Study Of Enzyme-amplified Lanthanide Luminescence Time-resolved Fluorescence Detection And Reagents For Nucleic Acid Hybridization Assays

Bioanalytical assay such as nucleic acid hybridization and immunoassays are extremely important in a variety of fields,for example clinical diagnostic medicine and drug testing. These assay require ultrasensitive detection methods to successfully observe the minute quantities of the analyte of interest in a biological sample, such as antibody or a specific nucleic acid sequence. The use of radioactive labels as tracers has achieved the necessary sensitivity, however, the hazards of radioisotope and limited shelf-life associated with the use of radioactive labels have limited their application. Recently , nonisotopic detection systems have been the center of the study work. Noniotopic detedtion system typically use enzymatic colorimetic, chemiluminescence-based, bioluminescence-based, or fluorescence-based detection. And in all of these nonisotopic detections, enzymatic colorimetic detection can not achieve necessary sensitivity; chemiluminescence-based detecion and bioluminescence-based detection have high background signals and labels are instable, so that these two detection methods also have low sensitivity.Enzyme-amplified lanthanide luminescence time-resolved fluorescence assay (EALL-TRFA) method, which is developed recently, is a method which use time-resolved detection to measure the enzyme-amplified lanthanide luminescence. This method combines the high affinity and amplification of biotin-streptavidin system, the amplification of enzyme, the inherent advantages of lanthanide chelate with the background elimination of time-resolved detection. 5-Fluorosalicyl phosphate is converted to 5- fluorosalicylic acid (5-FSA) by alkaline phosphatase. 5-Fluorosalicylic acid product forms a luminescent ternary chelate with Tb³⁺ and EDTA. By the measure of the luminescence (commonly referred to as fluorescence), we can calculate the quantity of the analyte of interest. This method is a new noniotopic and ultrasensitive detection method.The purpose of this article is to study and establish the new methods of EALL-TRFA system for nucleic acid hybridization, and to study the major reagents of this assay system.