A method for detecting a plant-specific molecular marker by chemical analysis

Abstract

The present application relates to the field of spectral detection technology, and particularly relates to a chemical analysis detection method for specific molecular markers of plants, target reaction rates and reference reaction rates of plant extract are obtained respectively; according to the difference of reaction rates in local window at each time, target rate difference feature sets and reference rate difference feature sets at each time are obtained, element pairing is performed on the feature sets, response intensity of target wavelength relative to reference wavelength at each time and credibility of target reaction rate at each time are obtained; according to the response intensity and the reference reaction rate, interference components are stripped from the target reaction rate, and the reaction rate is adjusted through the credibility, to obtain a corrected reaction rate; the concentration of specific molecular markers in the plant extract is obtained from the corrected reaction rate. The present application can maximally inhibit non-specific interference caused by complex matrix, so as to improve the accuracy of concentration acquisition of specific molecular markers in the plant extract.

Description

Technical Field

[0001] This invention relates to the field of spectroscopic detection technology, and more specifically to a chemical analysis and detection method for plant-specific molecular markers. Background Technology

[0002] In the field of plant active ingredient analysis, especially in the quality evaluation of rhizome medicinal materials (such as Astragalus membranaceus and Codonopsis pilosula), colorimetric reactions combined with spectrophotometry are often used to determine the content of specific molecular markers. This type of method is typically based on the reaction of a colorimetric reagent with the target molecule to generate a product that absorbs at a specific wavelength, and the concentration is calculated by measuring the absorbance.

[0003] However, plant extracts typically have high viscosity and complex matrix compositions, rich in polysaccharides and colloidal substances. During the chemical reaction initiated by the addition of a colorimetric reagent, changes in the ionic strength and pH of the solution system can easily lead to the precipitation of fine particles or the slow movement of microbubbles. This matrix-induced turbidity change is a nonlinear dynamic process, generating instantaneous, non-stationary scattering signal fluctuations in the optical path. Conventional methods for eliminating background interference in existing technologies include static blank subtraction or dual-wavelength subtraction. These methods are generally based on two assumptions: first, the background interference remains constant during the reaction; second, the response ratio of the interference at different wavelengths is a fixed linear relationship. However, in the aforementioned high-viscosity, complex systems, signal fluctuations caused by colloidal particle tumbling or bubble movement are random and time-varying, and the response ratio at different wavelengths dynamically fluctuates with changes in particle morphology. Conventional static or fixed-ratio subtraction methods cannot adapt to this dynamic interference, making it difficult to distinguish between gradual signal changes caused by chemical reactions and sudden signal jumps caused by matrix disturbances, resulting in positive errors in the detection results and affecting the accuracy of the analytical data. Summary of the Invention

[0004] The technical problem that this invention aims to solve is the low accuracy of existing chemical analysis and detection methods for plant-specific molecular markers.

[0005] The purpose of this invention is to provide a chemical analysis and detection method for specific molecular markers in plants, and the specific technical solution adopted is as follows: This invention provides a chemical analysis and detection method for plant-specific molecular markers, comprising: The target reaction rate and reference reaction rate of the plant extract were obtained separately; the target reaction rate is the rate of change of absorbance at a target wavelength, and the reference reaction rate is the rate of change of absorbance at a reference wavelength. Based on the rate difference of reaction rates between adjacent times within the sliding time window at each time, the target rate difference feature set and the reference rate difference feature set at each time are obtained respectively. Optimal element pairing is performed on the target rate difference feature set and the reference rate difference feature set to obtain the optimal set of matching pairs and the set of unmatched elements. Based on the optimal set of matching pairs and the set of unmatched elements, the response intensity of the target wavelength relative to the reference wavelength at each time step, as well as the confidence level of the target reaction rate at each time step, are obtained. Based on the response intensity and the reference response rate, the interference component is removed from the target response rate to obtain the decoupled response rate, and the decoupled response rate is adjusted by the confidence level to obtain the corrected response rate. The concentration of specific molecular markers in plant extracts was obtained by modifying the reaction rate. Wherein, the rate difference of the target reaction rate at adjacent moments within the sliding time window is the second-order difference of the target reaction rate within the sliding time window; the rate difference of the reference reaction rate at adjacent moments within the sliding time window is the second-order difference of the reference reaction rate within the sliding time window. The process of obtaining the response intensity includes: determining a first sum and a second sum, wherein the first sum is the sum of the absolute values ​​of the rate differences of all target response rates in the optimal matching pair set, and the second sum is the sum of the absolute values ​​of the rate differences of all reference response rates in the optimal matching pair set; and calculating the ratio of the first sum to the second sum to obtain the response intensity.

[0006] In an exemplary embodiment, the process of obtaining the target rate difference feature set includes: Determine whether the absolute value of the difference in the target reaction rate at each moment within the sliding time window is greater than a first preset difference threshold. Obtain a target rate difference feature set, which includes the rate differences of the target reaction rate at each time point that are greater than the first preset difference threshold; The process of obtaining the reference rate difference feature set includes: Determine whether the absolute value of the rate difference of the reference reaction rate at each moment within the sliding time window is greater than the second preset difference threshold. Obtain a reference rate difference feature set, which includes the rate difference of the reference response rate at each time point greater than the second preset difference threshold.

[0007] In an exemplary embodiment, the step of performing optimal element pairing on the target rate difference feature set and the reference rate difference feature set to obtain an optimal set of matched pairs and a set of unmatched elements includes: The target rate difference feature set is used as the left node of the bipartite graph, and the reference rate difference feature set is used as the right node of the bipartite graph. Construct a cost matrix, where each element of the cost matrix is ​​the time interval between any left node and any right node; With the goal of minimizing the total cost, the cost matrix is ​​solved using the minimum weight matching algorithm to obtain the best matching pair that satisfies the preset constraints, which constitutes the set of optimal matching pairs. The remaining unmatched rate differences in the target rate difference feature set and the reference rate difference feature set constitute the set of unmatched elements. The preset constraint is: both are positive or both are negative, and the time interval between the left node and the right node is less than the preset interval threshold.

