文档介绍：Time-resolved fluorescence immunoassay (TRFIA)
Main content
The definition of TRFIA
The principle of TRFIA
The advantages of TRFIA
The clinical application of detecting HBV infection
Definition
TRFIA is a analysis that use trivalent rare earth ions and chelating agents as a tracer, instead of fluorescent substances, isotopes or enzyme, marking up protein, hormone, antibody, nucleic acid probes or biological activity cells. When the reaction occurs, TRF instrument detect the fluorescent intensity of final product. According to the fluorescence intensity to determine concentration of the substances.
Unique fluorescent marker of rare earth metals- Lanthanide
Total 15 kinds of lanthanide elements(trivalent element), 4 elements used in TRFIA
铕(Eu)、钐Sm)、镝(Dy)、铽(Te)
Eu3+ is most widely application
Sm3+ as second option can be used for double labeling or labeling
Different trivalent rare earth ions have different emission spectra and fluorescence lifetime. It can be used to double labeling and multi-tag and detect two or more antigens or antibodies simultaneously.
Characteristics of lanthanide fluorescence :
Stokes shift is large
Eu: emission 613nm, excitation 340nm
Fluorescence near 280nm
Rare earth ion excitation and emission light and Stokes shift
Rare earth ion chelate
Excitation wavelength
(nm)
Emission wavelength
(nm)
Stokes
shift
(nm)
Eu chelate
340
613
273
Sm chelate
340
600
260
Tb chelate
295
490/543
195/248
Dy chelate
295
573
278
What is Time-resolved?
Fluorescent of biological products
Proteins fluorescence wavelength 400-600nm
Protein fluorescence decay time is about 1-10ns
Detecting by Ordinary fluorescent, interference is very large
Fluorescent of lanthanide chelates
Fluorescence intensity is strong
Fluorescence lifetime is long(10-1000us)
Higher 5-6 orders of magnitude than the ordinary fluorescent markers
Delaying measurement time
Fluorescence of short-lived sample pletely
Detection of rare earth ion chelate fluo