Time-resolved fluoroimmunoassay of progesterone by Eu-labelled protein-A

Summary A standard curve for progesterone using a solid phase time-resolved fluoroimmunoassay was obtained. The 11-hydroxyprogesterone hemisuccinate bonded to ovalbumin was adsorbed to the wells of polystyrene microtiter strips. After incubation of scalar quantities of free progesterone (from 12 to 5000 pg) with specific antibody and an excess of Eu-labelled protein-A, the wells were washed and the europium bound to the solid phase was measured as a 2-naphthoyltrifluoroacetone chelate using a time resolved fluorometer.

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Zeitaufgelöster Fluorescenzimmunoassay von Progesteron mit Hilfe von Eu-markiertem Protein-A

     

Time-resolved fluoroimmunoassay for ractopamine in swine tissue

This study describes the development and validation of a time-resolved fluoroimmunoassay (TR-FIA) for screening ractopamine (RAC) in swine tissue. The method is based on the direct competitive-type immunoassay using europium-labeled anti-RAC monoclonal antibody as a tracer and RAC–ovalbumin as a solid-phase antigen. When RAC was spiked at levels of 1–10 μg kg⁻¹, recoveries ranged from 88.2 to 118.5% for swine liver and muscle with coefficients of variation from 7.1 to 20.5%. The detection limit was 0.1 μg kg⁻¹. The proposed TR-FIA method was applied to the determination of RAC in an actual residue study and the applicability was confirmed by liquid chromatography–tandem mass spectrometry.     

Time-resolved fluoroimmunoassay for 19-nortestosterone residues in aquaculture tissues.

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of 19-nortestosterone (17beta-NT) residues in aquaculture tissues. The limit of detection (LOD) was determined to be 0.08 ng g-1 and the limit of quantification (LOQ) was less than 0.8 ng g-1. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS). This proposed technique could be applied to routine residue analysis.     

Time-resolved fluoroimmunoassay for quantitative determination of ampicillin in cow milk samples with different fat contents

In this paper, we have reported an immunoassay with time-resolved revelation system for ampicillin in raw milk samples. Immunological methods appear to be a promising approach in the analysis of β-lactam compounds, because they do not need previous sample pre-treatments. In fact, β-lactam ring is not very stable in extensive sample pre-treatment procedures requested in conventional analytical techniques. Specimens were collected from lactating cows bred in various conditions and assayed for the fat contents. Ampicillin was assayed in samples with different fat concentrations. The assay was performed using ampicillin-specific polyclonal antibody raised in rabbit; the immunogen was synthesized using bovine thyroglobulin conjugated to ampicillin by glutaraldehyde reaction; as fluorescent marker we used goat anti-rabbit IgG conjugated with a chelating molecule complexed with Eu³⁺. Bovine serum albumin (BSA) conjugated with ampicillin was synthesized and used to prepare a solid phase on polystyrene microtiter plates. The use of a lanthanide chelate as label allowed to achieve 1 ng mL⁻¹ sensitivity, which is four times more sensitive than limits requested from European Community. Fat contents did not affect the assay performance.     

Time-resolved electrochemiluminescence of platinum(II) coproporphyrin

Cathodic pulse polarisation of oxide-covered aluminium electrodes can generate electrochemiluminescence (ECL) from metalloporphyrins. This is based on the tunnel emission of hot electrons into aqueous electrolyte solution, which probably results in the generation of hydrated electrons as reducing mediators. These tunnel emitted electrons allow the production of highly reactive radicals, such as sulfate radicals from peroxodisulfate ions, which can induce strong redox luminescence from various organic chemiluminophores including metalloporphyrins. The work presented here illustrates the generation of ECL from platinum(II) coproporphyrin (PtCP) and its bovine serum albumin (BSA) conjugate. This allows the detection of these molecules below nanomolar concentrations and over several orders of magnitude of concentration. The relatively long luminescence lifetime of PtCP allows discrimination from the background ECL signal using time resolved measurements, leading to higher sensitivity and the detection of PtCP-BSA indicates the potential use of metalloporphyrins as labels in ECL-based bioassays such as immunoassays and DNA-binding assays.     

