Fluorescence quenching competitive immunoassay in micro droplets

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-microm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 x 512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen-antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.     

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.     

Fluorescence quenching studies with proteins

A review is presented on the use of the technique of solute fluorescence quenching to study the structure and dynamics of proteins. A number of factors are discussed that must be considered in analyzing such data. Among these factors are the efficiency of the quenching process, the relative importance of static quenching, the heterogeneity of the emission, and the tendency of the quencher to interact with the protein.     

Fluorescence quenching studies on binding fluoreno-9-spiro-oxazolidinedione to human serum albumin.

Human serum albumin fluorescence quenching by fluorene-9-spiro-oxazolidinedione has been analyzed as a function of temperature. Such temperature dependence suggests that the mechanism of the quenching process is static in origin. This type of quenching implies that a non-fluorescent complex between oxazolidinedione and serum albumin has been formed and following the Stern-Volmer relationship we have calculated the binding constant. Thermodynamic parameters were also determined. The positive and large values of entropy and the negative value for enthalpy suggest that both hydrophobic and electrostatic interactions may play an important role in the stabilization of the complex. Finally, the irreversible changes in the spectral properties of HSA are interpreted in binding terms.     

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol^?1 and 1.9·10⁹ 1·mol^?1·s^?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol^?1 and 092·10⁹ 1·mol^?1·^?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.     

Fluorescence quenching studies of Trp repressor-operator interaction

Steady-state quenching and time-resolved fluorescence measurements of L-tryptophan binding to the tryptophan-free mutant W19/99F of the tryptophan repressor of Escherichia coli have been used to observe the coreperessor microenvirnment changes upon ligand binding. Using iodide and acrylamide as quenchers, we have resolved the emission spectra of the corepressor into two components. The bluer component of L-tryptophan buried in the holorepressor exhibits a maximum of the fluorescence emission at 336 nm and can be characterized by a Stern–Volmer quenching constant equal to about 2.0–2.3 M^–1. The second, redder component is exposed to the solvent and possesses the fluorescence emission and Stern–Volmer quenching constant characteristic of L-tryptophan in the solvent. When the Trp holorepressor is bound to the DNA operator, further alterations in the corepressor fluorescence are observed. Acrylamide quenching experiments indicate that the Stern–Volmer quenching constant of the buried component of the corepressor decreases drastically to a value of 0.56 M^–1. The fluorescence lifetimes of L-tryptophan in a complex with Trp repressor decrease substantially upon binding to DNA, which indicates a dynamic mechanism of the quenching process.     

Fluorescence polarization immunoassay employing immobilized antibody

The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is 10(-10) mol l-1 and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time.     

Fluorescence quenching studies of nitrated polycyclic aromatic hydrocarbons

The analysis of nitrated polycyclic aromatic hydrocarbons (NPAHs) is of great importance because of the mutagenicity and possible carcinogenic activity of these compounds, which are distributed widely in the environment. Nitro‐substituents in aromatic compounds are known to quench fluorescence and NPAHs have no intrinsic fluorescence, but they can be determined using their quenching effects on other fluorophores. The quenching effects of several important NPAHs on 1,2,3,4‐ tetrahydro‐1‐naphthol,5,6,7,8‐tetrahydro‐1‐naphthol,4‐(2‐hydroxy‐4‐sulfo‐1‐naphthylazo)‐2‐naphthalene carboxylic acid and 7‐amino‐4‐methyl coumarin have been studied. The singlet emission of these fluorophores is efficiently quenched by all the NPAHs, the quenching following the Stern–Volmer relationship. Quenching constants and the limits of detection and linear ranges of the quenchers have been determined in each case: the limits of detection are ca 1 µm . Copyright © 2010 John Wiley & Son, Ltd.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

Fluorescence polarization competition immunoassay for tyrosine kinases

To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used (32)PO(4) transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the (32)PO(4) transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3, 4-d]pyrimidine in these two FP assays was similar to that obtained in the (32)PO(4) transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.     

Simultaneous enzyme immunoassay of cortisol and dehydroepiandrostone-sulphate from a single serum sample using a transferable solid phase system

An enzyme immunological methodology for the direct and simultaneous estimation of serum cortisol and dehydroepiandrosterone-sulphate (DHEA-S) useful for the biochemical differential diagnosis of Cushing's syndrome has been developed. The combined estimation of both steroids is more economical in time, work and materials than two separate assays. Two solid phases, a microtitre plate and a covering transferable needle lid system were used in the present procedure. Both solid phases are first coated with anti-rabbit IgG and then each with a specific antiserum. Horseradish peroxidase was used as marker enzyme and tetramethylbenzidine as the chromogen for measuring enzyme activity. No extraction or deproteinization steps are involved. The turn around time for 41 samples (in duplicate) is 3 h. The detection limit of the assay is 5 pg/well for cortisol and 10 pg/well for DHEA-S. Results of the present method correlated well (cortisol, r = 0.95; DHEA-S, r = 0.98) with those of commercial radioimmunoassays using iodinated labels. Thus, this technique offers a convenient non-isotopic procedure in the routine clinical laboratory.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Fluctuation domains in myoglobin. Fluorescence quenching studies

The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenching of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal-free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns).     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Enzyme immunoassay of serum testosterone.

An enzyme immunoassay of serum testosterone using the testosterone-glucoamylase complex is described. Testosterone was estimated by the enzyme immunoassay after extraction with hexan: ether (4:1) for serum from men and additional thin layer chromatographic step for serum from women. The within and between assay errors, measured as the coefficient of variation were 11.1 percent (n=8) and 12.0 percent (n=12). The sensitivity of this assay was 0.25 ng. The mean testosterone concentration (+/- SD) in 19 normal men and 4 normal cycling women were 5.3 +/- 1.8 and 0.52 +/- 0.12 ng/ml, respectively. The level of testosterone found by the present assay compared favorably with those obtained by other methods.