Method for defining effective working temperature parameter of fluorescent probe
阅读说明：本技术 一种荧光探针有效工作温度参数的界定方法 (Method for defining effective working temperature parameter of fluorescent probe ) 是由 石吉勇 李文亭 邹小波 黄晓玮 李志华 胡雪桃 郭志明 史永强 石海军 于 2019-08-26 设计创作，主要内容包括：本发明属于分析测试技术领域,涉及一种荧光探针有效工作温度参数的界定方法；具体包含温度偏移情况下探针信号的检测、荧光探针对温度偏移响应规律解析、以及探针有效工作温度的定量界定三个步骤；本发明通过解析荧光探针信号对温度正向偏移和负向偏移的响应规律,分别构建了荧光信号正向偏移幅度函数和荧光信号负向偏移幅度函数,实现了允许检测误差范围内荧光探针对应工作温度参数的精确界定,可为荧光探针在实际应用中的有效工作温度范围界定提供参考依据,从而实现待测分析物的高效、精准荧光法检测。(The invention belongs to the technical field of analysis and test, and relates to a method for defining an effective working temperature parameter of a fluorescent probe; the method specifically comprises three steps of detecting a probe signal under the condition of temperature deviation, analyzing a response rule of a fluorescent probe to the temperature deviation and quantitatively defining the effective working temperature of the probe; according to the invention, by analyzing the response rule of the fluorescent probe signal to positive deviation and negative deviation of the temperature, a positive deviation amplitude function and a negative deviation amplitude function of the fluorescent signal are respectively constructed, so that the accurate definition of the corresponding working temperature parameter of the fluorescent probe in the allowable detection error range is realized, a reference basis can be provided for the effective working temperature range definition of the fluorescent probe in practical application, and the efficient and accurate fluorescence detection of the analyte to be detected is realized.)
1. A method for defining effective working temperature parameters of a fluorescent probe is characterized by comprising the following steps:
detection of probe signal under temperature excursion:
for an optimum working temperature of T0The temperature forward deviation amplitude of the fluorescent probe is set to be delta T1DEG C, negative temperature offset amplitude Delta T2DEG C; sequentially measuring the fluorescent signals of the corresponding fluorescent probes at different temperatures in the positive deviation amplitude and the negative deviation amplitude;
analyzing the temperature deviation response rule by the fluorescent probe:
corresponding fluorescent signals of the fluorescent probe to the optimal working temperature T at different temperatures within the positive deviation amplitude and the negative deviation amplitude of the temperature0Subtracting the corresponding fluorescence signals at the temperature of DEG C and taking an absolute value to obtain a fluorescence signal offset amplitude; temperature deviation amplitude is used as an independent variable, and fluorescence signal deviation amplitude is usedThe value is a dependent variable, and a forward shift amplitude function Y ═ F of the fluorescence signal is established1(X) and a negative offset magnitude function V ═ F2(U); simultaneously establishing the inverse function X-F of the forward offset amplitude function of the fluorescence signal1 -1(Y) and the inverse of the negative offset magnitude function U ═ F2 -1(V)；
Quantitative definition of the effective operating temperature of the probe:
setting the positive and negative allowance errors of the fluorescence signal to obtain the optimal working temperature T of the fluorescence probe0Calculating the positive allowable maximum deviation amplitude and the negative allowable maximum deviation amplitude of the fluorescent signal by taking the corresponding fluorescent signal as a reference point;
respectively substituting the positive allowable maximum deviation amplitude and the negative allowable maximum deviation amplitude of the fluorescent signal into the inverse function of the positive deviation amplitude function and the inverse function of the negative deviation amplitude function of the signal, and calculating the corresponding positive deviation temperature T when the positive allowable maximum deviation amplitude of the fluorescent signal is reached+Temperature T at negative offset corresponding to maximum allowable negative offset-(ii) a Thereby determining the corresponding effective working temperature T within the allowable error range of the fluorescent probe signal0-T-～T0+T+℃。
2. The method of claim 1, wherein the method for detecting the fluorescence signal of the fluorescence probe at the temperature within the positive offset amplitude and the negative offset amplitude comprises the following steps:
when the temperature is shifted in the positive direction, the detection frequency of the fluorescence signal is n +1, and the single amplification is delta T1N ℃, measuring the temperature T in sequence0，T0+ΔT1/n，T0+2ΔT1/n，……，T0+(n-1)ΔT1/n，T0+nΔT1Fluorescence signal A corresponding to/n DEG C0，A1，A2，……，An-1，An(ii) a When the temperature is shifted negatively, the detection frequency of the fluorescence signal is m, and the single amplitude is reduced by delta T2M, measuring the temperature T in sequence0-ΔT2/m，T0–2ΔT2/m，……，T0-(m-1)ΔT2/m，T0–mΔT2Fluorescence signal B corresponding to m DEG C1，B2，……，Bm-1，Bm(ii) a Wherein n and m are integers greater than 0.
