sodium 3-bromopropane sulfonate in ionic liquids (ILs) is a benign alternative to 1,3-propane sultone for introducing the N-sulfopropyl group in chemiluminescent acridinium ester labels. The sultone reagent can be eliminated in the synthesis of N-sulfopropyl acridinium dimethylphenyl ester labels by taking advantage of the increased reactivity of acridan esters toward nontoxic sodium 3-bromopropane sulfonate in [BMIM][BF₄]. Sodium 3-bromopropane sulfonate in ILs is also potentially a nontoxic alternative to 1,3-propane sultone for introducing the water-soluble, three-carbon sulfobetaine moiety in other molecules as well.     

Zwitterionic reagents for labeling, cross-linking and improving the performance of chemiluminescent immunoassays

Improving reagent performance in immunoassays both to enhance assay sensitivity and to minimize interference are ongoing challenges in clinical diagnostics. We describe herein the syntheses of a new class of hydrophilic reagents containing sulfobetaine zwitterions and their applications. These zwitterionic reagents are potentially useful for improving the properties of immunoassay reagents. We demonstrate for the first time that zwitterion labeling is a general and viable strategy for reducing the non-specific binding of proteins to microparticles and, to improve the aqueous solubility of hydrophobic peptides. We also describe the synthesis of zwitterionic cross-linking reagents and demonstrate their utility for peptide conjugation. In automated, chemiluminescent immunoassays, improved assay performance was observed for a hydrophobic, small analyte (theophylline) using an acridinium ester conjugate with a zwitterionic sulfobetaine linker compared to a hexa(ethylene)glycol linker. Sandwich assay performance for a large analyte (thyroid stimulating hormone) was similar for the two acridinium ester labels. These results indicate that zwitterions are complementary to poly(ethylene)glycol in improving the aqueous solubility and reducing the non-specific binding of chemiluminescent acridinium ester conjugates.     

Chemiluminescent reductive acridinium triggering (CRAT)--mechanism and applications

Acridinium esters traditionally are triggered using basic hydrogen peroxide. By serendipity, we have found that acridinium esters can also be triggered with emission of chemiluminescence by reductive triggering, e.g., by zinc metal or reduced forms of ferric and cupric salts. Furthermore, organic reducing compounds like dithiothreitol, tricarboxyethylphosphine or glutathione could be used in combination with organic oxidants like quinones or inorganic ferric or cupric salts. Mechanisms are proposed which involve the intermediacy of superoxide. Two forms of reactive oxygen species (i.e., hydrogen peroxide and superoxide) could be discriminated based on differences in kinetics. Some applications (improved detection of acridinium ester, use of acridinium ester as redox probes) are discussed.     

Chemiluminescence of Electrochemically Oxidized 9‐(Phenylthiophosphoryloxymethylidene)‐10‐methylacridan Derivatives

The electrochemistry and electrochemiluminescence (ECL) properties of acridan phosphate ester are reported. Electrochemical oxidation of 9‐(phenylthiophosphoryloxymethylidene)‐10‐methylacridan disodium salt (Compound 1) yields the corresponding acridinium ester. The latter undergoes a fast reaction with hydrogen peroxide forming an intermediate, which produces electronically excited 9‐methyl acridone and emits blue light after relaxation to the ground state. The electrochemical oxidation of this compound appears to occur in two one‐electron steps and light emission is observed for both steps. The chemiluminescence reaction could also be triggered by electrochemical oxidation of Compound 1 in the absence of H₂O₂ when the solution was saturated with O₂. Mechanisms for these reactions based on ECL, voltammetry and in situ UV‐vis identification of the oxidation products are proposed. Due to the low electrode potential required to achieve ECL emission and the occurrence of light emission in the absence of hydrogen peroxide, this compound is proposed as a label for rapid and sensitive determination of biomolecules in automated analysis.     

Chemiluminescent Detection of Carbohydrates in the Tumoral Breast Diseases

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-d-mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.     

Development of a Novel Chemiluminescent Probe with a Higher Efficiency than LucigeniiT

A many efforts have been made to take advantage of the chemiluminesce of lucigenin and acridine derivatives, especially for analytical purposes. Aryl acridinium esters have been used as labels for the immunoassay of both large and small molecules. Lucigenin is more stable and higher quantum yield than acridinium ester,but it has not been used as labels in immunoassay and DNA probes,because it dose not have a label moiety group, so for.     