[0008] In one exemplary embodiment, the process of obtaining the credibility includes: The third sum and the fourth sum are determined. The third sum is the sum of the squares of the rate differences of each optimal matching pair in the optimal matching pair set. The process of obtaining the fourth sum is as follows: calculate the squares of all rate differences in the unmatched element set, multiply by a preset penalty coefficient, and then add the third sum. The confidence level is obtained by calculating the ratio of the third sum to the fourth sum.

[0009] In an exemplary embodiment, the process of obtaining the decoupling reaction rate includes: The interference component is obtained based on the response intensity and the reference response rate; the interference component is positively correlated with both the response intensity and the reference response rate. The decoupled reaction rate is obtained by subtracting the interference component from the target reaction rate.

[0010] In an exemplary embodiment, the process of obtaining the modified reaction rate includes: The median of the decoupling response rates at all times within a preset repair window of the candidate time is determined as the trend benchmark response rate of the candidate time; the candidate time can be any time. Based on the confidence level, the weight values ​​of the decoupling response rate and the trend baseline response rate at the candidate time are obtained; the weight value of the decoupling response rate is positively correlated with the confidence level, and the weight value of the trend baseline response rate is negatively correlated with the confidence level. Based on the weight values ​​of the decoupled reaction rate and the trend benchmark reaction rate, the decoupled reaction rate and the trend benchmark reaction rate at the candidate time are weighted and summed to obtain the corrected reaction rate at the candidate time.

[0011] In one exemplary embodiment, obtaining the concentration of a specific molecular marker in the plant extract by modifying the reaction rate includes: The cumulative absorbance value for the target time period is obtained based on the corrected reaction rate at each time point within the target time period. The concentration of a specific molecular marker in the plant extract is obtained from the cumulative absorbance value according to a preset conversion algorithm.

[0012] In an exemplary embodiment, the process of obtaining the cumulative absorbance value includes: Determine the average corrected reaction rate for every two adjacent moments within the target time period; The cumulative absorbance value is obtained by summing the average corrected reaction rate over the target time period.

[0013] This invention offers the following advantages: the rate of change in absorbance directly reflects the dynamic changes in absorbance; by calculating the rate difference between adjacent moments using a sliding time window, abnormal fluctuations in absorbance can be captured, providing a data foundation for subsequent interference elimination; through the optimal element pairing algorithm, the most temporally and morphologically related interference events in two feature sets are dynamically searched to form the optimal matching pair, which can characterize the magnitude of rate fluctuations caused by the same physical disturbance (such as particle flipping) at a certain moment at the target wavelength and reference wavelength respectively. This dynamic registration completely abandons the assumption of a fixed proportional coefficient and achieves adaptive dynamic response relationships of interference across different wavelengths. Registration; by using the response intensity calculated from the registration result and combining it with the reaction rate at the reference wavelength, the component of the target wavelength signal contributed by matrix interference at the current moment can be accurately calculated. Subtracting this component from the original target reaction rate achieves decoupling, resulting in a purer chemical reaction signal. This achieves precise decoupling of the signal and interference and quantitative assessment of signal reliability. After eliminating dynamic interference, the decoupling reaction rate is adjusted according to the reliability of the signal to obtain the corrected reaction rate. This dual protection mechanism minimizes non-specific interference caused by complex matrices, thereby improving the accuracy of obtaining the concentration of specific molecular markers in plant extracts. Attached Figure Description

[0014] Figure 1 This is a flowchart of a chemical analysis and detection method for plant-specific molecular markers provided in one embodiment of the present invention. Detailed Implementation

[0015] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the specific implementation methods, structures, features, and effects of the present invention are described in detail below with reference to the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All data and information collected in this application have been obtained with full consent.

[0017] This embodiment provides a chemical analysis and detection method for plant-specific molecular markers, used to obtain the concentration of specific molecular markers in plant extracts.

[0018] The process of the chemical analysis and detection method for plant-specific molecular markers provided in this embodiment is as follows: Figure 1 As shown, it includes the following steps: Step S1: Obtain the target reaction rate and reference reaction rate of the plant extract, respectively; Step S2: Based on the rate difference of reaction rates between adjacent times within the sliding time window at each time, obtain the target rate difference feature set and the reference rate difference feature set at each time. Step S3: Perform optimal element pairing on the target rate difference feature set and the reference rate difference feature set to obtain the optimal set of matching pairs and the set of unmatched elements; Step S4: Based on the optimal set of matching pairs and the set of unmatched elements, obtain the response intensity of the target wavelength relative to the reference wavelength at each time step, as well as the confidence level of the target reaction rate at each time step. Step S5: Based on the response intensity and the reference response rate, remove the interference component from the target response rate to obtain the decoupled response rate, and adjust the decoupled response rate through the confidence level to obtain the corrected response rate. Step S6: Obtain the concentration of specific molecular markers in the plant extract by correcting the reaction rate.

[0019] The following is a detailed explanation of each step.

[0020] Step S1: Obtain the target reaction rate and reference reaction rate of the plant extract.

[0021] First, plant extracts (such as astragalus extract, codonopsis extract, etc.) are obtained. The core of obtaining plant extracts is to break down the plant cell walls, allowing the active ingredients to dissolve in a solvent (such as water or ethanol). The extract is then obtained through separation and purification. In one exemplary embodiment, the cavitation effect of ultrasound can be used to break down the plant cell walls, combined with solvent extraction to obtain the plant extract. It should be understood that the specific molecular markers in the plant extracts extracted in this embodiment are related to the colorimetric method. Different colorimetric agents can target different molecular markers. For example, adding aluminum chloride reagent to the plant extract is mainly for detecting the total flavonoid content.

[0022] This embodiment sets a target time period, which is a monitoring period for the plant extract to obtain the concentration of a specific molecular marker in the plant extract. The duration of the target time period is set according to the actual situation; in this embodiment, it is set to 900 seconds.