3. The method of claim 1, wherein the fluorescence signal offset amplitude is calculated as follows:
when the temperature is shifted in the positive direction, the temperature T is adjusted0+ΔT1/n，T0+2ΔT1/n，……，T0+(n-1)ΔT1/n，T0+nΔT1Fluorescence signal A corresponding to/n DEG C1，A2，……，An-1，AnAnd a temperature of T0Fluorescent signal A of0Subtracting and taking the absolute value to obtain the temperature deviation amplitude delta T1/n，2ΔT1/n，……，(n-1)ΔT1/n，nΔT1Fluorescence signal offset amplitude | A corresponding to/n DEG C1-A0|，|A2-A0|，……，|An-1-A0|，|An-A0L, |; when the temperature is negatively shifted, the temperature T is adjusted0-ΔT2/m，T0–2ΔT2/m，……，T0-(m-1)ΔT2/m，T0–mΔT2Fluorescence signal B corresponding to m DEG C1，B2，……，Bm-1，BmAnd a temperature of T0Fluorescent signal A of0Subtracting and taking the absolute value to obtain the temperature deviation amplitude delta T2/m，2ΔT2/m，……，(m-1)ΔT2/m，mΔT2Fluorescence signal offset amplitude | B corresponding to m DEG C1-A0|，|B2-A0|，……，|Bm-1-A0|，|Bm-A0|；
Wherein n and m are integers greater than 0.
4. The effective operating temperature of a fluorescent probe of claim 1Method for defining parameters, characterized in that said forward offset magnitude function Y ═ F1(X) and a negative offset magnitude function V ═ F2The establishing method of (U) is specifically operated as follows:
when the temperature is shifted in the positive direction, the temperature is shifted by the amplitude 0, Delta T1/n，2ΔT1/n，……，(n-1)ΔT1/n，nΔT1The/n is independent variable X, and the fluorescence signal deviation amplitude is 0, | A1-A0|，|A2-A0|，……，|An-1-A0|，|An-A0I is dependent variable Y, and a fluorescence signal forward deviation amplitude function Y is established as F1(X); when the temperature is shifted in the negative direction, the temperature is shifted by the amplitude 0, delta T2/m，2ΔT2/m，……，(m-1)ΔT2/m，mΔT2M is an independent variable U, and the fluorescence signal shifts the amplitude value 0, | B1-A0|，|B2-A0|，……，|Bm-1-A0|，|Bm-A0L is a dependent variable V, and a negative deviation amplitude function V ═ F of the fluorescence signal is established2(U)。
5. The method of claim 1, wherein the positive shift amplitude function and the negative shift amplitude function are obtained by Excel linear fitting.
6. The method of claim 1, wherein the maximum allowable positive and negative offset magnitudes of the fluorescence signal are calculated as follows: setting a forward allowable error C1% of the optimum operating temperature T of the fluorescent probe0Corresponding fluorescence signal A0Calculating the maximum allowable forward deviation amplitude A of the fluorescent signal as a reference point1＝|A0*C1Percent, |; set negative allowable error-C2% of the optimum operating temperature T of the fluorescent probe0Corresponding fluorescence signal A0Calculating the maximum allowable negative deviation amplitude A of the fluorescence signal as a reference point2＝|-A0*C2％|。
Technical Field
The invention belongs to the technical field of analysis and test, and relates to a method for defining an effective working temperature parameter of a fluorescent probe.