Clinical applications of chemiluminescence

This article reviews the clinical applications of chemiluminescence in routine testing and surveys the diverse applications of chemiluminescence in clinical research. In routine clinical testing, chemiluminescent labels (acridinium ester, acridinium sulfonamide) and detection reactions for peroxidase and alkaline phosphatase enzyme labels (luminol and adamantyl 1,2-dioxetane-based reactions, respectively) are widely used in immunoassay and nucleic acid probe assays (e.g. hybridization protection assay, Hybrid Capture^® assay). In clinical research the sensitivity, dynamic range and diversity of chemiluminescent assays has led to a vast range of applications, notably in protein and nucleic acid blotting, microarray-based assays, monitoring reactive oxygen species, and as detection reactions for substances separated by HPLC, capillary electrophoresis (CE), and flow-injection analysis.     

Chemiluminogenic features of 10-methyl-9-(phenoxycarbonyl)acridinium trifluoromethanesulfonates alkyl substituted at the benzene ring in aqueous media

10-Methyl-9-(phenoxycarbonyl)acridinium trifluoromethanesulfonates bearing alkyl substituents at the benzene ring were synthesized, purified, and identified. In the reaction with OOH(-) in basic aqueous media, the cations of the compounds investigated were converted to electronically excited 10-methyl-9-acridinone, whose relaxation was accompanied by chemiluminescence (CL). The kinetic constants of CL decay, relative efficiencies of light emission, chemiluminescence quantum yields, and resistance toward alkaline hydrolysis were determined experimentally under various conditions. The mechanism of CL generation is considered on the basis of thermodynamic and kinetic parameters of the reaction steps predicted at the DFT level of theory. The chemiluminescence efficiency is the result of competition of the electrophilic center at C(9) between nucleophilic substitution by OOH(-) or OH(-) and the ability of the intermediates thus formed to decompose to electronically excited 10-methyl-9-acridinone. Identification of stable and intermediate reaction products corroborated the suggested reaction scheme. The results obtained, particularly the dependency of the "usefulness" parameter, which takes into account the CL quantum yield and the susceptibility to hydrolysis, on the cavity volume of the entity removed during oxidation, form a convenient framework within which to rationally design chemiluminescent 10-methyl-9-(phenoxycarbonyl)acridinium cations.     

Solvent Effect on the Chemiluminescence of Acridinium Thioester: A Computational Study

Chemiluminescent labelling, which is one of the promising procedures of modern immunodiagnostics, is increasingly carried out using acridinium derivatives, an oxidant, and an alkaline aqueous environment. However, the efficiency of the chemiluminescence of luminol or acridinium esters is higher in non-aqueous solvents such as dimethyl sulfoxide or acetonitrile. Therefore, the search for a new environment for the chemiluminescence reaction, especially the one characterized by a higher quantum yield of chemiluminescence, is one of the aims of current research. Using computational methods (DFT and TD DFT with PCM model of solvent), we examined thermodynamic and kinetic data concerning the chemiluminescence and competitive dark pathways. Our results suggest that better characteristics of the chemiluminescence reaction of acridinium thioester are observed in nonpolar solvents, such as methylcyclohexane, n-hexane and n-pentane, than in aqueous media used so far. Further experimental verification is necessary to confirm the possible application of proposed nonpolar solvents in chemiluminescent labelling and hence in immunodiagnostics.     

Capillary electrophoresis chemiluminescent detection system equipped with a two-step postcolumn flow interface for detection of some enkephalin-related peptides labeled with acridinium ester

Despite its low equipment cost and simple design, as one of the sensitive detectors for CE, the chemiluminescence (CL) detector was less developed compared to the detectors of MS and LIF. The main reasons were the limitation of CL reagents, the repeatability problems and the relatively low sensitivity compared to LIF. In this paper, a highly sensitive CE-CL detection system was developed for detection of some enkephalin-related peptides labeled with acridinium ester. A new detection interface was designed for CE with CL detection of acridinium ester and its labeled analytes. The interface included two sections: one was used to acidify the capillary outflow so that the corresponding acridinium pseudo-base form can be changed into acridinium ester form by adding excess acid to the system; the other was designed to provide a suitable solution to produce the CL from acridinium ester. The effect factors, such as pH, the concentration of reaction reagents and the flow rates of the reagents, were investigated. The results showed that acridinium ester had similar CL properties in this interface when pH values of CE BGE were changed from 2.0 to 10.8. The interface was used to detect acridinium ester and three acridinium ester-labeled enkephalin-related peptides, the corresponding LODs were found to be in the attomole range. This CL detection system proved to be of high sensitivity, good repeatability, and relatively low cost.     