[0023] This embodiment specifically involves a UV-Vis spectrophotometer, and initializes the kinetic scanning parameters of the UV-Vis spectrophotometer. The sampling period (i.e., sampling interval) in this embodiment... The setting should be adjusted according to the actual situation; this example uses 0.5 seconds as an example. Therefore, the total number of sampling points within the target time period, i.e., the number of time points, is 1800.

[0024] A colorimetric reagent (e.g., aluminum chloride reagent) is added to the plant extract and quickly mixed until homogeneous. The starting time of this homogeneous mixture is recorded as the starting time of the target time period, and the data acquisition program is initiated simultaneously. To capture the entire reaction process and monitor background changes in real time, this embodiment uses a discrete time index... (Time Index) Indicates the first [number]th [time period] within the target time period At that moment, , This indicates the number of moments within the target time period. The photometer is controlled to synchronously trigger signal readings from two optical channels. These two optical channels are the target detection channel and the background reference channel. The target detection channel sets the monochromator wavelength to the characteristic absorption peak position of the analyte molecular marker (the wavelength corresponding to the characteristic absorption peak position is used to define the target wavelength, e.g., 415 nm), collects absorbance readings at each moment, defines them as the target absorbance at each moment, and stores them in a target absorbance sequence according to time sequence, thus obtaining the target absorbance sequence for the target time period. Background reference channel: The monochromator wavelength is set to a wavelength position where the analyte molecular marker has no absorption but is sensitive to turbidity scattering (this wavelength is defined as the reference wavelength, e.g., 700 nm). Absorbance readings at various times are collected and defined as the reference absorbance at each time point. These are then stored in the reference absorbance sequence according to time sequence to obtain the reference absorbance sequence for the target time period. .

[0025] It should be understood that the target absorbance sequence and reference absorbance sequence The sequence lengths are all and the same index The target absorbance and reference absorbance belong to the same point in time, and this strict time alignment is the basis for subsequent data analysis.

[0026] Obtain the target absorbance sequence The rate of change of the target absorbance at each time step is defined as the target response rate at that time step. A reference absorbance sequence is obtained. The rate of change of the reference absorbance at each time step is defined as the reference reaction rate at that time step. In an exemplary embodiment, the target absorbance sequence is obtained. The first derivative of the target absorbance at each time step is used as the target reaction rate at that time step; similarly, the reference absorbance sequence is obtained. The first derivative of the reference absorbance at each time step is used as the reference reaction rate at each time step.

[0027] It should be understood that plant extracts inherently contain pigments and colloids, resulting in an initial absorbance background. To eliminate this constant term interference and provide standardized time-series data for subsequent feature analysis, this step involves simultaneous data acquisition and smoothing differential transformation. This is because the target absorbance sequence... and reference absorbance sequence The absorbance signal may not only contain the absorbance signal but may also be superimposed with the high-frequency electronic noise inherent in the photometer (such as white noise). To suppress this high-frequency noise while calculating the rate of change and prevent noise amplification during differential operations from degrading the signal-to-noise ratio, this embodiment can also perform analysis on the target absorbance sequence. and reference absorbance sequence For noise reduction, in one exemplary embodiment, a Savitzky-Golay digital filter is used. The specific parameter configuration of the Savitzky-Golay digital filter is as follows: Window width The selection principle is to suppress high-frequency electronic noise and preserve mid-frequency turbidity fluctuations. Preferably, the window width is set... It can cover approximately 0.5 to 1.0 times the typical bubble or flocculation disturbance cycle. In this embodiment, the window width is set. With seven time points (i.e., a 3.5-second time window), this width is sufficient to smooth out instantaneous electron pulses while preserving the morphological fluctuation characteristics caused by colloidal motion. Polynomial order. The order is set to 2, which is sufficient to fit the local response curve trend and avoid overfitting to high-frequency noise. (Derivative order) Set to order 1 for calculating the rate of change.

[0028] Target absorbance sequence and reference absorbance sequence Perform convolution operations separately for the target absorbance sequence. Any point in, with the first Taking the first moment as an example, the first... The window of time is The polynomial is fitted using the data within the window, and the polynomial is calculated at the center point (i.e., the nth polynomial). The first derivative value at time (i.e., the value of the first derivative at time i) is used as the first derivative value. The target reaction rate at each time point is calculated, and the target reaction rate at each time point is then arranged in chronological order to form a target reaction rate sequence. Similarly, for the reference absorbance sequence... Any point in, with the first Taking the first moment as an example, the first... The window of time is The polynomial is fitted using the data within the window, and the polynomial is calculated at the center point (i.e., the nth polynomial). The first derivative value at time (i.e., the value of the first derivative at time i) is used as the first derivative value. The reference reaction rate at each time step is obtained by taking the reference reaction rate at each time step, and then arranged in chronological order to form a reference reaction rate sequence. .

[0029] It should be understood that, based on the above window setting method, this will result in several starting positions in the reaction rate sequence, i.e., from the first time step to the second time step. The nth time point, and several end positions of the sequence, i.e., the nth... From the nth time to the Nth time, a complete window cannot be obtained. In order to keep the length N of the reaction rate sequence constant, these edge positions need to be filled. This embodiment can adopt a nearest neighbor numerical filling strategy: for several starting positions in the target reaction rate sequence, the calculated nth time... The target reaction rate for the next time step is assigned to the values ​​from the first time step to the second time step. The target reaction rate at time n; similarly, for several starting positions in the reference reaction rate sequence, the calculated target reaction rate at time n... The reference reaction rate for the next time step is assigned to the values ​​from time step 1 to time step 2. The reference reaction rate at time n; for several end positions in the target reaction rate sequence, the calculated first... The target reaction rate of the previous time step is assigned to the first time step. The target reaction rate from time 1 to time N; similarly, for several end positions in the reference reaction rate sequence, the calculated [response rate] will be [calculated]. The reference reaction rate from the previous time step is assigned to the first time step. The reference reaction rate from time 1 to time 2.

[0030] By using the above-mentioned complementary values, the target reaction rate sequence is made possible. and reference reaction rate sequence All are complete sequences containing N values, and all have the same index. The target reaction rate and the reference reaction rate are reaction rates at the same point in time, and the time bases are perfectly aligned.

