Enzyme-amplified Lanthanide Luminescence Research Articles

Development of a Fluorescent Method for Determining Esterase Activity with Terbium/BAPTA-AM System

Enzyme-amplified lanthanide luminescence (EALL) is a promising method which has been developed for enzymatically amplified signal detection. A signal is generated by enzymatically transforming a substrate into a product, which forms a fluorescent lanthanide chelate. With the aim of establishing a new EALL detection system, we focused on O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (BAPTA), which is known to be a specific chelator for calcium. BAPTA also forms some energy-transfer fluorescent complexes with lanthanide ions at physiological pH, and functions as a good sensitizer, especially for terbium(III) ion. Thus, we examined whether esterase activity can be determined by EALL detection where BAPTA and its tetraacetoxymethyl ester (BAPTA-AM) are used as the Tb(III) chelate and the reaction substrate, respectively. Regarding the rate of the Tb(III) fluorescent intensity changing per unit time (ΔI/Δt) as an initial rate of the enzymatic reaction, the determination of esterase activity was successful in the range of 0.010∼0.10 u/mL. Within this work, the suitability of the Tb(III)/BAPTA-AM system for EALL detection has been proven for determining esterase activity.

Lysozyme-Enhanced Europium(III)-Metacycline Luminescence and Its Application to the Determination of Metacycline

A new spectrofluorometric method is described for the determination of metacycline (MC), based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu3+-MC forms a ternary complex with lysozyme in close proximity. Then lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu3+ at 612 nm in metacycline-Eu3+ binary complex. The enhanced fluorescence intensity is in proportion to the concentration of MC. The limit of detection is 1.6 x 10(-8) mol L(-1), with a linear range from 6.2 x 10(-6) to 1.7 x 10(-5) mol L(-1). Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of MC in serum and urine samples. The mechanism of fluorescence enhancement was also studied.

Detection scheme for bioassays based on 2,6-pyridinedicarboxylic acid derivatives and enzyme-amplified lanthanide luminescence

2,6-Pyridinedicarboxylic acid (PDC) and its derivatives are introduced as a new sensitizer system for enzyme-amplified lanthanide luminescence (EALL), a detection scheme for bioassays, which combines enzymatic amplification with time-resolved luminescence measurements of lanthanide chelates. Various PDC esters have been synthesized as esterase substrates that are cleaved to PDC in the presence of the enzyme. PDC forms luminescent complexes with Tb(III) or Eu(III), and the evaluation of the reaction is used for the selective and sensitive detection of esterases. For an esterase from hog liver a limit of detection of 10 −3 u/mL (equivalent to 10 −9 mol/L) and a limit of quantification of 3 × 10 −3 u/mL (equivalent to 3 × 10 −9 mol/L) could be achieved. As a second model reaction, xanthine oxidase (XOD) catalyzes the oxidation of 2,6-pyridinedicarboxaldehyde to PDC. Here, the limit of detection was 3 × 10 −3 u/mL and the limit of quantification 10 −2 u/mL for XOD from microorganisms. Major advantage of the tridentate PDC ligand is the possibility to perform all steps of the assay within or close to the physiological pH range, while the established EALL schemes based on bidentate salicylates or bisphenols have to be carried out at strongly alkaline pH to ensure sufficient complexation with the lanthanides.

Detection strategies for bioassays based on luminescent lanthanide complexes and signal amplification.

Two attractive detection strategies for bioassays are reviewed in this article. Both approaches use the highly sensitive time-resolved luminescence detection of lanthanide complexes in combination with a signal amplification scheme. While enzyme-amplified lanthanide luminescence (EALL) has been an established technique for more than a decade, nanoparticles doped with luminescent lanthanide complexes have been introduced very recently. In this paper, the basic properties and major applications of both techniques are presented, and their future perspectives are discussed critically.

Enzyme-amplified lanthanide luminescence based on complexation reaction—a new technique for the determination of doxycycline

A new spectrofluorimetric method is described for the determination of doxycycline, based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu 3+–doxycycline forms a ternary complex with lysozyme in close proximity and lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu 3+ at 612 nm in doxycycline–Eu 3+ binary complex. The enhanced fluorescence intensity is in proportion to the concentration of doxycycline. The limit of detection is 1.28×10 −8 mol l −1, with a linear range from 1.7×10 −7 to 1.7×10 −6 mol l −1. Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of doxycycline in serum, urine and real samples. The mechanism of fluorescence enhancement was also studied.

A tripod ligand as new sensitiser for the enzyme amplified lanthanide luminescence determination of esterase.