Background
The fluorescent probe changes the self fluorescent characteristic after reacting with a target analyte to be detected, thereby realizing qualitative or quantitative detection of the analyte to be detected, having the advantages of high analysis sensitivity, low detection cost, simple operation and the like, and being widely applied to the fields of food detection, environmental monitoring and biological imaging. The high sensitivity of the fluorescent probe also means that the fluorescent probe is susceptible to environmental parameters of a detection system, so that the definition of the effective working environment of the fluorescent probe is crucial to the effectiveness, reliability and stability of the actual detection result of the fluorescent probe.
The test environment temperature is one of the important factors influencing the detection effectiveness of the fluorescent probe. In a laboratory environment, the fluorescent probe has high test precision after being optimized by the test environment temperature. In the practical application process of the fluorescent probe, the corresponding test environment temperature cannot be strictly consistent with the optimal test environment temperature. It is important to accurately define the temperature of the operating environment of the fluorescent probe within the allowable detection accuracy error.
The existing method for defining the effective working temperature of the fluorescent probe comprises the steps of measuring a fluorescent signal corresponding to a limited temperature value near the optimal working temperature, and comparing the fluorescent signal intensity of the limited temperature value with the fluorescent signal intensity corresponding to the optimal working temperature so as to determine the effective working temperature range corresponding to the given error. The disadvantage of this method is that the boundary of the resulting effective operating temperature range must be one of a limited number of measured temperature values, the boundary values of which cannot be precisely defined.
Disclosure of Invention
The invention aims to solve one of the problems in the prior art, and provides a method for defining the effective working temperature parameter of a fluorescent probe, thereby providing a reference basis for determining the practical application range of the fluorescent probe.
The invention relates to a method for defining effective working temperature parameters of a fluorescent probe, which specifically comprises the following three steps of detecting probe signals under the condition of temperature deviation, analyzing the response rule of the fluorescent probe to the temperature deviation and quantitatively defining the effective working temperature of the probe:
detection of probe signal under temperature excursion:
for an optimum working temperature of T0The temperature forward deviation amplitude of the fluorescent probe is set to be delta T1DEG C, negative temperature offset amplitude Delta T2DEG C; sequentially measuring the fluorescent signals of the corresponding fluorescent probes at different temperatures in the positive deviation amplitude and the negative deviation amplitude;
the detection method of the fluorescence signal corresponding to the fluorescent probe at the temperature within the positive deviation amplitude and the negative deviation amplitude comprises the following specific steps:
when the temperature is shifted in the positive direction, the detection frequency of the fluorescence signal is n +1, and the single amplification is delta T1N ℃, measuring the temperature T in sequence0，T0+ΔT1/n，T0+2ΔT1/n，……，T0+(n-1)ΔT1/n，T0+nΔT1Fluorescence signal A corresponding to/n DEG C0，A1，A2，……，An-1，An(ii) a When the temperature is shifted negatively, the detection frequency of the fluorescence signal is m, and the single amplitude is reduced by delta T2M, measuring the temperature T in sequence0-ΔT2/m，T0–2ΔT2/m，……，T0-(m-1)ΔT2/m，T0–mΔT2Fluorescence signal B corresponding to m DEG C1，B2，……，Bm-1，Bm(ii) a Wherein n and m are integers greater than 0.
Analyzing the temperature deviation response rule by the fluorescent probe:
corresponding fluorescent signals of the fluorescent probe to the optimal working temperature T at different temperatures within the positive deviation amplitude and the negative deviation amplitude of the temperature0Subtracting the corresponding fluorescence signals at the temperature of DEG C and taking an absolute value to obtain a fluorescence signal offset amplitude; temperature deviation amplitude is used as an independent variable, and fluorescence is usedThe optical signal offset amplitude is used as a dependent variable, and a fluorescence signal forward offset amplitude function Y-F is established1(X) and a negative offset magnitude function V ═ F2(U); simultaneously establishing the inverse function X-F of the forward offset amplitude function of the fluorescence signal1 -1(Y) and the inverse of the negative offset magnitude function U ═ F2 -1(V)；
The fluorescence signal offset amplitude is calculated as follows: when the temperature is shifted in the positive direction, the temperature T is adjusted0+ΔT1/n，T0+2ΔT1/n，……，T0+(n-1)ΔT1/n，T0+nΔT1Fluorescence signal A corresponding to/n DEG C1，A2，……，An-1，AnAnd a temperature of T0Fluorescent signal A of0Subtracting and taking the absolute value to obtain the temperature deviation amplitude delta T1/n，2ΔT1/n，……，(n-1)ΔT1/n，nΔT1Fluorescence signal offset amplitude | A corresponding to/n DEG C1-A0|，|A2-A0|，……，|An-1-A0|，|An-A0L, |; when the temperature is negatively shifted, the temperature T is adjusted0-ΔT2/m，T0–2ΔT2/m，……，T0-(m-1)ΔT2/m，T0–mΔT2Fluorescence signal B corresponding to m DEG C1，B2，……，Bm-1，BmAnd a temperature of T0Fluorescent signal A of0Subtracting and taking the absolute value to obtain the temperature deviation amplitude delta T2/m，2ΔT2/m，……，(m-1)ΔT2/m，mΔT2Fluorescence signal offset amplitude | B corresponding to m DEG C1-A0|，|B2-A0|，……，|Bm-1-A0|，|Bm-A0|；
Wherein n and m are integers greater than 0.