[0031] Through the above processing, the transformation from static to dynamic is achieved: the pigment background of the constant term becomes zero after differentiation, while the chemical reaction manifests as a slowly changing rate baseline, and the turbidity interference manifests as mid-frequency fluctuations superimposed on the baseline.

[0032] Step S2: Based on the rate difference of reaction rates between adjacent times within the sliding time window at each time, obtain the target rate difference feature set and the reference rate difference feature set at each time.

[0033] Step S2 involves the target reaction rate sequence. and reference reaction rate sequence The analysis aimed to separate signal abrupt changes caused by turbidity from a background of smooth chemical reactions, and to analyze the interference through dual-channel matching.

[0034] Step S2 uses any moment within the target time period as the analysis object for subsequent data analysis, using the first... Taking the first moment as an example. Determine the first... The sliding time window at the nth moment, the th The sliding time window at the nth moment is the time window with respect to the nth moment. Centered at a time, with a radius of The sliding time window. Among them, The size of the sliding time window is set according to actual needs. In this embodiment, taking 7 as an example, the length of the sliding time window is 15 moments. It should be understood that for the first 7 moments and the last 7 moments in the target time period, a complete sliding time window cannot be obtained. In this case, the actual window size can be used as the corresponding sliding time window.

[0035] Determine the target reaction rate sequence The first in The target reaction rate at each time point within the sliding time window at time point n is obtained. The rate difference of the target reaction rate at each time point within a sliding time window. The rate difference characterizes the rate difference between adjacent target reaction rates within the sliding time window. In an exemplary embodiment, to separate mid-to-high frequency turbidity interference from a signal containing low-frequency chemical reaction trends, this embodiment utilizes second-order difference operations to extract morphological abrupt change features of the signal. Second-order difference mathematically corresponds to curvature or acceleration, effectively suppressing linear reaction baselines while significantly amplifying sudden non-stationary fluctuations. Accordingly, the rate difference of the target reaction rate is specifically the second-order difference value of the target reaction rate. (The text then repeats the first instance, which is likely an error in the original.) The first moment within the sliding time window at the nth moment Taking a specific time point as an example, calculate its second-order difference value: ; in, Indicates the first The rate difference of the target reaction rate at each moment. Indicates the first The target reaction rate at each moment Indicates the first The target reaction rate at each moment Indicates the first The target reaction rate at each moment.

[0036] It should be understood that this embodiment no longer calculates the rate difference of the target reaction rate at the first time step and the rate difference of the target reaction rate at the last time step within the sliding time window. The rate difference of the target reaction rate at these two time steps does not participate in subsequent judgments. Thus, the [previous calculation] is obtained. The rate difference of the target reaction rate at each time point within the sliding time window. For any rate difference, it may be positive, negative, or 0. The larger the absolute value of the rate difference at time step n, the greater the rate difference at time step n. The greater the difference in the target reaction rate between the first moment and the moments before and after, the greater the difference in the target reaction rate between the first moment and the moments before and after. The greater the fluctuation in the target reaction rate at any given moment, the more pronounced the fluctuation characteristics.

[0037] Similarly, the above process is used to determine the reference reaction rate sequence. The first in The rate difference of the reference reaction rate at each time point within the sliding time window is specifically the second-order difference of the reference reaction rate. The specific acquisition process is exactly the same as the acquisition process of the rate difference of the target reaction rate mentioned above.

[0038] Regarding the rate difference of the target reaction rate, this embodiment sets a first preset difference threshold. This first preset difference threshold is used to compare with the absolute value of the rate difference of the target reaction rate at each moment within the sliding time window to determine whether the absolute value of the rate difference of the target reaction rate at each moment within the sliding time window is high, thereby obtaining whether the fluctuation degree of the target reaction rate at each moment within the sliding time window is high. This first preset difference threshold is set according to actual needs, such as a preset fixed threshold based on the static baseline of the photometer for the target absorbance in a laboratory environment.

[0039] Judge the first Within a sliding time window, the absolute value of the rate difference of the target reaction rate at each time step is determined. If the absolute value of the rate difference is greater than the first preset difference threshold, the rate difference of the target reaction rate at each time step corresponding to the absolute value of the rate difference greater than the first preset difference threshold is obtained, thus constituting the rate difference of the target reaction rate at each time step. The target rate difference feature set at time n, then, the nth The target rate difference feature set at time n includes the first Within a sliding time window of time 1, the rate difference of the target reaction rate at each time corresponding to the absolute value of the rate difference greater than the first preset difference threshold. Then, the rate difference of the target reaction rate at each time 1. Each element in the target rate difference feature set at each moment records the time when the fluctuation occurs and the fluctuation intensity, which is the rate difference of the target reaction rate.

[0040] To address the rate difference in the reference reaction rate, this embodiment sets a second preset difference threshold. This second preset difference threshold is used to compare with the absolute value of the rate difference of the reference reaction rate at each moment within the sliding time window, in order to determine whether the absolute value of the rate difference of the reference reaction rate at each moment within the sliding time window is high, thereby determining whether the fluctuation degree of the reference reaction rate at each moment within the sliding time window is high. This second preset difference threshold is set according to actual needs, such as in a laboratory environment, based on a preset fixed threshold of the static baseline of the photometer for the reference absorbance.

[0041] Judge the first The absolute value of the rate difference of the reference reaction rate at each time point within the sliding time window is determined by whether it is greater than a second preset difference threshold. The absolute values ​​of rate differences greater than the second preset difference threshold are then used to obtain the rate differences of the reference reaction rate at each time point corresponding to these absolute values, thus forming the first... Given the reference rate difference feature set at time n, then, the i-th The reference rate difference feature set at time n includes the nth time n. Within a sliding time window of time 1, the rate difference of the reference reaction rate at each time corresponding to the absolute value of the rate difference greater than the second preset difference threshold. Then, the rate difference of the reference reaction rate at each time 2. Each element in the reference rate difference feature set at each moment records the time when the fluctuation occurred and the fluctuation intensity, which is the rate difference of the reference reaction rate.

