Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay

A new nonadentate ligand, N, N, N-1, N-1-[2,6-bis(3'-aminomethyl-1 1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb3+ fluorescent complex was synthesized. The Tb3+ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifetime of 2.681 ms in 0.05 M berate buffer of pH 9.1. Streptavidin (SA) was labeled with SPTA by using its succinimidyl monoester, and the BPTA-Tb3+-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of alpha -fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb3+-labeled SA was also used in competitive type TR-FIA of bensulfuron- methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simultaneous measurement method for AFP and CEA in a human serum sample was developed by using 4,4'-bis(1 " ,1 " ,1 " ,2 " ,2 " ,3 " ,3 " -heptafluoro-4 " ,6 " -hexanedion-6 " -yl)chlorosulfo-o-terphenyl ((BHHCT)-Eu3+-labeled anti-AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled SA. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.

Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb3+ fluorescent chelate with N,N,N',N'-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb3+) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb3+ labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb3+ labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ng ml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement. (C) 2001 Elsevier Science B.V. All rights reserved.

Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles

New silica-based europium fluorescent nanoparticles having surface amino groups were prepared by a covalent binding-copolymerization technique. In the nanoparticles, the fluorescent Eu3+ chelate molecules were covalently bound to silicon atoms to protect the nanoparticles from dye leaking in bio-applications. The amino groups on the surface of nanoparticles made the surface modification and bioconjugation of nanoparticles easier. The nanoparticles were characterized and developed as a new type of fluorescence probe for a highly sensitive time-resolved fluoroimmunoassay (TR-FIA) of human hepatitis B surface antigen (HBsAg).

Time-Resolved Synchronous Fluorescence for Biomedical Diagnosis.

This article presents our most recent advances in synchronous fluorescence (SF) methodology for biomedical diagnostics. The SF method is characterized by simultaneously scanning both the excitation and emission wavelengths while keeping a constant wavelength interval between them. Compared to conventional fluorescence spectroscopy, the SF method simplifies the emission spectrum while enabling greater selectivity, and has been successfully used to detect subtle differences in the fluorescence emission signatures of biochemical species in cells and tissues. The SF method can be used in imaging to analyze dysplastic cells in vitro and tissue in vivo. Based on the SF method, here we demonstrate the feasibility of a time-resolved synchronous fluorescence (TRSF) method, which incorporates the intrinsic fluorescent decay characteristics of the fluorophores. Our prototype TRSF system has clearly shown its advantage in spectro-temporal separation of the fluorophores that were otherwise difficult to spectrally separate in SF spectroscopy. We envision that our previously-tested SF imaging and the newly-developed TRSF methods will combine their proven diagnostic potentials in cancer diagnosis to further improve the efficacy of SF-based biomedical diagnostics.

Space and time resolved rotational state populations and gas temperatures in an inductively coupled hydrogen RF discharge

Gas temperature is of major importance in plasma based surface treatment, since the surface processes are strongly temperature sensitive. The spatial distribution of reactive species responsible for surface modification is also influenced by the gas temperature. Industrial applications of RF plasma reactors require a high degree of homogeneity of the plasma in contact with the substrate. Reliable measurements of spatially resolved gas temperatures are, therefore, of great importance. The gas temperature can be obtained, e.g. by optical emission spectroscopy (OES). Common methods of OES to obtain gas temperatures from analysis of rotational distributions in excited states do not include the population dynamics influenced by cascading processes from higher electronic states. A model was developed to evaluate this effect on the apparent rotational temperature that is observed. Phase resolved OES confirmed the validity of this model. It was found that cascading leads to higher apparent temperatures, but the deviation (similar or equal to 25 K) is relatively small and can be ignored in most cases. This analysis is applied to investigate axially and radially resolved temperature profiles in an inductively coupled hydrogen RF discharge.