The forward offset magnitude function Y ═ F1(X) and a negative offset magnitude function V ═ F2The establishing method of (U) is specifically operated as follows: when the temperature is shifted in the positive direction, the temperature is shifted by the amplitude 0, Delta T1/n，2ΔT1/n，……，(n-1)ΔT1/n，nΔT1The/n is independent variable X, and the fluorescence signal deviation amplitude is 0, | A1-A0|，|A2-A0|，……，|An-1-A0|，|An-A0I is dependent variable Y, and a fluorescence signal forward deviation amplitude function Y is established as F1(X); when the temperature is shifted in the negative direction, the temperature is shifted by the amplitude 0, delta T2/m，2ΔT2/m，……，(m-1)ΔT2/m，mΔT2M is an independent variable U, and the fluorescence signal shifts the amplitude value 0, | B1-A0|，|B2-A0|，……，|Bm-1-A0|，|Bm-A0L is a dependent variable V, and a negative deviation amplitude function V ═ F of the fluorescence signal is established2(U)。
The positive offset amplitude function and the negative offset amplitude function are obtained through Excel linear fitting.
Quantitative definition of the effective operating temperature of the probe:
setting the positive and negative allowance errors of the fluorescence signal to obtain the optimal working temperature T of the fluorescence probe0Calculating the positive allowable maximum deviation amplitude and the negative allowable maximum deviation amplitude of the fluorescent signal by taking the corresponding fluorescent signal as a reference point;
the calculation method of the positive allowable maximum deviation amplitude and the negative allowable maximum deviation amplitude of the fluorescence signal is as follows: setting a forward allowable error C1% of the optimum operating temperature T of the fluorescent probe0Corresponding fluorescence signal A0Calculating the maximum allowable forward deviation amplitude A of the fluorescent signal as a reference point1＝|A0*C1Percent, |; set negative allowable error-C2% of the optimum operating temperature T of the fluorescent probe0Corresponding fluorescence signal A0Calculating the maximum allowable negative deviation amplitude A of the fluorescence signal as a reference point2＝|-A0*C2|
Forward allowing maximum deviation amplitude A of fluorescent signal1Substituting the inverse of the forward offset magnitude function of the signal X ═ F1 -1(Y) calculating the maximum allowable forward shift amplitude A of the fluorescence signal1Forward direction of time correspondenceOffset temperature T+＝F1-1(ΔA1) (ii) a Negative-going allowable maximum shift amplitude A of fluorescent signal2Substituting the inverse U-F of the forward offset magnitude function of the signal2 -1(V) calculating the maximum allowable negative shift amplitude A of the fluorescence signal2Time corresponding negative offset time temperature T-＝F2 -1(ΔA2) (ii) a From this, the allowable error range-C of the fluorescent probe signal was determined2％～C1% corresponding to an effective operating temperature of T0-T-～T0+T+℃。
The invention has the beneficial effects that:
according to the invention, by analyzing the response rule of the fluorescent probe signal to positive deviation and negative deviation of the temperature, a positive deviation amplitude function and a negative deviation amplitude function of the fluorescent signal are respectively constructed, so that the accurate definition of the corresponding working temperature parameter of the fluorescent probe in the allowable detection error range is realized, a reference basis can be provided for the effective working temperature range definition of the fluorescent probe in practical application, and the efficient and accurate fluorescence detection of the analyte to be detected is realized.