[0042] Step S3: Perform optimal element pairing on the target rate difference feature set and the reference rate difference feature set to obtain the optimal set of matching pairs and the set of unmatched elements.

[0043] Since actual detection may involve non-homogeneous noise (such as unilateral bubbles or electron pulses) that only appear in a single channel, it is necessary to filter out fluctuations that appear simultaneously in two channels and have the same morphology in order to pinpoint the true turbidity interference. This step uses a bipartite graph matching algorithm to establish a correlation between the target rate difference feature set and the reference rate difference feature set, thereby performing optimal element pairing on the target rate difference feature set and the reference rate difference feature set to obtain the optimal set of matching pairs and the set of unmatched elements.

[0044] If we consider the target rate difference feature set as the left-hand nodes of the bipartite graph and the reference rate difference feature set as the right-hand nodes, then the bipartite graph consists of multiple left-hand nodes, each representing an element from the target rate difference feature set, and multiple right-hand nodes, each representing an element from the reference rate difference feature set.

[0045] A cost matrix is ​​constructed, where each element represents the matching resistance between feature pairs. In this embodiment, each element is the time interval between any left-hand node and any right-hand node. It should be understood that the cost matrix needs to be expanded into a square matrix, the size of which is the maximum of the number of elements in the target rate difference feature set and the number of elements in the reference rate difference feature set. The cost matrix is ​​initialized with all elements set to a very large value (e.g., much larger than the length of the sliding time window), indicating that matching is not allowed. It should be understood that through the above cost matrix expansion, the elements in the cost matrix can be divided into real elements and virtual elements. The cost value of a real element is the actual time interval between two nodes, while the cost value of a virtual element is a constant much larger than all possible real costs (e.g., set to a constant much larger than the length of the sliding time window), indicating that the cost is abandoned.

[0046] The predefined constraints for matching left and right nodes in a bipartite graph are as follows: both nodes must be positive or both must be negative, and the time interval between the left and right nodes must be less than a predefined interval threshold. Here, both being positive or both being negative indicates polarity consistency, meaning the values ​​of the two nodes in the matching pair are both positive or both are negative, i.e., the signs of the two rate differences are the same (this embodiment may exclude the case where both are 0). The time interval between the left and right nodes being less than the predefined interval threshold indicates temporal synchronization. The predefined interval threshold is set according to actual needs. In this embodiment, the predefined interval threshold is the duration of one sampling period or two sampling periods. This predefined interval threshold not only allows for sampling jitter but also compensates for the slight time lag that may be caused by instrument optical path switching.

[0047] To minimize the total cost of the cost matrix, a minimum weight matching algorithm (such as the Hungarian algorithm) is used to solve the cost matrix, resulting in several optimal matching pairs that satisfy the aforementioned preset constraints. In each optimal matching pair, the two nodes have the same polarity, their time interval is less than a preset interval threshold, and the sum of their overall time intervals is minimized.

[0048] All the best matching pairs obtained are added to the optimal matching pair set, thus obtaining the first set of best matching pairs. The optimal matching pair set at time n is obtained. Simultaneously, the remaining unmatched rate differences in the target rate difference feature set and the reference rate difference feature set are obtained; these are the rate differences not included in the optimal matching pair set. These rate differences not included in the optimal matching pair set are then added to the unmatched element set, thus obtaining the nth... The set of unmatched elements at each time step. Then, all elements in the target rate difference feature set and the reference rate difference feature set are divided into two sets: the set of optimal matching pairs and the set of unmatched elements. This ensures that subsequent calculations are based only on the strongly correlated homologous fluctuations of the two channels, eliminating random noise.

[0049] Step S4: Based on the optimal set of matching pairs and the set of unmatched elements, obtain the response intensity of the target wavelength relative to the reference wavelength at each time step, as well as the confidence level of the target reaction rate at each time step.

[0050] According to the The optimal set of matching pairs and the set of unmatched elements at time i are obtained to obtain the set of... The response intensity of the target wavelength relative to the reference wavelength at time n, and the response intensity of the target wavelength relative to the reference wavelength at time n. The credibility of the target reaction rate at each moment.

[0051] Among them, the The response intensity of the target wavelength relative to the reference wavelength at time t is quantized at time t. At time 1, the ratio of the turbidity interference response intensity at the target wavelength to the reference wavelength is calculated using only the optimal matching pair set. The calculation process for the response intensity of the target wavelength relative to the reference wavelength at each moment is as follows: Calculate the first The sum of the absolute values ​​of the rate differences of all target response rates in the optimal matching pair set at time n is defined as the first sum; the calculation of the second sum is... The sum of the absolute values ​​of the rate differences among all reference reaction rates in the optimal matching pair set at time n is defined as the second sum. The ratio of the first sum to the second sum is calculated to obtain the second sum. The response intensity of the target wavelength relative to the reference wavelength at each moment: ; in, Indicates the first The response intensity of the target wavelength relative to the reference wavelength at time t is, based on the above calculation method, essentially a dual-wavelength turbidity response ratio; M represents the response intensity of the target wavelength relative to the reference wavelength at time t. The set of optimal matching pairs at each time step. This indicates that in the set of optimal matching pairs, the pair consisting of the first matching pair is the one that matches the second matching pair. The rate difference of the target reaction rate and the first target reaction rate The optimal matching pair is formed by the rate difference of a reference reaction rate. Indicates the first The rate difference (i.e., fluctuation intensity) of the target reaction rate. This represents the function that takes the absolute value. Indicates the first The rate difference between the reference reaction rates; This represents the first sum, i.e., the first... The sum of the absolute values ​​of the rate differences of all target response rates in the set of optimal matching pairs at each time step. This represents the second sum, i.e., the th sum. The sum of the absolute values ​​of the rate differences of all reference reaction rates in the set of optimal matching pairs at each time point; To prevent extremely small positive numbers from being divided by zero, such as The dimensions are the same as the denominator.