Thermally cleavable surfactants based on furan-maleimide Diels-Alder adducts

Two new surfactant molecules are reported that contain thermally labile Diels-Alder adducts connecting the hydrophilic and hydrophobic sections of each molecule. The two surfactants possess identical hydrophobic dodecyl tail segments but have phenol and carboxylic acid hydrophilic headgroups, respectively. Deprotonation with potassium hydroxide affords the formation of water-soluble surfactants. Room temperature aqueous solutions of both surfactants exhibit classical surface-active agent behavior similar to common analagous alkylaryl surfactant molecules. Critical micelle concentrations have been determined for each surfactant through dynamic surface tension and dye solubilization techniques. Small-angle neutron scattering measurements of the aqueous surfactant solutions indicate the presence of spherical micelles with radii of 16.5 angstroms for the carboxylate and 18.8 angstroms for the phenolate. When these surfactants are exposed to elevated temperatures (>50 degrees C), the retro Diels-Alder reaction occurs, yielding hydrophilic and hydrophobic fragments. Aqueous solutions of each surfactant subsequently exhibit a loss of all surface-active behavior and the micellar aggregates are no longer detectable.     

Aggregation and pH responsive disassembly of a new acid-labile surfactant synthesized by thiol-acrylate Michael addition reaction

Nucleophilic thiol-acrylate Michael reaction between a hydrophobic thiol and hydrophilic acrylate derivative generated a nonionic surfactant with acid-labile β-thiopropionate linker. Micellization of the surfactant, its ability to encapsulate hydrophobic dye, acid-induced disruption of the aggregate and pH-selective dye release profile have been revealed in this report. The micellar aggregates were found to be stable under neutral conditions, but they could be disrupted in acidic pH (5.3), and thus the encapsulated hydrophobic dye molecules could be selectively released. Appropriate control experiments revealed that the sulfur atom in the β-position is essential for acidic hydrolysis of the ester functionality of the surfactant.     

Chemiluminescence as diagnostic tool. A review

The principles of chemiluminescence and its applications as diagnostic tool are reviewed. After an introduction to the theoretical aspects of luminescence and energy transfer, the different classes of chemiluminogenic labels including luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their derivatives are described emphasizing the molecules which best fulfil the requirements of today's clinical chemistry. Applications of chemiluminescence and enhanced chemiluminescence to immunoassays, receptor assays, DNA probes, biosensors and oxygen metabolism are discussed as well as the role of enzymes in the selectivity and the sensitivity of these reactions.     

Electrochemiluminescence assay for the detection of acridinium esters

An electrochemiluminescence (ECL)-based method for rapid and sensitive detection of acridinium ester in neutral solution was described. Strong ECL emission was observed when a positive voltage over 2.0 V (versus Ag/AgCl) was applied to the working electrode (Pt) immersed in the acridinium ester solution of 2.0 mol l⁻¹ KNO₃ (pH 7.0). The possible ECL mechanism was discussed. It was proposed that the ECL emission came out of N-methylacridone, the oxidization product of acridinium ester by the nascent oxygen generated on the surface of working electrode in the course of oxidization of water. Other influenced factors including the electrochemical parameters, the ECL reaction medium and pH value, were investigated in detail. Under the optimal conditions, ECL intensity has a linear relationship with the concentration of acridinium ester in the range of 0.24-96 ng ml⁻¹ (r=0.9999). The relative standard deviation for 24 ng ml⁻¹ acridinium ester was 4.6% (n=11). The limit of detection was 0.16 ng ml⁻¹.     

Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.     

