Time-resolved fluorescence immunoassay
Time-resolved Fluorescence Immunoassay


Time-resolved Fluorescence Immunoassay Technology

Time-resolved fluorescence immunoassay(TRFIA) is a non-isotopic immunoassay technique, which uses lanthanide elements to label antigens or antibodies. According to the luminescence characteristics of lanthanide chelates, time-resolved technology is used to measure fluorescence, and the two parameters of wavelength and time are simultaneously detected for signal resolution. The interference of non-specific fluorescence is effectively eliminated, and the analysis sensitivity is greatly improved.



Time-resolved Fluorescence Immunoassay Principle

Many complexes and proteins in biological fluids and serum can fluoresce by themselves, thus the sensitivity of fluorescence detection with traditional chromophores will be severely reduced. Most of the background fluorescence signal exists for a short period of time. Therefore, the combination of long-decay-life markers and time-resolved fluorescence technology can minimize transient fluorescence interference.

Time-resolved fluorescence immunoassay analysis (TRFIA) is actually developed on the basis of fluorescence immunoassay analysis (FIA), which is a special type of fluorescence analysis. Fluorescence analysis utilizes the huge difference between the wavelength of fluorescence and its excitation wavelength in order to overcome the influence of variegated light in ordinary ultraviolet-visible spectroscopy. At the same time, fluorescence analysis is different from ordinary spectroscopy. The photoelectric receiver and the excitation light are not on the same line. The excitation light cannot directly reach the photoelectric receiver, thus greatly improving the sensitivity of optical analysis.

However, when performing ultra-micro analysis, the influence of the stray light of the excitation light becomes serious. Therefore, solving the influence of the stray light of the excitation light has become a bottleneck for improving the sensitivity. The best way to solve the influence of stray light is that there is no excitation light when measuring. However, the fluorescence lifetime of ordinary fluorescent markers is very short (10-100ns), the excitation light disappears, and the fluorescence also disappears. However, there are very few rare earth metals (Eu, Tb, Sm, Dy) that have a long fluorescence lifetime, up to 1 to 2 ms, which can meet the measurement requirements. Therefore, a time-resolved fluorescence analysis method was developed, that is, the use of long-lasting fluorescent markers, The analytical method of measuring the fluorescence intensity after turning off the excitation light.



Time-resolved Signal Principle

The commonly used lanthanides are mainly Eu (europium) and Tb (terbium). The fluorescence lifetime of Eu is about 1ms, which is unstable in water, but it can be overcome by adding an enhancer. The fluorescence lifetime of Tb is 1.6ms, stable in water, but its fluorescence wave Long and short, serious scattering, and high energy are easy to decompose the components. Therefore, Tb is very good from the measurement methodology, but it is not suitable for biological analysis, so Eu is the most commonly used.

The fluorescence lifetime of europium can reach about 1ms. In the detection, only the method of delaying the measurement time after each excitation light pulse is adopted, after the short-lived (10-100ns) background fluorescence decay disappears, the sampling door light source is turned on for detection. It can completely avoid the interference of the background fluorescence of the background itself, which greatly improves the specificity of the detection. The sample is tested 1000 times per second, and the results are averaged, which is beneficial to improve the accuracy of the detection.

Time Resolved Immunochromatography



Principle of Wavelength Resolution

The fluorescence spectrum of ordinary substances is divided into excitation spectrum and emission spectrum. When selecting fluorescent substances as markers, the wavelength difference between the excitation spectrum and the emission spectrum, that is, the magnitude of the Stokes shift, must be considered. If the Stokes shift is small, the excitation spectrum and emission spectrum often overlap and interfere with each other, which affects the accuracy of the detection results. 

The excitation light wavelength of Europium is 340nm, and the emission light wavelength is 613nm. The wavelength difference between the two, that is, the Stokes shift is 273nm, so the excitation light and the emission light are easily separated by interference filters.


In the field of application and development of quantitative real-time detection technology and infection control technology, Lumigenex has formed a strategic partnership with Kimberly-Clark of the United States. It is the only company in the world that legally uses time-resolved fluorescence immunochromatography technology platform.



Nanosphere Technology

On the basis of traditional time resolution technology, Lumigenex adopts exclusive patented nano-microsphere technology to embed thousands of Eu3+ in nano-microspheres, and then combine with antibodies. Compared with ordinary time resolution, it not only solves the problem of Eu3+ in water The problem of instability, the fluorescence signal can be amplified tens of thousands of times, thereby greatly improving the sensitivity of detection。

Lumigenex Bio-Nano-Microsphere Patented Technology

Mar. 2014

CFDA approvals for five immunoassay reagents adaptable to most chemical analyzers were obtained

Feb. 2014

CFDA approvals for time-resolved fluorescence immunochromatographic analyzer were obtained

Sep. 2013

Lumigenex passed The first Food and Drug Administration by the Suzhou Municipal Quality Management System Assessment