[0052] It should be understood that the response intensity may be different at different times, and the response intensity is time-varying. If the colloidal particles aggregate or change their morphology, the scattering spectrum characteristics will change accordingly, and the response intensity will automatically change and update.

[0053] In addition, if the first The set of optimal matching pairs at time t is empty. To ensure the temporal order of the response strength, the set of optimal matching pairs at time t is [missing information]. The response strength at time step 1 remains at the level of time step 2. The response strength at time 1. In addition, if the set of best matching pairs at time 1 is empty, the response strength at time 1 can be set to a preset initial value, such as 1.0.

[0054] No. The reliability of the target reaction rate at time 1 needs to be combined with the first time step. The set of optimal matching pairs at time n and the set of best matching pairs at time n The calculation is performed on the set of unmatched elements at each time point, specifically: Calculate the first The sum of squared rate differences for each optimal matching pair in the set of optimal matching pairs at time n is calculated, and then the sum of squared rate differences for the th optimal matching pair is calculated. The sum of the squared rate differences of all optimal matching pairs in the set of optimal matching pairs at time n is defined as the third sum.

[0055] Calculate the first The sum of the squares of all rate differences in the set of unmatched elements at time t is multiplied by a preset penalty coefficient, and then added to a third sum. The result is defined as the fourth sum.

[0056] Calculate the ratio of the third sum to the fourth sum to obtain the third sum. The reliability of the target reaction rate at each moment: ; in, Indicates the first The reliability of the target reaction rate at a given time point. Represents the optimal matching pair The sum of squared rate differences, E represents the first... The set of unmatched elements at each moment. Indicates the first The first unmatched element in the set at time n One rate difference, Indicates the first The sum of squares of all rate differences in the set of unmatched elements at time n; e is the sum of squares of the rates in the set of unmatched elements at time n. The index of the rate difference in the set of unmatched elements at each moment; This indicates the preset penalty coefficient. The range of values ​​is to The specific value should be set according to the actual situation. For example, when there are many air bubbles in the sample or the matrix is ​​extremely uneven, a larger value should be used. (e.g., 1.5). In When the value is large, the non-matching terms in the denominator (i.e.) The weights of the ) are amplified, resulting in a significant decrease in the calculation results. This means that once one-sided noise appears, the reliability will be severely reduced. To prevent extremely small positive numbers from being divided by zero, such as The dimensions are the same as the denominator.

[0057] As can be seen from the above calculation formula, the reliability value ranges from 0 to 1. The closer it is to 1, the more reliable the data is, indicating that the interference is mainly from the same source. The closer it is to 0, the more reliable the data is, indicating that there is a lot of non-same-source noise.

[0058] The response intensities at each time point are arranged in time sequence to obtain the response intensity sequence. The confidence scores obtained at each time point are arranged in chronological order to obtain a confidence score sequence. Response intensity sequence and credibility sequence These are two parameter sequences of equal length (length N) and strictly aligned. Response intensity sequence Provides dynamic scaling for background subtraction and a credibility sequence. It provides a reliability index for judging data quality.

[0059] Step S5: Based on the response intensity and the reference response rate, remove the interference component from the target response rate to obtain the decoupled response rate, and adjust the decoupled response rate through the confidence level to obtain the corrected response rate.

[0060] Based on the target reaction rate sequence and reference reaction rate sequence and response intensity sequence and credibility sequence The process involves background subtraction and signal restoration, ultimately outputting a quantitative absorbance result.

[0061] First, according to the The response strength at time t and the t The reference reaction rate at time t, from the t... After removing the interference component from the target reaction rate at time t, we obtain the t-th time. The decoupling reaction rate at each moment.

[0062] It should be understood that, assuming at a specific moment, i.e. the first... At any given moment, the response intensity of physical turbidity interference at the target wavelength and the reference wavelength follows a linear proportional relationship, i.e. .in, Indicates physical turbidity interference in the first... The response intensity of the target wavelength at each moment. Indicates the first The linear proportion at each moment, Indicates physical turbidity interference in the first... The response intensity at the reference wavelength at each moment. According to the linear law of calculus, if the original function satisfies a linear ratio... ( If the change is relatively slow, then the first derivative (i.e., the rate of change) and the second derivative (i.e., the acceleration) of the interference signal approximately satisfy the same proportionality coefficient between the two wavelengths. Therefore, the response intensity obtained in step S4 can be mathematically applied to the decoupling correction of the first-order rate signal in this step.

[0063] In an exemplary embodiment, according to the first The response strength at time t and the t The reference reaction rate at time n is obtained to get the nth time. The disturbance component at time step n. The disturbance component is positively correlated with both the response intensity and the reference response rate; then, the disturbance component at time step n... The target reaction rate at time n minus the first The interference component at time i is obtained. The decoupling reaction rate at each time point is calculated using the following formula: ; in, Indicates the first The decoupling reaction rate at time 1 Indicates the first The target reaction rate at each moment Indicates the first The reference reaction rate at that time point Indicates the first The formula represents the response intensity sequence at each time step. It performs a dynamic subtraction operation. Indicates the first The interference components at time t were estimated. The turbidity interference rate component hidden in the target signal at a given time moment is obtained by subtracting this component. Theoretically, it only includes chemical reaction rates and residual non-homogeneous noise.

[0064] Although the decoupled reaction rate obtained after the above processing removes common turbidity interference, it may still contain one-sided random strong noise (such as unmatched isolated bubble spikes). In order to eliminate these residual noises and maintain the continuity of the reaction curve, this step also needs to perform adaptive repair using the confidence level of the target reaction rate. In order to prevent residual strong noise from skewing the repair benchmark (i.e., solving the logic deadlock problem), this embodiment specifically uses a moving median filtering algorithm to construct a trend benchmark, rather than a traditional moving average.

