A time-resolved fluorescence detection method based on the principle of phase-equilibrium frequency doubling modulation

Abstract

The invention relates to a time-resolved fluorescence detection method based on the phase equilibrium frequency multiplication modulating theory. A base frequency signal modulating excitation light source acts on subject matter to be detected to trigger fluorescent light and enable periodical enhancement and decay of the fluorescent light, the sampling period is controlled through a double-frequency square wave signal to divide the rising period and the decaying period into two parts, and then the sampling difference value of the two parts are calculated and added together to obtain the intensity representation value of the fluorescence signal after respectively independent sampling so as to acquire the concentration value of the subject matter to be detected. The method can not only eliminate fluorescence interference of a substrate in a sample but also signals, such as environmental bias lighting, space electromagnetic wave power frequency interference and the like, improves the fluorescence measurement signal strength of the detected sample, has the advantages that cannot be achieved by the conventional time-resolved fluorescence method and can be used for fluorescence strength detection of subject matters in the fields of biology, chemistry, medicine and the like.

Description

technical field [0001] The invention belongs to the technical field of testing or analyzing materials by means of measuring the chemical or physical properties of materials, in particular to a method that can eliminate fluorescence interference of substrates in samples, eliminate environmental background light, eliminate space electromagnetic wave power frequency interference and other signals to improve A time-resolved fluorescence detection method based on the principle of phase-equilibrium frequency-doubling modulation to detect sample fluorescence measurement signal strength and improve the accuracy of fluorescence detection. Background technique [0002] At present, the rapid detection strips of immunochromatography (lateral flow immunoassay, LFIA) mostly use colloidal gold or fluorescent dyes as markers. In recent years, technological breakthroughs have been made in the field of immunofluorescence detection, and time-resolved fluorescence (TRF) immunoassay technology h...

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Problems solved by technology

[0006] The technical problem solved by the present invention is that in the prior art, in the process of exciting fluorescence by excitation light irradiation, in addition to measuring the fluorescence emitted by the target, that is, the fluorescence information of the fluorescent pigment marked on the sample, the environmental background Fluorescence and impurity-excited fluorescence interfere with fluorescence. Although in the traditional time-resolved fluorescence detection method, the time point of the maximum fluorescence emission can be delayed by about 200 μs during the process of the excitation light irradiating the sample to stimulate the fluorescence emission decay. time, wait for the impurity to excite the fluorescence to be quenched, and then start to detect the fluorescence, so as to eliminate the influence of the second type of interference fluorescence above, to obtain the fluorescence intensity emitted by the dyed fluorochrome, so as to obtain relatively accurate information about the measurement target, but due to the interference Substances are uncertain, and the time delay of fluorescence detection is also uncertain. Therefore, it is impossible to completely eliminate all interfering substances to stimulate fluorescence, and it cannot eliminate the first type of environmental background light interference, such as background autofluorescence, light leakage from the detection system, and electromagnetic interference. interference, etc., and then provides an optimized time-resolved fluorescence detection method based on the principle of phase-equilibrium frequency-doubling modulation

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