A method, device and computer equipment for correcting chemiluminescence reaction curve

Abstract

The present invention provides a method, device and computer equipment for correcting a chemiluminescent reaction curve, and relates to the field of instrument detection technology. The method comprises: obtaining a photon counter characteristic curve, wherein the photon counter characteristic curve reflects the relationship between a standard light intensity value and a light intensity measurement value, wherein the light intensity measurement value refers to a light intensity value measured by a photon counter, and the standard light intensity value refers to a light intensity value obtained by controlling current conversion after calibration of a standard light source; obtaining a chemiluminescent reaction curve, wherein the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value; and correcting the chemiluminescent reaction curve in sections according to the photon counter characteristic curve to obtain a corrected reaction curve. The present invention indirectly improves the linear measurement range of the photon counter by using the photon counter characteristic curve to correct the chemiluminescent reaction curve in sections without changing the hardware device.

Description

Technical Field

[0001] The present invention relates to the technical field of instrument detection, and in particular to a correction method, device and computer equipment for a chemiluminescence reaction curve. Background Art

[0002] Chemiluminescent immunoassay is a technique that combines highly sensitive chemiluminescent assay technology with highly specific immune reactions. It is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Among them, the photon counter is the core part of chemiluminescent immunoassay. As a highly sensitive weak light detection device, the photon counter has a great influence on the repeatability, sensitivity and linear measurement range of the photon counter due to factors such as discrimination voltage, photomultiplier tube high voltage and pulse resolution time. The linear measurement range of the photon counter is limited. When the measured light intensity exceeds the linear detection range, the photon pulse pile-up effect will be significantly enhanced, resulting in serious distortion of the photon pulse counting results. However, the chemiluminescent immunoassay instrument has many measurement items and the individual differences of the items are large. The linear measurement range of the photon counter usually cannot meet the requirements of some detection items, such as human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP).

[0003] At present, in order to expand the measurement range, two methods are generally used in the market. One method uses pre-dilution and secondary measurement processing, which increases the cost and workload of sample measurement; the other method uses vacuum silicon photomultiplier tubes or dual-range mode photomultiplier tubes, which will increase the production cost and instability of the instrument. Summary of the invention

[0004] The problem solved by the present invention is that the prior art usually adopts a method of pre-dilution and secondary measurement processing of samples or a multiplier tube method when expanding the measurement range of photon counters, which has defects such as increased sample measurement cost and instrument production cost.

[0005] In order to solve the above problems, the present invention provides a method for correcting a chemiluminescent reaction curve, comprising:

[0006] Obtaining a photon counter characteristic curve, wherein the photon counter characteristic curve reflects the relationship between a standard light intensity value and a light intensity measurement value, wherein the light intensity measurement value refers to a light intensity value measured by a photon counter, and the standard light intensity value refers to a light intensity value obtained by converting a control current after calibration of a standard light source;

[0007] Acquire a chemiluminescent reaction curve, wherein the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value;

[0008] The chemiluminescence reaction curve is corrected in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

[0009] Preferably, the step of correcting the chemiluminescent reaction curve in sections according to the photon counter characteristic curve comprises:

[0010] Dividing the photon counter characteristic curve into a plurality of interval segments according to the light intensity measurement value;

[0011] Obtaining the interval segment where the light intensity measurement value of the chemiluminescent reaction curve is located;

[0012] Determine whether the light intensity measurement peak of the chemiluminescent reaction curve is concave,

[0013] When the light intensity measurement peak of the chemiluminescent reaction curve is concave, the chemiluminescent reaction curve is corrected using the photon counter characteristic curve according to the interval where the light intensity measurement value on the chemiluminescent reaction curve is located.

[0014] When the light intensity measurement peak of the chemiluminescent reaction curve does not show a depression, the chemiluminescent reaction curve is corrected using a peak shape fitting method.

[0015] Preferably, the interval segment includes a first interval, a second interval, a third interval, a fourth interval and a fifth interval, wherein the first interval is an interval in which the light intensity measurement value increases linearly and monotonically, the second interval is an interval in which the light intensity measurement value increases nonlinearly and monotonically, the third interval is an interval in which the light intensity measurement value is saturated and non-monotonic, the fourth interval is an interval in which the light intensity measurement value decreases nonlinearly and monotonically, and the fifth interval is an interval in which the light intensity measurement value tends to saturate;

[0016] When the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the fourth interval, the light intensity measurement peak value of the chemiluminescent reaction curve appears concave;

[0017] The method of correcting the chemiluminescent reaction curve by using the photon counter characteristic curve according to the interval segment where the light intensity measurement value on the chemiluminescent reaction curve is located comprises:

[0018] When the light intensity measurement value on the chemiluminescent reaction curve is in the first interval, the light intensity measurement value is equal to the first segment correction value, and a first correction curve is obtained according to the first segment correction value.

[0019] When the light intensity measurement value on the chemiluminescent reaction curve is in the fourth interval, the light intensity measurement value is corrected according to the first calibration equation to obtain a second segment correction value, and a second correction curve is obtained according to the second segment correction value, the light intensity measurement value is corrected according to the second calibration equation to obtain a fourth segment correction value, and a fourth correction curve is obtained according to the fourth segment correction value,

[0020] The second correction curve and the fourth correction curve are connected by a straight line,

[0021] A corrected reaction curve is obtained according to the first correction curve, the second correction curve, the straight line and the fourth correction curve.

[0022] Preferably, the first calibration equation is as follows:

[0023] y=a0*exp(a1*x)+a2;

[0024] The second calibration equation is as follows:

[0025] y=a0+a1*x+a2*x^2+a3*x^3;

[0026] Wherein, a0, a1, a2 and a3 are model coefficients, exp represents an exponential function with the natural constant e as the base, x represents the light intensity measurement value, and y represents the corrected light intensity measurement value.