Time-resolved photoion and photoelectron imaging of NO(2)

Time-resolved photoion and photoelectron velocity mapped images from NO(2) excited close to its first dissociation limit [to NO(X(2)Pi) + O((3)P(2))] have been recorded in a two colour pump-probe experiment, using the frequency-doubled and frequency-tripled output of a regeneratively amplified titanium-sapphire laser. At least three processes are responsible for the observed transient signals; a negative pump-probe signal (corresponding to a 266 nm pump), a very short-lived transient close to the cross-correlation of the pump and probe pulses but on the 400 nm pump side, and a longer-lived positive pump-probe signal that exhibits a signature of wavepacket motion (oscillations). These transients have two main origins; multiphoton excitation of the Rydberg states of NO(2) by both 266 and 400 nm light, and electronic relaxation in the 1(2)B(2) state of NO(2), which leads to a quasi-dissociated NO(2) high in the 1(2)A(1) electronic ground state and just below the dissociation threshold. The wavepacket motion that we observe is ascribed to states exhibiting free rotation of the O atom about the NO moiety. These states, which are common for loosely bound systems such as a van der Waals complex but unusual for a chemically-bound molecule, have previously been observed in the frequency domain by optical double resonance spectroscopy but never before in the time domain.     

Time-resolved fluorescence spectroscopy of polystyrene and poly(1-vinylnaphthalene)

An apparatus is described which enables the rapid recording of time-resolved emission spectra on a nanosecond time scale. The instrument permits detailed study of the time-dependent photo-physical processes occurring in many polymer systems. The technique has been used to investigate the kinetics of excimer formation in methylene chloride solutions of polystyrene and poly(1-vinyl-naphthalene). Unambiguous evidence for excimer dissociation is observed in poly(1-vinylnaphthalene) but the process appears unimportant in solutions of polystyrene.     

Time-resolved study of solvent-induced recombination in photodissociated IBr-(CO2)n clusters

We report the time-resolved recombination of photodissociated IBr-(CO2)n (n = 5-10) clusters following excitation to the dissociative IBr-A' 2Pi12 state of the chromophore via a 180 fs, 795 nm laser pulse. Dissociation from the A' state of the bare anion results in I- and Br products. Upon solvation with CO2, the IBr- chromophore regains near-IR absorption only after recombination and vibrational relaxation on the ground electronic state. The recombination time was determined by using a delayed femtosecond probe laser, at the same wavelength as the pump, and detecting ionic photoproducts of the recombined IBr- cluster ions. In sharp contrast to previous studies involving solvated I2-, the observed recombination times for IBr-(CO2)n increase dramatically with increasing cluster size, from 12 ps for n = 5 to 900 ps for n = 8,10. The nanosecond recombination times are especially surprising in that the overall recombination probability for these cluster ions is unity. Over the range of 5-10 solvent molecules, calculations show that the solvent is very asymmetrically distributed, localized around the Br end of the IBr- chromophore. It is proposed that this asymmetric solvation delays the recombination of the dissociating IBr-, in part through a solvent-induced well in the A' state that (for n = 8,10) traps the evolving complex. Extensive electronic structure calculations and nonadiabatic molecular dynamics simulations provide a framework to understand this unexpected behavior.     

Time-resolved photoelectron imaging of large anionic methanol clusters: (methanol)n-(n approximately 145-535)

The dynamics of an excess electron in size-selected methanol clusters is studied via pump-probe spectroscopy with resolution of approximately 120 fs. Following excitation, the excess electron undergoes internal conversion back to the ground state with lifetimes of 260-175 fs in (CH3OH)n- (n=145-535) and 280-230 fs in (CD3OD)n- (n=210-390), decreasing with increasing cluster size. The clusters then undergo vibrational relaxation on the ground state on a time scale of 760+/-250 fs. The excited state lifetimes for (CH3OH)n- clusters extrapolate to a value of 157+/-25 fs in the limit of infinite cluster size.     

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1 Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was log Y = 3.65786 + 0.43863·log X (R = 0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL⁻¹. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.     