[0065] Set any time as a candidate time, and use the first time as the candidate time. Taking the first moment as an example, let's take the first moment as an example. Centered on the [time], determine the [time]th [time]. The preset repair window is set for each time period. The size of the preset repair window is set according to actual needs. In this embodiment, the size of the preset repair window is taken as 5 time periods, that is, the length of the preset repair window is 5. It should be understood that for the first 2 time periods and the last 2 time periods in the target time period, a complete preset repair window cannot be obtained. In this case, the actual length of the preset repair window obtained shall prevail.

[0066] Extract the first The decoupling reaction rate at each time step within the preset repair window at time step 1 is obtained, thereby acquiring the 1st time step 2. The median of the decoupling reaction rates at all times within the preset repair window at time n is used as the median of the decoupling reaction rates at time n. The median filter exhibits excellent anti-impulse interference characteristics. Even if there are large-amplitude one-sided noise spikes (outliers) within the preset repair window, the median can still be stably anchored on the normal baseline of the reaction kinetics and will not be skewed by noise, ensuring that the trend baseline reaction rate is always a pure reference.

[0067] According to the The credibility at time n is obtained respectively. The decoupling reaction rate at time step 1, and the decoupling reaction rate at time step 2. The weighted value of the trend baseline reaction rate at time step n. It should be understood that when the nth time step n... The higher the confidence level at time step 1 (i.e., the closer it is to 1), the purer the signal. The more reliable the decoupling reaction rate at each moment, the higher its weight, thus preserving the subtle details of the actual chemical reaction; when the... The lower the confidence level at a given moment (i.e., the closer it is to 0), the stronger the interference in the signal, and the more effectively the signal is shielded. The measured value at the first moment (because this value may have been contaminated by noise) was instead used as the primary reference. The trend baseline reaction rate at each time point. Since the trend baseline reaction rate is a robust value generated by median filtering, this replacement effectively fills in the data gaps caused by noise with the dynamic trend of the reaction curve, thus achieving smooth repair. Therefore, the weight values ​​of the decoupled reaction rate are positively correlated with confidence, while the weight values ​​of the trend baseline reaction rate are inversely correlated with confidence.

[0068] In an exemplary embodiment, due to the first If the confidence level at the moment is a dimensionless value ranging from 0 to 1, then in this embodiment, the confidence level at the moment is directly... The credibility at the moment is used as the _th_ moment The weighted value of the decoupling reaction rate at time 1 is used to compare the value 1 with the weighted value of the decoupling reaction rate at time 2. The difference in credibility at time 1 is used as the first time. The weight value of the trend benchmark reaction rate at each moment.

[0069] Finally based on the first The decoupling reaction rate at time t and the t The weight value of the trend baseline reaction rate at time t, for the t... The decoupled reaction rate at time step i is weighted and summed with the trend baseline reaction rate to obtain the i-th time step. The corrected reaction rates at each time point are as follows: ; in, Indicates the first The corrected reaction rate at time 1. Indicates the first The trend baseline reaction rate at each moment; Indicates the first The credibility of the target reaction rate at each moment.

[0070] Using the above process, the corrected reaction rate at each moment within the target time period is obtained. The corrected reaction rate is used as a high-quality rate after decoupling and repair, thus obtaining the corrected reaction rate sequence of the target time period in time sequence. The modified reaction rate sequence Background turbidity and random noise have been eliminated, accurately reflecting the kinetic process of the chemical reaction itself.

[0071] Step S6: Obtain the concentration of specific molecular markers in the plant extract by correcting the reaction rate.

[0072] Based on the corrected reaction rate sequence for the target time period This process reduces the reaction rate from the rate domain to the absorbance domain, and ultimately calculates the concentration of specific molecular markers in the plant extract. This is achieved by modifying the reaction rate sequence. This represents the absorbance increment (i.e., the rate of change of absorbance) at each moment within the target time period. To obtain the total net absorbance generated within the target time period, it is necessary to sum the values ​​over the entire time domain of the target time period. Therefore, based on the corrected response rate at each moment within the target time period, the cumulative absorbance value for the target time period is calculated through cumulative summation.

[0073] In one exemplary embodiment, the modified reaction rate sequence Perform cumulative calculations. To avoid the minor errors caused by edge padding affecting the results, the interval is preferably set to avoid the effective data segments in the beginning and end padding areas, i.e., starting from the index. arrive This serves as the valid data segment for the target time period. It should be understood that, due to... Typically very small (such as the 3.5 seconds set above), relative to the entire reaction process, i.e. the duration of the target time period (such as 900 seconds), the few seconds of data truncated at the beginning and end have a negligible impact on the total (accounting for less than 1%). Therefore, ignoring marginal data will not affect the accuracy of quantitative analysis.

[0074] Within the valid data segment, the average corrected reaction rate is calculated for every two adjacent time points. Then, the average corrected reaction rates within the valid data segment are summed to obtain the cumulative absorbance value. The calculation formula is as follows: ; in, The cumulative absorbance value for the target time period represents the total net light absorption produced solely by the colorimetric reaction after deducting background and noise. This indicates the start time within the target time period, i.e., the start time of the valid data segment. ; This indicates the end time within the target time period, i.e., the end time of the valid data segment. ; Indicates the first The corrected reaction rate at time 1. This represents the sampling interval, which is 0.5 in this embodiment of the invention.

[0075] Finally, the concentration of a specific molecular marker in the plant extract is obtained from the cumulative absorbance value over the target time period using a preset conversion algorithm. The conversion algorithm between absorbance and marker concentration is a conventional technique; in this embodiment, the preset conversion algorithm is a pre-established standard curve equation, which typically takes the form of… ,in, This is the output quantity, representing the cumulative absorbance value. This is the input quantity, representing the concentration of the marker. Indicates the slope. This indicates the intercept. It should be understood that the conversion between absorbance and marker concentration here... and The value is a pre-set known value. The calculated cumulative absorbance value for the target time period will be used. Substitute into the equation: ; in, This indicates the calculation result, namely the concentration of a specific molecular marker in the plant extract. Through the above-described complete process, this invention achieves accurate chemical analysis results from high-viscosity, high-turbidity plant extracts using only mathematical algorithms, without the need for physical impurity removal (such as centrifugation or filtration).