[0027] Preferably, when the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the third interval, the light intensity measurement peak value of the chemiluminescent reaction curve does not appear concave;

[0028] The method of correcting the chemiluminescent reaction curve by using a peak shape fitting method comprises:

[0029] When the light intensity measurement value on the chemiluminescence reaction curve is in the first interval, the first segment correction value is obtained, and the first segment correction value is equal to the light intensity measurement value.

[0030] When the light intensity measurement value on the chemiluminescence reaction curve is in the second interval, the light intensity measurement value is corrected according to the first calibration equation to obtain the second segment correction value,

[0031] When the light intensity measurement value on the chemiluminescence reaction curve is in the third interval, the first-segment correction value and the second-segment correction value are used as basic data points of the fitting model to obtain a fitting curve, and the value in the fitting curve located in the third interval is used as the third-segment correction value. Based on the curve formed by the first-segment correction value, the second-segment correction value and the third-segment correction value, the corrected reaction curve is obtained.

[0032] Preferably, the step of using the first segment correction value and the second segment correction value as basic data points of the fitting model to obtain a fitting curve includes:

[0033] The fitting model is constructed based on the first segment correction value and the second segment correction value as basic data points, wherein the fitting model is as follows;

[0034]

[0035] Among them, I represents the light intensity change value, t represents the reaction time, parameters a, b, c, d are all coefficients to be optimized in the model, and e is a natural constant;

[0036] The fitting curve is obtained according to the fitting model.

[0037] Preferably, obtaining a characteristic curve of a photon counter comprises:

[0038] The standard light source is calibrated by using the strong light attenuation method, and the standard light intensity value is obtained by converting the control current of the standard light source;

[0039] A photon counter characteristic curve is obtained according to the light intensity measurement value and the standard light intensity value.

[0040] Preferably, obtaining a chemiluminescent reaction curve comprises:

[0041] The initial curve of the chemiluminescent reaction of a set duration is collected by a photon measurement system, and the initial curve is processed by median filtering and Savitzky-Golay filtering to obtain the chemiluminescent reaction curve.

[0042] The advantages of the present invention compared to the prior art are:

[0043] The method for correcting a chemiluminescent reaction curve of the present invention corrects the chemiluminescent reaction curve in sections according to the photon counter characteristic curve, wherein the photon counter characteristic curve reflects the relationship between the standard light intensity value and the light intensity measurement value, and the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value. The chemiluminescent reaction curve is corrected by using the photon counter characteristic curve, and the light intensity measurement value in the chemiluminescent reaction curve is corrected, so that the chemiluminescent reaction curve can accurately reflect the actual light intensity of the luminescent substance in the chemiluminescent reaction process, avoiding the situation where the chemiluminescent reaction curve is distorted due to the light intensity being too large and exceeding the linear range of the photon counter itself, and indirectly improving the linear measurement range of the photon counter.

[0044] The present invention corrects the chemiluminescence reaction curve in sections according to the photon counter characteristic curve, thereby ensuring the correction effect and making the data obtained from the chemiluminescence reaction curve more accurate. Without changing the hardware device of the photon counter, the chemiluminescence reaction curve is corrected in sections using the photon counter characteristic curve, thereby increasing the linear measurement range of the photon counter and meeting the requirements of wide-range detection projects.

[0045] The present invention also provides a device for correcting a chemiluminescent reaction curve, comprising:

[0046] A first acquisition unit, the first acquisition unit is used to acquire a photon counter characteristic curve, the photon counter characteristic curve reflects the relationship between the standard light intensity value and the light intensity measurement value, the light intensity measurement value refers to the light intensity value measured by the photon counter, and the standard light intensity value refers to the light intensity value obtained by controlling the current after calibration of the standard light source;

[0047] a second acquisition unit, the second acquisition unit being used to acquire a chemiluminescent reaction curve, the chemiluminescent reaction curve reflecting the relationship between the reaction time and the light intensity measurement value;

[0048] A correction unit is used to correct the chemiluminescence reaction curve in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

[0049] The advantages of the device for correcting a chemiluminescent reaction curve and the method for correcting a chemiluminescent reaction curve described in the present invention over the prior art are the same, and will not be described in detail herein.

[0050] The present invention also provides a computer device, comprising a memory and a processor, wherein the memory is used to store a computer program, and the processor is used to implement the method for correcting a chemiluminescent reaction curve as described in any one of the above items when executing the computer program.

[0051] The advantages of the computer device and the method for correcting the chemiluminescent reaction curve described in the present invention are the same as those of the prior art, and will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a flow chart of a method for correcting a chemiluminescent reaction curve in an embodiment of the present invention;

[0053] Figure 2 A curve diagram of light intensity and control current in calibration of a light source in an embodiment of the present invention;

[0054] Figure 3 is a characteristic curve diagram of a photon counter in an embodiment of the present invention;

[0055] Figure 4is a chemiluminescent reaction curve diagram of the HCG human chorionic gonadotropin sample in an embodiment of the present invention;

[0056] Figure 5 It is a schematic diagram of correcting the chemiluminescence reaction curve using the characteristic curve of the photon counter in an embodiment of the present invention;

[0057] Figure 6 This is a comparison diagram of the fitting effect of using different numbers of data points to fit the chemiluminescent reaction curve in the embodiment of the present invention;

[0058] Figure 7 The chemiluminescent reaction curve diagram after peak shape fitting correction in the embodiment of the present invention;

[0059] Figure 8 Schematic diagram of a device for correcting a chemiluminescent reaction curve in an embodiment of the present invention. DETAILED DESCRIPTION

