Chemiluminescence from alkoxy-substituted acridinium dimethylphenyl ester labels

Chemiluminescent acridinium dimethylphenyl ester labels are used in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered by alkaline peroxide in the presence of the cationic surfactant cetyltrimethylammonium chloride (CTAC). The surfactant plays a critical role in the chemiluminescence process of these labels by both accelerating their emission kinetics and increasing total light output enabling high throughout and improved assay sensitivity in automated immunoassays. Despite the surfactant's crucial role in the chemiluminescent reaction, no study has investigated how structural perturbations in the acridinium ring could impact the influence of the surfactant. We describe herein the synthesis and properties of three new alkoxy-substituted, acridinium dimethylphenyl esters where the nature of the alkoxy group in the acridinium ring was varied (hydrophobic or hydrophilic). Chemiluminescence measurements of these alkoxy-substituted labels indicate that hydrophilic functional groups in the acridinium ring, in particular sulfobetaine zwitterions, disrupt surfactant-mediated compression of emission times but not enhancement of light yield. These results support the hypothesis that surfactant-mediated effects require the binding of two different reaction intermediates to surfactant aggregates and, that surfactants influence light emission from acridinium esters by two separate mechanisms. Our studies also indicate that preservation of both surfactant effects on acridinium ester chemiluminescence and low non-specific binding of the label can be achieved with a relatively hydrophobic acridinium ring coupled to a hydrophilic phenolic ester leaving group.     

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.     

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.     

Chemiluminogenic labels,old and new

The essential features for successful chemiluminogenic labels immunoassays are discussed. After reviewing existing labels, some new acridinium labels are described.     

化学发光免疫分析试剂吖啶酯的合成新路线

设计了一种化学发光免疫分析试剂吖啶酯的合成新路线,以二苯胺为原料合成吖啶-9-羧酸,经酰氯化后与3-(4-羟基苯基)-丙酸-N-羟基琥珀酰亚胺酯反应得4-(2-琥珀酰亚胺基氧羰基乙基)苯基-9-吖啶羧酸酯,最后与氟磺酸甲酯反应即得4-(2-琥珀酰亚胺基氧羰基)苯基-10-甲基吖啶-9-羧酸酯氟磺酸盐(俗称吖啶酯).     

Flow-Injection immunoassays with acridinium ester-based chemiluminescence detection

A type of flow-injection immunoassay has been developed that makes use of acridinium ester-labelled antibodies for detection. The entire assay, including detection, takes place in a transparent 20-μl immunoreactor containing antibodies covalently immobilized to a rigid beaded support, Pierce Trisacryl GF-2000. An assay for mouse IgG in which the immunoreactor is prepared by the immobilization of anti-mouse IgG is presented as an example. A two-site immunoassay is accomplished by the consecutive injection of the sample, acridinium ester-labelled antibodies and alkaline hydrogen peroxide to initiate chemiluminescence. The light emission is collected directly from the transparent immunoreactor, which is then regenerated in preparation for the next sample injection. The detection sensitivity and time required per sample are dependent on the assay flow-rate and for times of 10, 12 and 18 min per sample the limits of detection are 500, 200 and 50 amol of mouse IgG, respectively. The precision of replicate measurements has a relative standard deviation of 3–4%, and the same 20- μl immunoreactor can be used for at least 1 week with an inter-assay relative standard deviation of 5.9%.     

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L⁻¹ and 1.3 ng L⁻¹ using two different up-converting phosphors and 0.16 ng L⁻¹ using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.     

Selective interactions of a few acridinium derivatives with single strand DNA: study of photophysical and DNA binding interactions

Novel acridinium derivatives 1-3, wherein steric factors have been varied systematically through substitution at the ninth position of the acridinium ring, were synthesized and their interactions with single strand and double strand DNA have been investigated through photophysical, biophysical, and microscopic techniques. The acridinium derivative 1 exhibited quantitative fluorescence yields (phi f approximately =1) and high lifetime of 35 ns, while significantly lower fluorescence yields of 0.11 and 0.02 and lifetimes of 3.5 and 1.2 ns were observed for 2 and 3, respectively. The derivatives 1 and 2 having 2-methylphenyl and 2,4-dimethylphenyl substituents at the ninth position of the acridinium ring showed selective interactions with single strand DNA (ssDNA) with association constants of KssDNA = 6.3-6.6 x 10(4) M(-1), while negligible interactions were observed with double strand DNA (dsDNA). In contrast, the derivative 3 with 2,6-dimethylphenyl substitution showed negligible interactions with both ssDNA and dsDNA. Studies with a series of 19-mer oligonucleotides indicate that these derivatives exhibit significant selectivity for the sequences rich in guanosine (ca. 3-fold) as compared to the cytosine-rich sequences. These derivatives with high water solubility and the ability to distinguish between ssDNA and dsDNA through changes in fluorescence emission can be used as fluorescent probes for understanding the role of ssDNA in various biological processes and to study various DNA-ligand interactions.     