[0076] This embodiment also provides a chemical analysis and detection system for plant-specific molecular markers, including: a memory and a processor; the memory is connected to the processor, and the memory is used to store program instructions; the processor is used to implement the steps in the above-described embodiment of the chemical analysis and detection method for plant-specific molecular markers when the program instructions are executed.

[0077] In one exemplary embodiment, the present invention provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps in the above-described embodiments of the chemical analysis and detection method for plant-specific molecular markers.

[0078] It should be noted that the order of the above embodiments of the present invention is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. The processes depicted in the accompanying drawings do not necessarily require a specific or sequential order to achieve the desired result. In some embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0079] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

Claims

1. A chemical analytical detection method for plant-specific molecular markers, characterized in that, include: The target reaction rate and reference reaction rate of the plant extract were obtained separately; the target reaction rate is the rate of change of absorbance at a target wavelength, and the reference reaction rate is the rate of change of absorbance at a reference wavelength. Based on the rate difference of reaction rates between adjacent times within the sliding time window at each time, the target rate difference feature set and the reference rate difference feature set at each time are obtained respectively. Optimal element pairing is performed on the target rate difference feature set and the reference rate difference feature set to obtain the optimal set of matching pairs and the set of unmatched elements. Based on the optimal set of matching pairs and the set of unmatched elements, the response intensity of the target wavelength relative to the reference wavelength at each time step, as well as the confidence level of the target reaction rate at each time step, are obtained. Based on the response intensity and the reference response rate, the interference component is removed from the target response rate to obtain the decoupled response rate, and the decoupled response rate is adjusted by the confidence level to obtain the corrected response rate. The concentration of specific molecular markers in plant extracts was obtained by modifying the reaction rate. Wherein, the rate difference of the target reaction rate at adjacent moments within the sliding time window is the second-order difference of the target reaction rate within the sliding time window; the rate difference of the reference reaction rate at adjacent moments within the sliding time window is the second-order difference of the reference reaction rate within the sliding time window. The process of obtaining the response intensity includes: determining a first sum and a second sum, wherein the first sum is the sum of the absolute values ​​of the rate differences of all target response rates in the optimal matching pair set, and the second sum is the sum of the absolute values ​​of the rate differences of all reference response rates in the optimal matching pair set; and calculating the ratio of the first sum to the second sum to obtain the response intensity. The method for obtaining the optimal set of matched pairs and the set of unmatched elements is as follows: the target rate difference feature set is used as the left node of the bipartite graph, and the reference rate difference feature set is used as the right node of the bipartite graph; a cost matrix is ​​constructed, where each element of the cost matrix is ​​the time interval between any left node and any right node; with the goal of minimizing the total cost, the cost matrix is ​​solved using the minimum weight matching algorithm to obtain the best matched pairs that satisfy the preset constraints, which constitute the optimal set of matched pairs. The remaining unmatched rate differences in the target rate difference feature set and the reference rate difference feature set constitute the set of unmatched elements; the preset constraints are: both are positive or both are negative, and the time interval between the left node and the right node is less than a preset interval threshold.

2. The chemical analysis and detection method for plant-specific molecular markers as described in claim 1, characterized in that, The process of obtaining the target rate difference feature set includes: Determine whether the absolute value of the difference in the target reaction rate at each moment within the sliding time window is greater than a first preset difference threshold. Obtain a target rate difference feature set, which includes the rate differences of the target reaction rate at each time point that are greater than the first preset difference threshold; The process of obtaining the reference rate difference feature set includes: Determine whether the absolute value of the rate difference of the reference reaction rate at each moment within the sliding time window is greater than the second preset difference threshold. Obtain a reference rate difference feature set, which includes the rate difference of the reference response rate at each time point greater than the second preset difference threshold.

3. The chemical analysis and detection method for plant-specific molecular markers as described in claim 1, characterized in that, The process of obtaining the credibility includes: The third sum and the fourth sum are determined. The third sum is the sum of the squares of the rate differences of each optimal matching pair in the optimal matching pair set. The process of obtaining the fourth sum is as follows: calculate the squares of all rate differences in the unmatched element set, multiply by a preset penalty coefficient, and then add the third sum. The confidence level is obtained by calculating the ratio of the third sum to the fourth sum.

4. The chemical analysis and detection method for plant-specific molecular markers as described in claim 1, characterized in that, The process of obtaining the decoupling reaction rate includes: The interference component is obtained based on the response intensity and the reference response rate; the interference component is positively correlated with both the response intensity and the reference response rate. The decoupled reaction rate is obtained by subtracting the interference component from the target reaction rate.

5. The chemical analysis and detection method for plant-specific molecular markers as described in claim 1, characterized in that, The process of obtaining the modified reaction rate includes: The median of the decoupling response rates at all times within a preset repair window of the candidate time is determined as the trend benchmark response rate of the candidate time; the candidate time can be any time. Based on the confidence level, the weight values ​​of the decoupling response rate and the trend baseline response rate at the candidate time are obtained; the weight value of the decoupling response rate is positively correlated with the confidence level, and the weight value of the trend baseline response rate is negatively correlated with the confidence level. Based on the weight values ​​of the decoupled reaction rate and the trend benchmark reaction rate, the decoupled reaction rate and the trend benchmark reaction rate at the candidate time are weighted and summed to obtain the corrected reaction rate at the candidate time.

6. The chemical analysis and detection method for plant-specific molecular markers as described in claim 1, characterized in that, The method of obtaining the concentration of specific molecular markers in plant extracts by modifying the reaction rate includes: The cumulative absorbance value for the target time period is obtained based on the corrected reaction rate at each time point within the target time period. The concentration of a specific molecular marker in the plant extract is obtained from the cumulative absorbance value according to a preset conversion algorithm.

7. The chemical analysis and detection method for plant-specific molecular markers as described in claim 6, characterized in that, The process of obtaining the cumulative absorbance value includes: Determine the average corrected reaction rate for every two adjacent moments within the target time period; The cumulative absorbance value is obtained by summing the average corrected reaction rate over the target time period.

Images