Time-resolved photoacoustics study of the ruthenium(II) bis(2,2'-bipyridine)(4,4'-dicarboxy-2,2'-bipyridine) complex

The formation and the decay of the triplet metal to ligand charge transfer state ((3)MLCT) of ruthenium(II) bis(2,2'-bipyridine)(4,4'-dicarboxy-2,2'-bipyridine) (Ru(bpy)(2)(dcbpy)) were characterized using photoacoustic calorimetry. At pH 6 and 2, the (3)MLCT state formation leads to a volume change of -8 mL mol(-1) and enthalpy changes of 17 kcal mol(-1) and 13 kcal mol(-1), respectively. We attribute the volume contraction to structural changes and to solvent electrostriction. At pH 4, the photoexcitation of the complex leads to an expansion of 14 mL mol(-1) and an enthalpy change of approximately 119 kcal mol(-1) due to protonation of the carboxyl group in the excited state.     

Time-resolved intraband electronic relaxation dynamics of Hg(n) (-) clusters (n=7-13,15,18) excited at 1.0 eV

Time-resolved photoelectron imaging has been used to study the relaxation dynamics of small Hg(n) (-) clusters (n=7-13,15,18) following intraband electronic excitation at 1250 nm (1.0 eV). This study furthers our previous investigation of single electron, intraband relaxation dynamics in Hg(n) (-) clusters at 790 nm by exploring the dynamics of smaller clusters (n=7-10), as well as those of larger clusters (n=11-13,15,18) at a lower excitation energy. We measure relaxation time scales of 2-9 ps, two to three times faster than seen previously after 790 nm excitation of Hg(n) (-), n=11-18. These results, along with size-dependent trends in the absorption cross-section and photoelectron angular distribution anisotropy, suggest significant evolution of the cluster anion electronic structure in the size range studied here. Furthermore, the smallest clusters studied here exhibit 35-45 cm(-1) oscillations in pump-probe signal at earliest temporal delays that are interpreted as early coherent nuclear motion on the excited potential energy surfaces of these clusters. Evidence for evaporation of one or two Hg atoms is seen on a time scale of tens of picoseconds.     

A phase separation fluoroimmunoassay of estradiol

A competitive indirect fluoroimmunoassay of free estradiol (E₂) was established based on the thermal sensitivity of hydrogel–‐poly‐N‐isopropylacrylamide. Free estradiol was covalently bound to bovine serum albumin (BSA) to form complete antigen (E₂‐BSA), which was in turn labeled by fluorescein isothiocyanate (FTTC) as the fluorescence probe. The anti‐ E₂ monoclonal antibody (McAb) was prepared by an in vivo method, and coupled with N‐isopropylacrylamide (NIPA) to make an immune copolymer, poly‐N‐isopropylacylamidemonoclonal antibody (pNIPA‐McAb), for the determination of free E₂. The immunoassay method was based on the competitive binding of free E₂ and fluoresceinated antigen (E₂‐BSA‐FTTC) with limited amount of pNIPA‐McAb. When the immunological reaction was over, precipitation and centrifugal procedures were carried out to separate pNIPA‐McAb‐E₂‐BSA‐FTTC from other constituents in solution. The precipitate pNIPA‐McAb‐E₂‐BSA‐FTTC was dissolved in solution and then the fluorescence intensity was measured. The calibration curve covered a range of 78–500 ng/mL for free E₂. The recoveries were 91.2–107.2%.     

Time-resolved emission studies of poly(n-v1nyl carbazole) using pulsed laser excitation

Time-resolved fluorescence spectroscope of poly(N-vinyl carbazole) in tetrahydrofuran solution has revealed the presence of three emitting species, namely a normal excimer emitting at λ_max = 420 nm, a higher-energy excimer site at λ_max = 370 nm which interconverts with the “normal” site, and a third species which emits at 370 nm but not at 420 nm. and which does not convert to the normal excimer. The identity of this species, and the kinetics of fluorescence of the polymer, are discussed.     

Phase-resolved fluoroimmunoassay of human serum albumin

In the homogeneous immunoassay of human serum albumin described, a difference in fluorescence lifetime is used along with a small difference in fluorescence intensity to discriminate between the free and the antibody-bound labelled antigen. The immunoassay is based on the use of phase-resolved fluorescence measurements, in which sinusoidally-modulated excitation is combined with phase-sensitive detection to generate time-dependent signals which are integrated over a pi-interval to produce phase-resolved intensities. Texas Red was used as the fluorescent label. Negligible matrix effects were observed from serum, and a comparison of values determined by using the phase-resolved fluoroimmunoassay with values provided by the hospital from which the samples were obtained yielded a correlation coefficient of 0.996.