Surfactant effect on the lower critical solution temperature of poly(organophosphazenes) with methoxy-poly(ethylene glycol) and amino acid esters as side groups

 The surfactant effect on the lower critical solution temperature (LCST) of thermosensitive poly(organophosphazenes) with methoxy-poly(ethylene glycol) and amino acid esters as side groups was examined in terms of molecular interactions between the polyphosphazenes and surfactants including various anionic, cationic, and nonionic surfactants in aqueous solution. Most of the anionic and cationic surfactants increased the LCST of the polymers: the LCST increased more sharply with increasing length and hydrophobicity of the hydrophobic part of the surfactant molecule. The ΔLCSTs (T _0.03M− T _0M), the change in the LCST by addition of 0 and 0.03 M sodium dodecyl sulfate (SDS), were found to be 7.0 and 14.5 °C for the polymers bearing ethyl esters of glycine and aspartic acid, respectively. The LCST increase of poly(organophosphazene) having a more hydrophobic aspartic acid ethyl ester was 2 times larger compared with that of the polymer having glycine ethyl ester as a side group. The binding behavior of SDS to the polymer bearing glycine ethyl ester as a hydrophobic group was explained from the results of titration of the polymer solutions containing SDS with tetrapropylammonium bromide. Graphic models for the molecular interactions of polymer/surfactant and polymer/surfactant/salt in aqueous solutions were proposed. Received: 17 February 2000/Accepted: 25 April 2000     

ANALOGS AND DERIVATIVES OF FIREFLY OXYLUCIFERIN, THE LIGHT EMITTER IN FIREFLY BIOLUMINESCENCE

Abstract— The 5-methyl analog of firefly oxyluciferin, two isomeric O-methyl ether derivatives of it and an O, O Ó-dimethyl ether derivative were synthesized and their UV absorption and fluorescence emission spectra were determined. Comparisons of the emission data with the emission wavelength in bioluminescence indicate that the mono-anions of firefly oxyluciferin are candidates for the light-emitters in bioluminescence. Further, we have found that the chemiluminescence of active esters of firefly luciferin produces (from the keto form of oxyluciferin) only red light emission under a variety of conditions; a yellow-green light emission (from the enolic forms of the oxyluciferin product) could not be elicited.     

发光标记分析及其在核酸检测中的应用

综述了化学发光,生物发光,电致化学发光中最重要的发光体系,增强鲁米诺体系,吖啶酯发光体系,碱性磷酸酶／1,2－二氧环乙烷体系,荧火虫荧光素／荧光素酶及联吡啶钌发光体系的原理,标记检测方法,最近的进展及在核酸领域的应用,对近年来新发展的发光标记方法作了介绍并分析了该领域今后可能的研究方向。     

Photoreversible DNA condensation using light-responsive surfactants

A means to control DNA compaction with light illumination has been developed using the interaction of DNA with a photoresponsive cationic surfactant. The surfactant undergoes a reversible photoisomerization upon exposure to visible (trans isomer, more hydrophobic) or UV (cis isomer, more hydrophilic) light. As a result, surfactant binding to DNA and the resulting DNA condensation can be tuned with light. Dynamic light scattering (DLS) measurements were used to follow lambda-DNA compaction from the elongated-coil to the compact globular form as a function of surfactant addition and light illumination. The results reveal that compaction occurs at a surfactant-to-DNA base pair ratio of approximately 7 under visible light, while no compaction is observed up to a ratio of 31 under UV light. Upon compaction, the measured diffusion coefficient increases from a value of 0.6 x 10(-8) cm2/s (elongated coil with an end-to-end distance of 1.27 microm) to a value of 1.7 x 10(-8) cm2/s (compact globule with a hydrodynamic radius of 120 nm). Moreover, the light-scattering results demonstrate that the compaction process is completely photoreversible. Fluorescence microscopy with T4-DNA was used to further confirm the light-scattering results, allowing single-molecule detection of the light-controlled coil-to-globule transition. These structural studies were combined with absorbance and fluorescence spectroscopy of crystal violet in order to elucidate the binding mechanism of the photosurfactant to DNA. The results indicate that both electrostatic and hydrophobic forces are important in the compaction process. Finally, a DNA-photosurfactant-water phase diagram was constructed to examine the effects of both DNA and surfactant concentration on DNA compaction. The results reveal that precipitation, which occurs during the latter stages of condensation, can also be reversibly controlled with light illumination. The combined results clearly show the ability to control the interaction between DNA and the complexing agent and, therefore, DNA condensation with light.