A method for calculating overall fluorescence intensity based on the theoretical model of direct chemiluminescence fluorescence spectrum

Abstract

The present invention provides a direct chemiluminescence fluorescence spectrum theoretical model, the model is as follows: due to the multiple detection errors CV variability caused by different injection speeds and angles of the excitation liquid, the present invention reduces it from the original 5% to 1%.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro diagnosis (IVD) of medical devices, and in particular relates to a direct chemiluminescence fluorescence spectrum theoretical model. Background Art

[0002] Direct chemiluminescence detection technology is currently the mainstream detection method for immunoassays, exemplified by acridinium esters as luminescent substrates. Because acridinium esters emit flashes of light, the luminescent energy is relatively concentrated within a short period of time. However, abnormal conditions such as slow injection speed or angle of the excitation solution can slow the diffusion of the excitation solution into the reagent solution, causing a decrease in the luminescence peak and a prolonged luminescence duration. However, the optical detection time of the PMT sensor in current chemiluminescence instruments is generally controlled within 0.5-1 second. If the peak becomes shorter and wider, the fluorescence signal collected within the effective time is reduced, resulting in a low final detection result and a large detection error. Summary of the Invention

[0003] Technical problem to be solved: The present invention starts with the model analysis of liquid diffusion and luminescence, theoretically derives the theoretical model of the fluorescence spectrum of direct chemiluminescence, and derives the model parameters based on the fluorescence signal detection data within the detection window time. The overall fluorescence signal intensity is then calculated based on the model, solving the problem of detection error caused by differences in diffusion rate due to differences in the injection speed and angle of the excitation liquid.

[0004] Technical solution: A direct chemiluminescence fluorescence spectroscopy theoretical model, the model is as follows: Preferably, the parameter K and the parameter τ are different coefficients, and the fluorescence signal acquisition is a discrete sampling process. The starting point is calculated from the rise of the fluorescence signal. According to the first 50 fluorescence signal acquisition values, the fitting iterative algorithm is used to calculate the parameter K and the parameter τ. When the correlation coefficient R between the theoretical value calculated each time and the true value of the discrete sampling reaches 0.99 or more, the iteration is stopped, and the obtained parameter K and parameter τ are calculated. Theoretical value.

[0005] Preferably, the C 底物 Obtained from a pre-set calibration curve.

[0006] Preferably, the preset calibration curve is obtained by calibrating each batch of reagents with a standard substance by the user.

[0007] The derivation method of the above-mentioned direct chemiluminescence fluorescence spectrum theoretical model is as follows:

[0008] (1) Analyze and deduce the liquid diffusion and luminescence model, assuming that the fluorescence signal intensity at a certain moment is:

[0009]

[0010] Where t is the measurement time point, τ A is the attenuation coefficient constant of the fluorescent substance, I0 is the initial fluorescence signal intensity after excitation;

[0011] (2) The I0(t) signal is proportional to the concentration of the luminescent substrate at that moment, and is also proportional to the volume of the excitation liquid within the detection window area at that moment. The formula is further derived as follows:

[0012] I0(t)=K1·C 底物 ·V 激 (t);

[0013] Among them C 底物 is the concentration to be solved, which is uniformly distributed in the solution, and V 激 is the volume of the excitation liquid that diffuses to the fluorescence detection window at this moment. The excitation liquid is the last reagent added. There is a diffusion process in the detection cup. The instantaneous volume of the excitation liquid satisfies the diffusion equation as follows:

[0014]

[0015] Where C0 is the initial concentration of the excitation solution, τ B Diffusion process coefficient, A is the area of ​​the detection window, V(t) is the liquid flow rate reaching the area at that moment. To simplify the model, assuming V(t) is a constant flow rate constant K2, the above formula can be simplified to:

[0016]

[0017] (4) By integrating the above formulas, we can obtain the following model:

[0018]

[0019] Among them, K1, K2, τ A , τ B To detect the constant coefficient related to the environment, C0 is the concentration of the initial added excitation solution;

[0020] (4) Simplifying the above formula, we can get:

[0021] in,

[0022] K=K1·A·K2·C0,τ=τ A +τ B ;

[0023] (5) In C 底物Under constant conditions, the same amount of excitation liquid, the total fluorescence energy emitted is fixed, and the collected fluorescence signals are a constant value, that is, the total fluorescence signal energy is related to C 底物 The concentration is positively correlated, and the theoretical model of direct chemiluminescence fluorescence spectrum is finally obtained:

[0024]

[0025] Beneficial effects:

[0026] 1. The present invention reduces the variability of the CV error caused by multiple detections due to different injection speeds and angles of the excitation liquid from the original 5% to 1%.

[0027] 2. The model of the present invention fully considers the differences in the liquid mixing process caused by mechanical structure and physical environmental variables, and establishes a characteristic model based on this difference. The model parameters can be solved based on the fluorescence signal curve fitting, and then the characteristic fluorescence signal value can be calculated. The entire process more realistically reflects the liquid mixing and luminescence process, and can effectively reduce system errors.

[0028] 3. The detection method established based on the model of the present invention can accurately determine the concentration of the substrate. In direct luminescence detection methodology, the substrate concentration is proportional to the concentration of the analyte, thereby enabling more accurate analysis of the detection concentration of the target substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The same concentration of substrate solution, the detection data of 5 different excitation liquid nozzle speeds, the detection time is 10ms per sampling point, the sampling time is 500ms, a total of 50 sampling points. In the figure, the excitation liquid nozzle speed of test 1 is 200ul / s, as shown in the fluorescence signal data table and Figure 1 middle As shown in the curve; the excitation liquid nozzle speed of test 2 is 190ul / s, as shown in the fluorescence signal data table and Figure 1 middle As shown in the curve; the excitation liquid nozzle speed of test 3 is 195ul / s, as shown in the fluorescence signal data table and Figure 1 middle As shown in the curve; the excitation liquid nozzle speed of test 4 is 195ul / s, as shown in the fluorescence signal data table and Figure 1 middle As shown in the curve; the excitation liquid nozzle speed of test 5 is 185ul / s, as shown in the fluorescence signal data table and Figure 1 middle As shown in the curve. DETAILED DESCRIPTION

[0030] Principle: In the chemiluminescent immunoassay method, the antibody is labeled with a luminescent substrate (typically acridinium or isoluminol). After a double antibody sandwich or competitive immunoreaction, the bound composite structure with the luminescent substrate is separated by magnetic particles, and then an excitation solution (typically sodium hydroxide NaOH and hydrogen peroxide H2O2 solution) is added to detect the intensity of the fluorescence signal. The intensity of the fluorescence signal represents the concentration of the luminescent substrate. Then, the concentration of the target antigen can be calculated based on the specific binding of the antigen and antibody.

[0031] A theoretical model of direct chemiluminescence fluorescence spectroscopy is as follows: The derivation method of the above-mentioned direct chemiluminescence fluorescence spectrum theoretical model is as follows:

[0032] (1) Analyze and deduce the liquid diffusion and luminescence model, assuming that the fluorescence signal intensity at a certain moment is:

[0033]

[0034] Where t is the measurement time point, τ A is the attenuation coefficient constant of the fluorescent substance, I0 is the initial fluorescence signal intensity after excitation;

[0035] (2) The I0(t) signal is proportional to the concentration of the luminescent substrate at that moment, and is also proportional to the volume of the excitation liquid within the detection window area at that moment. The formula is further derived as follows:

[0036] I0(t)=K1·C 底物 ·V 激 (t);

[0037] Among them C 底物 is the concentration to be solved, which is uniformly distributed in the solution, and V 激 is the volume of the excitation liquid that diffuses to the fluorescence detection window at this moment. The excitation liquid is the last reagent added. There is a diffusion process in the detection cup. The instantaneous volume of the excitation liquid satisfies the diffusion equation as follows:

[0038]

[0039] Where C0 is the initial concentration of the excitation solution, τ B Diffusion process coefficient, A is the area of ​​the detection window, V(t) is the liquid flow rate reaching the area at that moment. To simplify the model, assuming V(t) is a constant flow rate constant K2, the above formula can be simplified to:

[0040]

[0041] (5) By integrating the above formulas, we can obtain the following model:

[0042]

[0043] Among them, K1, K2, τ A , τ B To detect the constant coefficient related to the environment, C0 is the concentration of the initial added excitation solution;

[0044] (4) Simplifying the above formula, we can get:

[0045] in,

[0046] K=K1·A·K2·C0,τ=τ A +τ B ;

[0047] (5) In C 底物 Under constant conditions, the same amount of excitation liquid, the total fluorescence energy emitted is fixed, and the collected fluorescence signals are a constant value, that is, the total fluorescence signal energy is equal to C 底物 The concentration is positively correlated, and the theoretical model of direct chemiluminescence fluorescence spectrum is finally obtained:

[0048] In each excitation process, the parameters K and τ are different coefficients. Fluorescence signal acquisition is a discrete sampling process. The starting point is calculated from the rise of the fluorescence signal. According to the first 50 fluorescence signal acquisition values, the fitting iterative algorithm is used to calculate the parameters K and τ. When the correlation coefficient R between the theoretical value calculated each time and the actual value of the discrete sampling reaches above 0.99, the iteration is stopped and the obtained parameters K and τ are calculated. Theoretical value, and then obtain C from the preset calibration curve 底物 The calibration curve is obtained by calibrating each batch of reagents with standard substances.

[0049] Using the same sample concentration, that is, the same concentration C 底物 The test was repeated 5 times, and the fluorescence signal intensity of the first 50 times was collected as shown in Table 1 below. Figure 1 As shown:

[0050] Table 1

[0051]

[0052]

[0053] The values ​​in each column are summed to obtain the fluorescence signal for each test, and the CV of the five measurements can be calculated to be 4.9%.

[0054] The model of the present invention is used for fitting calculation, and Table 2 is obtained:

[0055] Table 2

[0056]

[0057] Substituting the above model parameters into the formula to generate the fluorescence fitting signal of 500 sampling points, the CV can be calculated to be 0.5%.

Claims

1. A method for calculating overall fluorescence intensity based on a direct chemiluminescence fluorescence spectrum theoretical model, characterized in that: The model is as follows: The derivation method is as follows: (1) Analyze and deduce the model of liquid diffusion and luminescence, assuming that the fluorescence signal intensity at a certain moment is: Where t is the measurement time point, τ A is the attenuation coefficient constant of the fluorescent substance, I0 is the intensity of the initial fluorescence signal after excitation; (2) The I0(t) signal is proportional to the concentration of the luminescent substrate at this moment, and is also proportional to the volume of the excitation liquid in the detection window area at this moment. The formula is further derived as follows: I0(t)=K1·C 底物 ·V 激 (t); Among them C 底物 is the concentration to be solved, which is uniformly distributed in the solution, and V 激 is the volume of the excitation liquid diffused to the fluorescence detection window at this moment. The excitation liquid is the last reagent added. There is a diffusion process in the detection cup. The instantaneous volume of the excitation liquid satisfies the diffusion equation as follows: Where C0 is the initial concentration of the excitation solution, τ B Diffusion process coefficient, A is the area of ​​the detection window, V(t) is the liquid flow rate reaching the area at that moment; to simplify the model, assuming that V(t) is a constant flow rate constant K2, the above formula can be simplified to: (3) By integrating the above formulas, we can obtain the following model: Among them, K1, K2, τ A , τ B To detect the constant coefficient related to the environment, C0 is the concentration of the initially added stimulating solution; (4) Simplifying the above formula, we can get: in, K=K1·A·K2·C0,τ=τ A +t B ; (5) In C 底物 Under constant conditions, the total fluorescence energy emitted by the same amount of excitation solution is fixed, and the total fluorescence energy of all fluorescence signals collected is a constant value, that is, the total fluorescence signal energy and C 底物 The concentration is positively correlated, and finally the theoretical model of direct chemiluminescence fluorescence spectrum is obtained: The parameter K and parameter τ are different coefficients. Fluorescence signal acquisition is a discrete sampling process. The starting point is calculated from the rise of the fluorescence signal. According to the first 50 fluorescence signal acquisition values, the parameters K and τ are calculated by using the fitting iteration algorithm. When the correlation coefficient R between the theoretical value calculated each time and the actual value of the discrete sampling reaches more than 0.99, the iteration is stopped and the obtained parameters K and τ are calculated. Theoretical value.

2. The method for calculating the overall fluorescence intensity according to the direct chemiluminescence fluorescence spectrum theoretical model according to claim 1, characterized in that: The C 底物 Obtained from a preset calibration curve.

3. The method for calculating the overall fluorescence intensity according to the direct chemiluminescence fluorescence spectrum theoretical model according to claim 2, characterized in that: The preset calibration curve is obtained by calibrating each batch of reagents with a standard substance by the user.

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