Screening of a small library of tripod ligands resulted in the discovery of bis(2-pyridylmethyl)-(2-hydroxybenzyl)amine (HL1) as a new sensitiser, which is able to transfer its excitation energy to terbium(III). After synthesis of the acetic acid ester of HL1, a highly selective method for the determination of porcine liver esterase by means of enzyme amplified lanthanide luminescence (EALL) was developed. Enzyme-catalysed cleavage of the ester results in the formation of HL1. After excitation at 297 nm, the characteristic emission of Tb(III) at 545 nm is observed and used to determine the esterase concentration. In contrast to existing EALL methods, this method may be carried out at neutral pH and without further additives. Limit of detection for porcine liver esterase is 10(-9) mol l(-1) and limit of quantification is 3 x 10(-9) mol l(-1). A linear calibration range of two decades starting at the limit of quantification is observed.

Enzyme-linked immunosorbent assays based on peroxidase labels and enzyme-amplified lanthanide luminescence detection.

The enzyme-amplified lanthanide luminescence (EALL) detection is developed and applied for the determination of peroxidase as marker in enzyme-linked immunosorbent assays (ELISA). The detection scheme is based on the peroxidase catalysed dimerization of 4-hydroxyphenylpropionic acid (pHPPA) and the subsequent formation of a ternary complex with Tb(III)EDTA. Quantum yields and fluorescence lifetimes of the luminescent species are presented to give an estimate of the potential of this procedure. Two different ELISA were performed with the EALL detection scheme. For the first, a model ELISA for the determination of goat anti-rabbit IgG, a limit of determination of 3 micrograms dm-3 (2 fmol) of the antibody could be achieved. As second model assay, a commercial ELISA kit was successfully validated for the new detection scheme. Photometric and EALL detection were in good agreement for the determination of human anti-gliadin IgA in serum.

Peroxidase enhanced lanthanide luminescence—a new technique for the evaluation of bioassays

A new technique for the enzyme-amplified lanthanide luminescence (EALL) quantification of peroxidase in bioassays is introduced. Several phenolic peroxidase substrates may be used. These are dimerised in the course of the enzymatic reaction and subsequently form luminescent ternary complexes with terbium(III) EDTA. The luminescence signals are enhanced by addition of CsCl and can be read out in a time resolved type of measurement. With the substrate p-hydroxyphenylpropionic acid, a detection limit for horseradish peroxidase of 2 × 10−12 mol dm−3 could be achieved.

Determination of tumor necrosis factor-α (TNF-α) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay

Objectives: To develop a highly sensitive enzyme amplified lanthanide luminescence (EALL) immunoassay for tumor necrosis factor-α (TNF-α). Methods: The method is based on the use of two monoclonal antibodies against TNF-α, one “capture” antibody and one labeled with biotin, in a “sandwich type” assay format. Alkaline phosphatase (ALP) conjugated to an antibiotin-polyclonal antibody is used as the enzyme label. ALP cleaves phosphate from diflunisal phosphate (DIFP) to produce diflunisal (DIF). The detection system is based on the combination of enzymatic amplification introduced by ALP and the formation of a highly fluorescent terbium complex that can be monitored by time resolved or conventional fluorimetry. Results: By using 50 μL of sample, the dynamic range of the assay extends up to 2000 ng/L of TNF-α, with a detection limit of 1 ng/L, within-run CVs ranging from 3 to 15% and recoveries of 97 ± 2%. By using 100 μL of sample the dynamic range of the assay extends up to 1000 ng/L of TNF-α with a detection limit of 0.2 ng/L, recoveries of 94 ± 13%, within-run CVs ranging from 2 to 6.5% and between-run CVs ranging from 5 to 15%, in a total incubation time of 3h. No interference by the presence of other cytokines (IL-1β, IL-2, IL-4, IL-6, IFN-γ) or by rheumatoid factors has been observed. The results obtained by the proposed method and by a commercially available kit (Medgenix TNF-α EASIA) correlated well (n = 26, r = 0.934). Conclusion: The proposed method is highly sensitive, simple and rapid and can reliably measure TNF-α in the ng range in biological specimens.