时间分辨荧光免疫分析法间接测定雌二醇

以氯磺酰基噻吩甲酰三氟丙酮（CTTA）为铕（Eu)的螯合剂,羊抗鼠（SAM）的IgG为二抗,用SAM－IgG-CTTA-Eu作标记二抗,建立了以竞争抑制为基础的时间分辨荧光免疫分析测定离雌二醇（E2）的新方法。同均相方法相比灵敏度有很大提高,测定雌二醇（E2）的线性范围为2．5－200pg/mL,检测限为2.5pg/mL。这一方法可望用于E2的临床检测。     

Immunoassays for aflatoxins

Immunoassays for aflatoxin analysis have been regarded as valuable supplements to existing and rapidly developing chromatographic techniques. We describe six types of aflatoxin immunogens and their characteristics, reported antibodies against aflatoxins, traditional and novel labeled materials for assay signaling, three immunoassay formats, assay devices (e.g., microtiter plate and reader, lateral flow strip, electronic and optical immunosensors, and a rapid tester dedicated to aflatoxins) and applications of immunoassay in agricultural products. We show trends towards sensitivity, simplification, intelligence and portability. After setting out five challenges in developing immunoassays, we predict that techniques involving novel nanoparticle labels and non-competitive assay may become the main trends in research and that immunoassay devices will be used in many fields.     

Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T₄). In this technique, a sample containing thyroxine was first combined with an excess of anti-T₄ antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T₄. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T₄ in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.     

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.     

Photoswitchable calix[4]arenes bearing dihydroacridine substituents at the upper rim

Eight new calix[4]arene host molecules bearing acridinium or the corresponding dihydroacridine substituents have been prepared. ¹H NMR and electrochemical studies reveal that the acridinium substituents block the host cavity. Both photochemical and chemical switching between acridinium and dihydroacridine hosts are demonstrated.     

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.     

Nafion膜修饰无分离步骤的IgM电化学免疫传感器研究

发展了一种基于Nation膜修饰电极构制的无分离步骤的异相免疫分析法,羊抗人IgM抗血清和邻氨基酚通过Nation膜固定在电极表面,形成生物敏感膜。采用竞争吸附免疫分析法,以HRP—IgM作标记物,只有结合在生物敏感膜的酶标物才能催化邻氨基酚与H2O2的反应,产生电分析信号,从而与游离在溶液里的酶标物区分开。因此分析过程勿需洗脱步骤,简化了分析过程,在优化的条件下,对IgM的检测范围为0．07～1．8mg／L。该分析技术操作简单、分析速度快。     

Some new acridinium trifluoromethanesulfonates as spectrophotometric derivatization reagents for aromatic and aliphatic primary amines

Some new 9-substituted 10-methylacridinium trifluoromethanesuifonate (triflate) salts have been synthesized and shown to react in methanol with the model aromatic and aliphatic amines, aniline and n-butylamine, to form derivatives which absorb strongly at 445 and 439 nm, respectively. The color development is affected by heat and heating time and by the quantity of acridinium triflate used. A 10-50-fold molar excess of the triflate should be used and the solution heated at 60 degrees for 30 min. The linearity and reproducibility of the assay are improved by the presence of pyridine (for aniline) and triethylamine (for n-butylamine) in the reaction mixture. Beer's law is obeyed over the range 0-1860 ng/ml for aniline and 0-1440 ng/ml for n-butylamine, with each of the new reagents. The relative error and the precision of determination depends on the acridinium triflate used.     

Electron-Poor Acridones and Acridiniums as Super Photooxidants in Molecular Photoelectrochemistry by Unusual Mechanisms

Electron-deficient acridones and in situ generated acridinium salts are reported as potent, closed-shell photooxidants that undergo surprising mechanisms. When bridging acyclic triarylamine catalysts with a carbonyl group (acridones), this completely diverts their behavior away from open-shell, radical cationic, ‘beyond diffusion’ photocatalysis to closed-shell, neutral, diffusion-controlled photocatalysis. Brønsted acid activation of acridones dramatically increases excited state oxidation power (by +0.8 V). Upon reduction of protonated acridones, they transform to electron-deficient acridinium salts as even more potent photooxidants (*E_1/2=+2.56–3.05 V vs SCE). These oxidize even electron-deficient arenes where conventional acridinium salt photooxidants have thusfar been limited to electron-rich arenes. Surprisingly, upon photoexcitation these electron-deficient acridinium salts appear to undergo two electron reductive quenching to form acridinide anions, spectroscopically-detected as their protonated forms. This new behaviour is partly enabled by a catalyst preassembly with the arene, and contrasts to conventional SET reductive quenching of acridinium salts. Critically, this study illustrates how redox active chromophoric molecules initially considered photocatalysts can transform during the reaction to catalytically active species with completely different redox and spectroscopic properties.     

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.