[0060] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0061] like Figure 1 As shown, the embodiment of the present invention provides a method for correcting a chemiluminescent reaction curve, comprising the following steps:

[0062] Step 110, obtaining a photon counter characteristic curve, wherein the photon counter characteristic curve reflects the relationship between a standard light intensity value and a light intensity measurement value, wherein the light intensity measurement value refers to a light intensity value measured by a photon counter, and the standard light intensity value refers to a light intensity value obtained by converting a control current after calibration of a standard light source;

[0063] Step 120, obtaining a chemiluminescent reaction curve, wherein the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value;

[0064] Step 130, correcting the chemiluminescence reaction curve in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

[0065] The correction method of the chemiluminescent reaction curve of the present invention corrects the chemiluminescent reaction curve in sections according to the characteristic curve of the photon counter, wherein the characteristic curve of the photon counter reflects the relationship between the standard light intensity value and the light intensity measurement value, and the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value. The chemiluminescent reaction curve is corrected by using the characteristic curve of the photon counter, and the light intensity measurement value in the chemiluminescent reaction curve is corrected, so that the chemiluminescent reaction curve can accurately reflect the actual light intensity of the luminescent substance in the chemiluminescent reaction process, and avoid the situation where the chemiluminescent reaction curve is distorted due to the light intensity exceeding the linear range of the photon counter itself. The linear measurement range of the photon counter is indirectly improved. The present invention corrects the chemiluminescent reaction curve in sections according to the characteristic curve of the photon counter, ensures the correction effect, and makes the data obtained from the chemiluminescent reaction curve more accurate. Without changing the hardware device of the photon counter, the chemiluminescent reaction curve is corrected in sections using the characteristic curve of the photon counter, so as to improve the linear measurement range of the photon counter and meet the needs of wide-range detection projects.

[0066] In some implementation modes, obtaining a characteristic curve of a photon counter includes:

[0067] The standard light source is calibrated using the strong light attenuation method to obtain the relationship between the current and the standard light intensity value. Specifically, the standard light source can use a light source lamp with a peak wavelength corresponding to the peak wavelength of the spectrum of the chemiluminescent substance used, and the light intensity of the light source lamp is controlled by the current. The light intensity of the light source lamp is measured using a photon counter. The so-called calibration is to obtain the curve relationship between light intensity and current by controlling the current. It can be understood that when the current gradually increases, the light intensity value of the light source lamp also increases accordingly, and the light intensity value is measured using a photon counter. When the light intensity controlled by the current exceeds the linear measurement range of the photon counter, the light intensity measurement value of the photon counter will inevitably be distorted. Therefore, this embodiment uses the strong light attenuation method to attenuate the output light intensity, and multiplies the light intensity measurement value by the attenuation multiple as the standard light intensity value, thereby expanding the linear range of the light intensity of the light source lamp and the control current.

[0068] By changing the control current of the light source lamp, the light intensity of the light source lamp is changed by controlling the current, and the light intensity value is measured by the photon counter, and the light intensity measurement value of the photon counter is directly output to obtain the relationship between the current and the light intensity measurement value. Combined with the relationship between the current and the standard light intensity value obtained by the strong light attenuation method, the relationship between the light intensity measurement value and the standard light intensity value is obtained, thereby obtaining the photon counter characteristic curve.

[0069] For example, this embodiment uses acridinium ester as the chemiluminescent substance, and its spectral peak wavelength is 430nm. Therefore, the standard light source uses a blue LED lamp with a peak wavelength of 430nm, and the light intensity of the light source is controlled by current. The strong light attenuation method is adopted, and the measured values ​​of the Hamamatsu CH326 series photon counting detector and the CH297 series counting unit are used as the standard light intensity value. The linear output range of the light source lamp is extended to 2×10 3 / s-3×10 8 / s. Within this range, the relationship between the standard light source control current and the standard light intensity value is as follows: Figure 2 As shown, Figure 2 is a curve diagram of standard light intensity value and control current, Figure 2 The horizontal axis is the light source control current, the unit is uA, and the vertical axis is the standard light intensity value, the unit is s -1 Then change the standard light source control current from 20uA to 5mA step by step. The theoretical light intensity value corresponding to 20uA is about 8×10 5 / s; the theoretical light intensity corresponding to 5mA is about 3×10 8 / s. Study the relationship between the light intensity measurement value of the photon counter and the standard light intensity value, and obtain the characteristic curve of the photon counter, such as Figure 3 As shown, Figure 3 This is the characteristic curve of the photon counter. The horizontal axis is the standard light intensity value and the vertical axis is the light intensity measurement value.

[0070] In some embodiments, obtaining the chemiluminescent reaction curve includes: collecting an initial curve of a chemiluminescent reaction of a set duration through a photon measurement system, and processing the initial curve using a median filter and a Savitzky-Golay filter to obtain the chemiluminescent reaction curve.

[0071] Exemplarily, first, an initial curve of 3 seconds is collected by a photon measurement system, and a single sampling duration is 10 ms, and the collection duration is 3 seconds, so a total of 300 data points are collected for the initial curve. Through multiple sampling, the error generated in the formation of the chemiluminescent reaction curve can be reduced. Then, the chemiluminescent reaction curve is obtained by filtering the initial curve and other processing.

[0072] In chemiluminescent reactions, luminescent substances are usually directly labeled on antigens or antibodies. After being treated with oxidants or catalysts, the luminescent substances will produce a luminescent effect when they return to the ground state from the excited state. Generally speaking, the higher the antigen concentration, the more luminescent markers there are, and the higher the luminescent intensity. Luminescent reactions can be divided into flash type and glow type. For example, the acridinium luminescent reaction system is a flash type, and its luminescence rises quickly, generally reaching the peak light intensity in 0.4s, and the luminescence decreases relatively slowly, with a total duration of about 1-3s. Figure 4 As shown, Figure 4The chemiluminescent reaction curve of HCG human chorionic gonadotropin sample is shown in Figure 2. Figure 4 It can be seen that as the sample concentration increases, the number of luminescent markers increases and the corresponding luminescent intensity is also higher, which may exceed the linear measurement range of the photon counter itself, resulting in distortion of the chemiluminescence reaction curve.

[0073] In order to prove that the photon counter has a high repeatability after exceeding its own linear measurement range, repeated measurements were performed under different levels of high light intensity. Table 1 shows the repeatability verification data of the photon counter light intensity measurement value under different light intensity levels. The coefficient of variation CV value is used to measure the repeatability difference. Table 1 shows that when the light source signal intensity is 3×10 8 The coefficient of variation CV value corresponding to the control current within / s is high, indicating that the measurement results of the photon counter have high repeatability. Figure 3 The photon counter characteristic curve is used to compensate for distortion.

[0074] Table 1

[0075]

[0076]

[0077] In some embodiments, the step of correcting the chemiluminescent reaction curve in sections according to the photon counter characteristic curve includes:

[0078] Dividing the photon counter characteristic curve into a plurality of interval segments according to the light intensity measurement value;

[0079] Obtaining the interval segment where the light intensity measurement value of the chemiluminescent reaction curve is located;

[0080] Determine whether the light intensity measurement peak of the chemiluminescent reaction curve is concave,

[0081] When the light intensity measurement peak of the chemiluminescent reaction curve is concave, the chemiluminescent reaction curve is corrected using the photon counter characteristic curve according to the interval where the light intensity measurement value on the chemiluminescent reaction curve is located.

[0082] When the light intensity measurement peak of the chemiluminescent reaction curve does not show a depression, the chemiluminescent reaction curve is corrected using a peak shape fitting method.

[0083] In this embodiment, based on the light intensity measurement values ​​of each sampling point on the chemiluminescence reaction curve, it is determined whether the light intensity measurement value is in the interval segment of the photon counter characteristic curve, and then it is determined whether the light intensity measurement peak of the chemiluminescence reaction curve is concave. Based on the interval segment where the light intensity measurement value of the chemiluminescence reaction curve is located, the reaction curve is corrected one by one according to the characteristics of each interval segment of the photon counter characteristic curve.

[0084] The shape of a normal chemiluminescent reaction curve generally rises monotonically to the peak value of the light intensity measurement and then decreases. If the reaction curve is concave at the peak position, it indicates that the measured light intensity has exceeded the saturation point measured by the photon counter, which will cause the phenomenon of the light intensity measurement peak being concave. Therefore, in this embodiment, different correction methods are selected according to whether the chemiluminescent reaction curve is concave at the peak value of the light intensity measurement, respectively using the photon counter characteristic curve correction and peak shape fitting correction methods, and the two methods are combined to ensure the full range correction of the chemiluminescent reaction curve.

[0085] In some of the embodiments, the interval segment is divided into five intervals according to the light intensity measurement value, and the interval segment includes a first interval, a second interval, a third interval, a fourth interval and a fifth interval, wherein the first interval is an interval in which the light intensity measurement value linearly and monotonically increases, the second interval is an interval in which the light intensity measurement value nonlinearly and monotonically increases, the third interval is an interval in which the light intensity measurement value is non-monotonic and saturated, the fourth interval is an interval in which the light intensity measurement value nonlinearly and monotonically decreases, and the fifth interval is an interval in which the light intensity measurement value tends to saturation.

[0086] When the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the fourth interval, it indicates that the light intensity measurement peak value of the chemiluminescent reaction curve appears concave. Among them, the theoretical maximum light intensity measurement value refers to the maximum light intensity value when the chemiluminescent reaction curve does not appear concave at the light intensity measurement peak value. It can be understood that within the linear measurement range of the photon counter itself, as the reaction time progresses, the light intensity measurement value gradually increases and reaches a maximum value, which is the theoretical maximum light intensity measurement value. However, when the light intensity increases to exceed the linear measurement range of the photon counter itself, the maximum light intensity measurement value on the chemiluminescent reaction curve does not increase but decreases, resulting in a concave at the light intensity measurement peak value.

[0087] The method of correcting the chemiluminescent reaction curve by using the photon counter characteristic curve according to the interval segment where the light intensity measurement value on the chemiluminescent reaction curve is located comprises:

[0088] When the light intensity measurement value on the chemiluminescent reaction curve is in the first interval, the light intensity measurement value is equal to the first segment correction value, and a first correction curve is obtained according to the first segment correction value.

[0089] When the light intensity measurement value on the chemiluminescent reaction curve is in the fourth interval, the light intensity measurement value is corrected according to the first calibration equation to obtain a second segment correction value, and a second correction curve is obtained according to the second segment correction value, the light intensity measurement value is corrected according to the second calibration equation to obtain a fourth segment correction value, and a fourth correction curve is obtained according to the fourth segment correction value,

[0090] The second correction curve and the fourth correction curve are connected by a straight line,

[0091] A corrected reaction curve is obtained according to the first correction curve, the second correction curve, the straight line and the fourth correction curve.

[0092] For example, a photon counter characteristic curve is obtained by using acridinium ester as the chemiluminescent substance, and the photon counter characteristic curve is divided into five intervals according to the light intensity value. The first interval is an interval in which the measured value increases linearly and monotonically, and the light intensity range is about 2×10 3 / s-1.5×10 7 / s; the second interval is the interval where the measured value increases nonlinearly and monotonically, and the light intensity range is about 1.5×10 7 / s-3.5×10 7 / s; the third interval is the interval where the measured value is saturated and non-monotonic, and the light intensity range is 3.5×10 7 / s-6.5×10 7 / s; the fourth interval is the interval where the measured value decreases nonlinearly and monotonically, and the light intensity range is about 6.5×10 7 / s-3×10 8 / s; The fifth interval is when the measured value tends to be saturated, and the light intensity value is >3×10 8 / s.

[0093] When the light intensity measurement value of the chemiluminescent reaction curve is within the first interval measurement value range of the photon counter characteristic curve, the first curve corresponding to the photon counter measurement value is retained; when the light intensity measurement value of the chemiluminescent reaction curve is within the second interval measurement value range of the photon counter characteristic curve, the second segment correction value is calculated according to the first calibration equation of the second interval to obtain the second correction curve; when the light intensity measurement value of the chemiluminescent reaction curve is within the fourth interval measurement value range of the photon counter characteristic curve, the fourth segment correction value is calculated according to the second calibration equation of the fourth interval to obtain the fourth correction curve; when the light intensity measurement peak value of the chemiluminescent reaction curve is within the second interval, the first curve and the second correction curve are connected to obtain a complete reaction curve; when the light intensity measurement peak value of the chemiluminescent reaction curve is within the fourth interval, a curve is drawn through the second correction curve and the fourth correction curve, and the third correction curve when the light intensity measurement value of the chemiluminescent reaction curve is within the third interval measurement value range of the photon counter characteristic curve is completed by connecting two points by straight lines, and the first curve is connected to obtain a complete reaction curve.

[0094] For example, Figure 5 As shown, Figure 5 To correct the example diagram using the photon counter characteristic curve, Figure 5In the fourth interval, the light intensity measurement value is in the first quadrant, the first quadrant is the chemiluminescence reaction curve, the horizontal axis is the reaction time, and the vertical axis is the light intensity measurement value; the second quadrant is the photon counter characteristic curve, the horizontal axis is the standard light intensity value, and the vertical axis is the light intensity measurement value; the third quadrant is the corrected reaction curve, the horizontal axis is the standard light intensity value, and the vertical axis is the reaction time; the fourth quadrant is the time proportional projection. By projecting the points on the chemiluminescence reaction curve in the first quadrant onto the photon counter characteristic curve in the second quadrant, the horizontal axis of the corrected reaction curve in the third quadrant is obtained, and then the vertical axis of the corrected reaction curve in the third quadrant is obtained by time proportional projection in the fourth quadrant. Point A is on the reaction curve, and is projected onto the characteristic curve of the photon counter, and is located in the second interval. The first calibration equation is used in this interval, and the first calibration equation is a monotonically increasing nonlinear calibration equation, and the second correction value is obtained, and the measured value of the photon counter is corrected to the standard light intensity value; Point B is on the reaction curve, and is projected onto the characteristic curve of the photon counter, and is located in the fourth interval. The second calibration equation is used in this interval, and the second calibration equation is a monotonically decreasing nonlinear calibration equation, and the fourth correction value is obtained, and the measured value of the photon counter is corrected to the standard light intensity value; Point C is on the reaction curve, and is projected onto the characteristic curve of the photon counter, and is located in the first interval. This interval is a linear interval, and the light intensity measurement value is the standard light intensity value. The third interval is in a saturated state, with low sensitivity and non-monotonicity, and cannot be repaired by the characteristic curve of the photon counter. Considering that the reaction curve changes close to a straight line in this interval, the segmentation points of the repaired second interval and fourth interval curves are connected by a straight line to form a complete reaction curve.

[0095] The correction method of the chemiluminescent reaction curve described in this embodiment divides the characteristic curve of the photon counter into segments to obtain characteristic curves with different light intensity ranges, and corrects the chemiluminescent reaction curve according to the characteristic segment of the photon counter, so that the correction result is more accurate. The chemiluminescent reaction curve is corrected by segmenting the characteristic curve of the photon counter to improve the measurement accuracy of the photon counter.

[0096] In some implementation modes, the first calibration equation is as follows:

[0097] y=a0*exp(a1*x)+a2;

[0098] The second calibration equation is as follows:

[0099] y=a0+a1*x+a2*x^2+a3*x^3;

[0100] Among them, a0, a1, a2 and a3 are model coefficients, exp represents an exponential function with the natural constant e as the base, x represents the light intensity measurement value, y represents the corrected light intensity measurement value, and x^2 and x^3 represent the square of x and the cube of x respectively.

[0101] The correction method of the chemiluminescent reaction curve described in this embodiment uses the first calibration equation to calculate the second correction value for the second interval, and uses the second calibration equation to calculate the fourth correction value for the fourth interval. Different calibration equations are used for different sections of the chemiluminescent reaction curve to obtain different correction results, so that the obtained data is more accurate.

[0102] In some embodiments, when the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the third interval, the light intensity measurement peak of the chemiluminescent reaction curve does not appear concave. The method of correcting the chemiluminescent reaction curve using the peak shape fitting method includes:

[0103] When the light intensity measurement value on the chemiluminescence reaction curve is in the first interval, the first segment correction value is obtained, and the first segment correction value is equal to the light intensity measurement value.

[0104] When the light intensity measurement value on the chemiluminescence reaction curve is in the second interval, the light intensity measurement value is corrected according to the first calibration equation to obtain the second segment correction value,

[0105] When the light intensity measurement value on the chemiluminescence reaction curve is in the third interval, the first-segment correction value and the second-segment correction value are used as basic data points of the fitting model to obtain a fitting curve, and the value in the fitting curve located in the third interval is used as the third-segment correction value. Based on the curve formed by the first-segment correction value, the second-segment correction value and the third-segment correction value, the corrected reaction curve is obtained.

[0106] The data points involved in the reaction curve fitting are the first and second correction values ​​of the photon counter characteristic curve. Obviously, in order to ensure the accuracy of the fitting, the number of fitting data points cannot be too small. Figure 6 As shown, Figure 6 The chemiluminescent reaction curve fitting effect diagram shows that the more fitting data points there are, the more accurate the result obtained. Figure 6 (a) The data points within 80% of the peak height were used for fitting, and the error between the fitted peak shape and the actual peak shape intensity was 1.8%; Figure 6 (b) The data points within 60% of the peak height were used for fitting, and the error between the fitted peak shape and the actual peak shape intensity was 2.6%; Figure 6 (c) The data points within 40% of the peak height are used for fitting, and the error between the fitted peak shape and the actual peak shape is 7.2%.

[0107] As the number of data points involved in fitting decreases, the degree of approximation between the fitted peak shape and the true peak shape gradually decreases. When the peak value of the light intensity measurement of the chemiluminescence reaction curve is in the third interval of the photon counter characteristic curve, the light intensity of the valid data point has reached 50% of the maximum light intensity. Therefore, when the light intensity measurement value on the chemiluminescence reaction curve does not exceed the third interval of the photon counter, 3.5×10 7 When fitting and compensating for light intensity data within / s, the error is within the controllable range. Figure 7 As shown, Figure 7 This is the chemiluminescence reaction curve after peak shape fitting correction.

[0108] The correction method of the chemiluminescent reaction curve described in this embodiment uses a peak shape fitting method to correct the chemiluminescent reaction curve, which is simple to calculate and accurately fits the chemiluminescent reaction curve. At the same time, it makes up for the shortcoming that the characteristic curve of the photon counter cannot perform curve repair on the third interval in a saturated state. By combining the two methods, the complete chemiluminescent reaction curve can be corrected across the board.

[0109] In some implementation modes, the first segment correction value and the second segment correction value are used as basic data points of the fitting model to obtain a fitting curve, including:

[0110] The fitting model is constructed based on the first segment correction value and the second segment correction value as basic data points, wherein the fitting model is as follows;

[0111]

[0112] Among them, I represents the light intensity change value, t represents the reaction time, parameters a, b, c, d are all coefficients to be optimized in the model, and e is a natural constant;

[0113] The fitting curve is obtained according to the fitting model.

[0114] In some implementations, the coefficient optimization algorithm uses Levenberg-Marquardt. To prevent local minimization from causing uncontrollable results, the coefficient iteration initial values ​​and constraint values ​​are given by empirical values. As shown in Table 2, Table 2 shows the optimization coefficient initial values ​​and constraint values.

[0115] Table 2

[0116] coefficient Initial Value Lower limit Upper limit a 7.0 0 20 b 1.5 1 2 c 0.8 0 2 d 0.1 0 1

[0117] The correction method of the chemiluminescent reaction curve described in this embodiment adopts an exponential Weibull equation approximation model, uses the Levenberg-Marquardt algorithm to optimize coefficients, and fits the data points to obtain a continuous and complete curve.

[0118] In some implementation modes, obtaining a characteristic curve of a photon counter includes:

[0119] The standard light source is calibrated by using the strong light attenuation method, and the standard light intensity value is obtained by converting the control current of the standard light source;

[0120] A photon counter characteristic curve is obtained according to the light intensity measurement value and the standard light intensity value.

[0121] The method for correcting the chemiluminescence reaction curve described in this embodiment uses the strong light attenuation method to obtain the relationship between the light source lamp control current and the standard light intensity value, and obtains the standard light intensity value by changing the light source lamp control current, thereby obtaining a photon counter characteristic curve that can reflect the relationship between the standard light intensity value and the light intensity measurement value.

[0122] In some embodiments, obtaining a chemiluminescent reaction curve includes:

[0123] The initial curve of the chemiluminescent reaction of a set duration is collected by a photon measurement system, and the initial curve is processed by median filtering and Savitzky-Golay filtering to obtain the chemiluminescent reaction curve.

[0124] Among them, the median filter takes out an odd number of data from a sampling window in the image and sorts them, and replaces the data to be processed with the sorted median. The Savitzky-Golay filter determines the appropriate filtering parameters according to the average trend of the curve, and uses a polynomial to achieve the least squares fitting within the sliding window.

[0125] The correction method of the chemiluminescent reaction curve described in this embodiment uses median filtering and Savitzky-Golay filtering to process the chemiluminescent reaction curve, which has a good filtering effect on pulse noise and can also protect the edge information of the signal so that it is not blurred.

[0126] The method for correcting the chemiluminescent reaction curve described in this embodiment uses the characteristic curve of the photon counter to correct the chemiluminescent reaction curve in sections, which can improve the linear measurement range of the photon counter.

[0127] The present invention is described in detail below through specific embodiments.

[0128] Example 1

[0129] Standard light source calibration: The measuring equipment uses Hamamatsu CH326 series photon counting detectors and CH297 series counting units, and the quasi-light source is a blue LED light source with a peak wavelength of 430nm. A replaceable optical attenuation sheet is installed at the light source output port to adjust the light source control current and the attenuation coefficient of the attenuation sheet to ensure that the light intensity detected by the measuring equipment is always maintained within the linear measurement range.

[0130] Photon counter calibration: The measured values ​​of the photon counter in each measurement system within the nonlinear measurement range are calibrated using a standard light source. The second interval and the fourth interval of the photon counter characteristic curve are used as calibration intervals to obtain the first calibration equation, the second calibration equation and the calibration coefficient, as shown in Table 3. Table 3 is the correction model for the second interval and the fourth interval, where y is the theoretical light intensity value, x is the original measurement value, and the correction range refers to the theoretical value range.

[0131] Table 3

[0132]

[0133] Chemiluminescence reaction curve data collection and filtering: The photon measurement system was used to collect 3-second chemiluminescence reaction curves. The single sampling duration was 10 ms. A total of 300 data points were collected for the chemiluminescence reaction curves. The median filter and Savitzky-Golay filter were used to smooth the chemiluminescence reaction curves.

[0134] Obtain the characteristic curve of the photon counter: Use the standard light source to calibrate the control current to obtain the standard light intensity value, and then use the photon counter to measure the light intensity measurement value. When the light intensity controlled by the current exceeds the linear measurement range of the photon counter, the strong light attenuation method is used to attenuate the output light intensity, and the measured value is multiplied by the attenuation multiple as the standard light intensity value, so as to expand the linear range of the photon counter.

[0135] Correcting the chemiluminescence reaction curve according to the characteristic curve of the photon counter: judging the interval of the theoretical maximum light intensity measurement value of the chemiluminescence reaction curve according to whether the light intensity measurement peak value of the chemiluminescence reaction curve is concave. When the theoretical maximum light intensity measurement value of the chemiluminescence reaction curve is in the second interval and the fourth interval, the light intensity measurement peak value of the chemiluminescence reaction curve is concave, and the chemiluminescence reaction curve is corrected by using the characteristic curve of the photon counter; when the theoretical maximum light intensity measurement value of the chemiluminescence reaction curve is in the third interval, the light intensity measurement peak value of the chemiluminescence reaction curve is not concave, and the chemiluminescence reaction curve is corrected by using the peak shape fitting method.

[0136] Specifically: according to the light intensity measurement value of each sampling point on the chemiluminescence reaction curve, determine the interval segment of the light intensity measurement value in the photon counter characteristic curve, and correct the reaction curve data points one by one according to the characteristics of each interval segment of the photon counter;

[0137] When the light intensity measurement value of the chemiluminescent reaction curve is in the first interval, the first segment correction value is equal to the first segment measurement value, and the first interval curve is retained as the first segment correction curve;

[0138] As the measured value gradually increases, when the light intensity measurement value of the chemiluminescence reaction curve is in the second interval, the second correction value is calculated according to the first calibration equation in Table 3 to obtain the second correction curve; if the light intensity measurement value never enters the second interval, the first correction curve is retained as the corrected reaction curve;

[0139] When the light intensity measurement value of the chemiluminescence reaction curve is in the third interval and is not in the fourth interval, the first section correction value and the second section correction value are used as the basic data points for fitting, and the third correction curve is obtained according to the fitting model and the initial value and constraint value of the optimization coefficient in Table 2, and the first correction curve, the second correction curve and the third correction curve are used to form a corrected reaction curve; if the light intensity measurement value does not enter the third interval, the corrected reaction curve is formed according to the first correction curve and the second correction curve;

[0140] When the light intensity measurement value of the chemiluminescent reaction curve is in the fourth interval, the fourth correction value is calculated according to the second calibration equation in Table 3 to obtain a fourth correction curve, and the third correction curve is completed with a straight line by connecting the second correction curve and the fourth correction curve at the beginning and the end, and the first correction curve, the second correction curve, the straight line and the fourth correction curve are used to form a corrected reaction curve;

[0141] The corrected reaction curve is integrated over time, and the integral value is used as the relative light intensity value of this chemiluminescence reaction.

[0142] Corresponding to the correction method of the chemiluminescent reaction curve, the present invention also provides a correction device for the chemiluminescent reaction curve, such as Figure 8 As shown, including:

[0143] A first acquisition unit, wherein the first acquisition unit is used for the photon counter characteristic curve to reflect the relationship between the standard light intensity value and the light intensity measurement value, wherein the light intensity measurement value refers to the light intensity value measured by the photon counter, and the standard light intensity value refers to the light intensity value obtained by converting the control current after the standard light source is calibrated;

[0144] a second acquisition unit, the second acquisition unit being used to acquire a chemiluminescent reaction curve, the chemiluminescent reaction curve reflecting the relationship between the reaction time and the light intensity measurement value;

[0145] A correction unit is used to correct the chemiluminescence reaction curve in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

[0146] The advantages of the device for correcting a chemiluminescent reaction curve and the method for correcting a chemiluminescent reaction curve described in the present invention over the prior art are the same, and will not be described in detail herein.

[0147] The present invention also provides a computer device, comprising a memory and a processor, wherein the memory is used to store a computer program, and the processor is used to implement the method for correcting a chemiluminescent reaction curve as described in any one of the above items when executing the computer program.

[0148] The advantages of the computer device and the method for correcting the chemiluminescent reaction curve described in the present invention are the same as those of the prior art, and will not be described in detail here.

[0149] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for correcting a chemiluminescent reaction curve, characterized in that: The steps include: Obtaining a photon counter characteristic curve, wherein the photon counter characteristic curve reflects the relationship between a standard light intensity value and a light intensity measurement value, wherein the light intensity measurement value refers to a light intensity value measured by a photon counter, and the standard light intensity value refers to a light intensity value obtained by converting a control current after calibration of a standard light source; Acquire a chemiluminescent reaction curve, wherein the chemiluminescent reaction curve reflects the relationship between the reaction time and the light intensity measurement value; The chemiluminescence reaction curve is corrected in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

2. The method for correcting a chemiluminescent reaction curve according to claim 1, characterized in that: The step of correcting the chemiluminescent reaction curve in sections according to the photon counter characteristic curve comprises: Dividing the photon counter characteristic curve into a plurality of interval segments according to the light intensity measurement value; Obtaining the interval segment where the light intensity measurement value of the chemiluminescent reaction curve is located; Determine whether the light intensity measurement peak of the chemiluminescent reaction curve is concave, When the light intensity measurement peak of the chemiluminescent reaction curve is concave, the chemiluminescent reaction curve is corrected using the photon counter characteristic curve according to the interval where the light intensity measurement value on the chemiluminescent reaction curve is located. When the light intensity measurement peak of the chemiluminescent reaction curve does not show a depression, the chemiluminescent reaction curve is corrected using a peak shape fitting method.

3. The method for correcting a chemiluminescent reaction curve according to claim 2, characterized in that: The interval segment includes a first interval, a second interval, a third interval, a fourth interval and a fifth interval, wherein the first interval is an interval in which the light intensity measurement value increases linearly and monotonically, the second interval is an interval in which the light intensity measurement value increases nonlinearly and monotonically, the third interval is an interval in which the light intensity measurement value is saturated and non-monotonic, the fourth interval is an interval in which the light intensity measurement value decreases nonlinearly and monotonically, and the fifth interval is an interval in which the light intensity measurement value tends to saturate; When the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the fourth interval, the light intensity measurement peak value of the chemiluminescent reaction curve appears concave; The method of correcting the chemiluminescent reaction curve by using the photon counter characteristic curve according to the interval segment where the light intensity measurement value on the chemiluminescent reaction curve is located comprises: When the light intensity measurement value on the chemiluminescent reaction curve is in the first interval, the light intensity measurement value is equal to the first segment correction value, and a first correction curve is obtained according to the first segment correction value. When the light intensity measurement value on the chemiluminescent reaction curve is in the fourth interval, the light intensity measurement value is corrected according to the first calibration equation to obtain a second segment correction value, and a second correction curve is obtained according to the second segment correction value, the light intensity measurement value is corrected according to the second calibration equation to obtain a fourth segment correction value, and a fourth correction curve is obtained according to the fourth segment correction value, The second correction curve and the fourth correction curve are connected by a straight line, A corrected reaction curve is obtained according to the first correction curve, the second correction curve, the straight line and the fourth correction curve.

4. The method for correcting a chemiluminescent reaction curve according to claim 3, characterized in that: The first calibration equation is as follows: y=a0*exp(a1*x)+a2; The second calibration equation is as follows: y=a0+a1*x+a2*x^2+a3*x^3; Wherein, a0, a1, a2 and a3 are model coefficients, exp represents an exponential function with the natural constant e as the base, x represents the light intensity measurement value, and y represents the corrected light intensity measurement value.

5. The method for correcting a chemiluminescent reaction curve according to claim 4, characterized in that: When the theoretical maximum light intensity measurement value of the chemiluminescent reaction curve is in the third interval, the light intensity measurement peak value of the chemiluminescent reaction curve does not appear concave; The method of correcting the chemiluminescent reaction curve by using a peak shape fitting method comprises: When the light intensity measurement value on the chemiluminescence reaction curve is in the first interval, the first segment correction value is obtained, and the first segment correction value is equal to the light intensity measurement value. When the light intensity measurement value on the chemiluminescence reaction curve is in the second interval, the light intensity measurement value is corrected according to the first calibration equation to obtain the second segment correction value, When the light intensity measurement value on the chemiluminescent reaction curve is in the third interval, the first segment correction value and the second segment correction value are used as basic data points of the fitting model to obtain a fitting curve, and the value in the third interval of the fitting curve is used as the third segment correction value. The corrected reaction curve is obtained according to a curve formed by the first-segment correction value, the second-segment correction value and the third-segment correction value.

6. The method for correcting a chemiluminescent reaction curve according to claim 5, characterized in that: The step of using the first segment correction value and the second segment correction value as basic data points of the fitting model to obtain a fitting curve comprises: The fitting model is constructed based on the first segment correction value and the second segment correction value as basic data points, wherein the fitting model is as follows; Among them, I represents the light intensity change value, t represents the reaction time, parameters a, b, c, d are all coefficients to be optimized in the model, and e is a natural constant; The fitting curve is obtained according to the fitting model.

7. The method for correcting a chemiluminescent reaction curve according to claim 1, characterized in that: The obtaining of the characteristic curve of the photon counter comprises: The standard light source is calibrated by using the strong light attenuation method, and the standard light intensity value is obtained by converting the control current of the standard light source; A photon counter characteristic curve is obtained according to the light intensity measurement value and the standard light intensity value.

8. The method for correcting a chemiluminescent reaction curve according to claim 1, characterized in that: The obtaining of the chemiluminescent reaction curve comprises: The initial curve of the chemiluminescent reaction of a set duration is collected by a photon measurement system, and the initial curve is processed by median filtering and Savitzky-Golay filtering to obtain the chemiluminescent reaction curve.

9. A device for correcting a chemiluminescent reaction curve, characterized in that: include: A first acquisition unit, the first acquisition unit is used to acquire a photon counter characteristic curve, the photon counter characteristic curve reflects the relationship between the standard light intensity value and the light intensity measurement value, the light intensity measurement value refers to the light intensity value measured by the photon counter, and the standard light intensity value refers to the light intensity value obtained by controlling the current after calibration of the standard light source; a second acquisition unit, the second acquisition unit being used to acquire a chemiluminescent reaction curve, the chemiluminescent reaction curve reflecting the relationship between the reaction time and the light intensity measurement value; A correction unit is used to correct the chemiluminescence reaction curve in sections according to the photon counter characteristic curve to obtain a corrected reaction curve.

10. A computer device, characterized in that: The method comprises a memory and a processor, wherein the memory is used to store a computer program, and the processor is used to implement the method for correcting a chemiluminescent reaction curve according to any one of claims 1 to 8 when executing the computer program.

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