A Highly Sensitive Enzyme-amplified Lanthanide Luminescence Immunoassay for Interleukin 6

Interleukin 6 (IL-6) is a cytokine that is produced by a wide variety of cells and has pleiotropic biological functions, such as the induction of acute phase proteins in hepatocytes, the terminal differentiation and growth promotion of B cells, the differentiation and activation of T cells and macrophages, and the regulation of expression of other cytokines (1). IL-6 has been implicated in the pathology of several diseases, and its role in inflammation, viral infection, autoimmunity, and cancer has been reviewed recently (1)(2). Serum IL-6 increases markedly in many pathological conditions, and its accurate and precise determination in biological fluids is of great importance for the early diagnosis of many diseases (3)(4)(5)(6)(7). Measurement of IL-6 in biological fluids is mainly based on bioassays and immunoassays (8)(9)(10)(11)(12)(13). The presence of natural compounds known to bind IL-6 in biological fluids (14)(15)(16)(17)(18) and the effect of preanalytical factors (19), as well as the use of an internationally accepted IL-6 standard preparation (20)(21), is of great importance for the performance of IL-6 immunoassays. Here we describe a highly sensitive enzyme immunoassay for the determination of IL-6 in serum and plasma, based on the enzyme-amplified lanthanide luminescence (EALL) detection approach (22)(23)(24)(25). An ultra-low detection limit is obtained by the powerful detection system that is based on the combination of enzymatic amplification introduced by the enzyme alkaline phosphatase (ALP) and the formation of a highly fluorescent terbium complex (26)(27), which is monitored by time-resolved or conventional fluorometry. A monoclonal antibody (anti-IL-6, CLB IL-6/16) and a biotinylated goat polyclonal antibody against human IL-6 were kindly donated by Prof. L. …

Comparative study of fluorescent ternary terbium complexes. Application in enzyme amplified fluorimetric immunoassay for α-fetoprotein

A systematic spectrofluorimetric study of the formation of ternary complexes of terbium ions (Tb 3+) with ethylenediamine tetraacetic acid (EDTA) and a variety of bidentate organic ligands has been performed. Fourteen ligands (salicylic acid, quinolones and hydroxybenzene sulfonic acid derivatives) were examined as efficient energy donating chelators for Tb 3+. The fluorescence properties of the ligands, alone as well as of the ternary complexes with Tb 3+ and EDTA have been examined. Optimization studies, including the effect of pH, buffer systems and the use of EDTA analogs, were performed. A possible relationship between the structure and the ability of the ligand to act as an efficient light absorbing and energy donating chelator for Tb 3+ is discussed. The phosphate ester of diflunisal, one of the most sensitive ligands for Tb 3+, was used as an alkaline-phosphatase substrate for the determination of α-fetoprotein (AFP) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay by means of second derivative synchronous fluorescence spectroscopy as a detection technique. By using this substrate, AFP was determined in serum with a limit of detection (3 × s.d. blank) of 5 pg ml −1, coefficients of variation in the 2.4–5.3% range and mean recovery from four pooled serum samples at four concentration levels (5, 40, 100 and 250 ng ml −1) equal to (99 ± 3)%.

Time-resolved detection of lanthanide luminescence for ultrasensitive bioanalytical assays

The principles and practice of the application of time-resolved lanthanide chelate luminescence (or fluorescence) as a detection method for ultrasensitive bioanalytical assays such as immunoassays and nucleic acid hybridization assays are reviewed. The various lanthanide chelate-based detection systems which have been developed for use in heterogeneous and homogeneous assay formats are described, including reagents, assay methods, and instrumentation, along with recent improvements in these methods. Detection systems described include those based on dissociative enhancement of lanthanide ions, direct labeling with luminescent chelates, enzyme-amplified lanthanide luminescence, lanthanide luminescence quenching, and energy transfer.

Second derivative synchronous scanning fluorescence spectrometry as a sensitive detection technique in immunoassays. Application to the determination of α-fetoprotein

Second derivative synchronous (scanning) fluorescence spectrometry (SDSFS) has been used for the first time as an alternative to time-resolved fluorescence for the detection of Tb 3+ chelates. This approach minimizes the background signal by taking advantage of the large Stokes shift properties of Tb 3+ chelates and offers high sensitivity by narrowing the spectral bands. The analytical performance of this detection technique has been evaluated by choosing the well-defined enzyme-amplified lanthanide luminescence (EALL) immunoassay of α-fetoprotein (AFP) as a model. Monoclonal “capture” antibodies and monoclonal biotin-labelled antibodies in a “sandwich-type” assay configuration in a microwell format have been used. Alkaline phosphatase (ALP) conjugated to an antibiotin antibody was used as an enzyme label. ALP cleaves phosphate from salicylphosphate to produce salicylic acid which forms a highly fluorescent ternary complex with Tb 3+ and EDTA, which is monitored by SDSFS. The method allows the measurement of AFP with a limit of detection of 2 pg ml −1, coefficients of variation in the range 4.5–9.9% and mean recovery from four pooled serum samples at two concentration levels equal to 94 ± 11%.

Enzyme-amplified lanthanide luminescence for detection of enzyme labels in ultrasensitive bioanalytical assays

Enzyme-amplified lanthanide luminescence for detection of enzyme labels in ultrasensitive bioanalytical assays

Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays

A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.

Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays

Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications.