A method for quantitatively analyzing aldose reductase activity and its inhibition

Abstract

The present application relates to aldose reductase activity analysis technical field, specifically relates to a kind of aldose reductase activity and its inhibitory effect quantitative analysis method.Local substrate concentration regulation is constructed by dividing multiple detection segments, combining bypass sampling channel, electrochemical detection cavity and compensation injection point, and the substrate environment of each detection segment is maintained consistent;Enzyme-free reference channel parallel to microfluidic reaction channel is set, and the total absorbance change amount background is separated, to obtain the real enzymatic absorbance change amount;Based on sliding time window, extract local dynamic enzyme reaction rate, combined with wall adsorption correction to eliminate the influence of enzyme activity attenuation along the way, identify stable reaction interval through local fluctuation coefficient of variation, determine local half response time and local onset time;Local onset time is used as the integration starting point, to calculate comprehensive inhibition intensity, realize inhibitor onset order and inhibition strength spatial quantitative analysis, improve the accuracy, stability and spatial comparability of detection result.

Description

Technical Field

[0001] This invention relates to the field of aldose reductase activity analysis technology, specifically to a quantitative analysis method for aldose reductase activity and its inhibitory effect. Background Technology

[0002] The detection of aldose reductase activity and its inhibitory effects is of great significance in research on diabetic complications, inhibitor screening, and enzyme kinetic analysis. Especially within microfluidic reaction channels, substrates are continuously consumed along the flow direction, naturally resulting in gradients in local substrate concentrations at different locations. Simultaneously, samples within the channels are affected by dead volume, diffusion distortion, transport delays, and local flow field disturbances, leading to inconsistent reaction conditions at different locations at the same time. Directly placing the detection structure within the reaction channel may further alter the local flow field distribution, residence time, and enzymatic kinetics, causing the detection results to deviate from the true reaction state. Furthermore, aldose reductase reactions are usually accompanied by changes in NADPH absorbance, while the background signal in the microfluidic chip is superimposed with non-enzymatic factors such as coenzyme natural decay, optical path differences, channel optical drift, and baseline drift. Without an independent background channel, it is difficult to separate the true enzymatic changes from background fluctuations. In addition, aldose reductase may adsorb and remain on the channel walls, causing a gradual decrease in the effective enzyme concentration along the path, making direct comparison of the local dynamic responses of different detection segments impossible. Furthermore, the combined effects of inhibitor transport attenuation, diffusion loss, and onset lag within the channel can easily include periods that have not yet truly entered a stable inhibitory state in the statistics, which can affect the accuracy of the determination of inhibitory intensity. Summary of the Invention

[0003] To address the aforementioned shortcomings of existing technologies, this invention provides a quantitative analysis method for aldose reductase activity and its inhibitory effect. This method effectively solves the problems in existing technologies where inconsistent local substrate concentrations within microfluidic reaction channels, aldose reductase adsorption attenuation on the reaction wall, and asynchronous inhibitor activation lead to difficulties in accurately comparing segmented enzymatic responses and quantifying inhibitory intensity.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a quantitative analytical method for aldose reductase activity and its inhibitory effect, comprising at least: The microfluidic reaction channel is divided into multiple detection segments, and a bypass sampling channel and an electrochemical detection chamber are set up. Fluid is drawn out through a bypass sampling channel to the electrochemical detection chamber to detect local substrate concentration; The local substrate concentration is compared with the preset target substrate concentration to determine the substrate injection amount at the corresponding compensation injection point; An enzyme-free reference channel was set up for each detection segment. The total absorbance change and the background natural attenuation absorbance change were collected separately in combination with the reaction channel. The actual enzymatic absorbance change for the corresponding detection segment was calculated. Based on the sliding time window, the actual change in absorbance between consecutive time points is calculated, and the local dynamic enzyme reaction rate of each detection segment is obtained by combining the Lambert-Beer law. The local dynamic enzyme reaction rate is calculated by the local fluctuation variation coefficient of each detection segment within the sliding time window, and the corresponding local stability indicator is determined when the local fluctuation variation coefficient is lower than the preset stability threshold. Within the stable interval indicated by the local stability marker, the local half-response time of each detection segment is identified, and the true local onset time is determined as the integration starting boundary. The local dynamic enzyme reaction rate corresponding to the inhibited state and the reference state is integrated to obtain the comprehensive inhibition intensity corresponding to each detection segment.

[0005] Furthermore, including: A bypass sampling channel is set up in each detection segment, and the bypass sampling flow rate is controlled to be less than the flow rate of the reaction channel formed by the total liquid volume in the corresponding detection segment. A constant-volume transport section is set up. Based on the channel length and fluid flow velocity of the bypass sampling channel, the transmission delay time is determined. Combined with the analysis time of the reaction channel, the concentration reading time is determined, and the local substrate concentration is output. Based on the electrode sensitivity coefficient and background current, the local substrate concentration corresponding to the detection segment is obtained.

[0006] Furthermore, when determining the substrate injection volume at the compensation injection point, it also includes ensuring uniform substrate mixing by controlling the compensation injection point: A mixing section is set after each compensation injection point; Multiple sampling locations are set at the outlet of the mixing section, and the local substrate concentrations at the multiple sampling locations are measured to calculate the average substrate concentration. Determine the difference between the local substrate concentration at each sampling location and the average substrate concentration at the outlet of the mixing section; Establish the standard deviation of the substrate concentration at the outlet of the mixing section; The concentration variation coefficient at the outlet of the mixing section is obtained by using the standard deviation and the average substrate concentration at the outlet of the mixing section; When the concentration variation coefficient at the outlet of the mixing section is less than or equal to the threshold, the current mixing section is determined to meet the mixing conditions.

[0007] Furthermore, after obtaining the local dynamic enzyme reaction rate of each detection segment using the Lambert-Beer law, wall adsorption correction is also included, specifically: Calculate the path length from the center of each detection segment to the inlet of the microfluidic reaction channel; Divide the path length by the total geometric length of the microfluidic reaction channel to obtain the normalized path position; The normalized path position and the preset wall adsorption coefficient are used to obtain the corresponding wall adsorption correction coefficient. The adsorption-corrected local dynamic enzyme reaction rate is obtained by comparing the local dynamic enzyme reaction rate with the wall adsorption correction coefficient.

[0008] Furthermore, the method for determining the local onset time is as follows: Within a sliding time window, the local dynamic enzyme reaction rates after adsorption correction at multiple time points are summed, and the average value of the window is obtained by the ratio of the summation value to the number of sampling points. Calculate the difference between the local dynamic enzyme reaction rate after each adsorption correction and the window average value. Sum the squares of each difference, divide by the number of sampling points, and take the square root to obtain the window standard deviation. Based on the aforementioned standard deviation and mean, a local fluctuation variation coefficient is constructed; When the local fluctuation variation coefficient is lower than the preset stability threshold, the corresponding time period is marked as a local stability indicator; The adsorption-corrected local dynamic enzyme reaction rates at multiple moments within a preset time window before the addition of the inhibitor are summed and divided by the number of sampling points to obtain the average enzyme reaction rate. A half-response threshold is established by comparing the average enzyme reaction rate with a preset efficacy determination coefficient. The time point at which the local dynamic enzyme reaction rate, after adsorption correction, first drops to the half-response threshold is identified and determined as the local half-response time. Subtracting the inhibitor addition time from the local half-response time yields the local inhibition hysteresis index; The time decay coefficient is combined with the local inhibition hysteresis exponent for exponential decay. The effective inhibitor concentration is then established by multiplying the initial inhibitor injection concentration with the exponential decay result. The local onset time is obtained by using the local inhibition hysteresis index, the effective inhibitor concentration, and the time of inhibitor addition.

[0009] Further, the method for obtaining the overall suppression strength includes: The local onset time is used as the starting boundary for integration; Within the stable range defined by the local stability marker, the local dynamic enzyme reaction rate after adsorption correction under the inhibited state and the local dynamic enzyme reaction rate after adsorption correction under the reference state are obtained, a ratio is established, and the instantaneous degree of inhibition corresponding to each time point is calculated. The instantaneous inhibition level at each moment is combined with the corresponding time interval and then summed to obtain the integral value of the inhibition effect. By combining the local stability markers at each moment with the corresponding time intervals, a normalized reference integral value is established. The ratio of the integral value of the inhibition effect to the integral value of the normalized reference value constitutes the overall inhibition strength.

[0010] The technical solution provided by this invention has the following advantages compared with the known prior art: By dividing the microfluidic reaction channel into multiple detection segments and setting up a bypass sampling channel, an electrochemical detection chamber, a compensation injection point, and an enzyme-free reference channel in each detection segment, the local substrate concentration can be monitored and compensated in real time, thereby weakening the substrate gradient along the process from the source and keeping each detection segment in a relatively consistent reaction environment as much as possible.

[0011] By controlling the sampling volume and transmission stability through a constant-volume transport section and a capillary flow-limiting structure, interference from dead volume, diffusion distortion, and detection delay on local substrate concentration readings is reduced. By using an enzyme-free reference channel to subtract the effects of natural background decay, the true enzymatic absorbance change can be separated from the total absorbance change, avoiding contamination by coenzyme decay, optical drift, and baseline fluctuations.

[0012] Local dynamic enzyme reaction rates are calculated, and wall adsorption correction is used to eliminate biases caused by the decrease in effective enzyme concentration along the reaction path, making reaction rates at different locations more comparable. Local stability markers are identified using the local fluctuation coefficient of variation, eliminating unstable regions caused by initial mixing transitions and later depletion drift. The local onset time is determined based on the local half-response time, local inhibition hysteresis index, and effective inhibitor concentration. The integration starting point is aligned to the true inhibition start time, and the local dynamic enzyme reaction rates of the inhibited state and the reference state are weighted and integrated to obtain the comprehensive inhibition intensity. This improves the accuracy of inhibitor onset sequence determination, the consistency of inhibition results at different spatial locations, and the stability and repeatability of overall quantitative analysis. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0014] Figure 1 This is a flowchart of the overall method of the present invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0016] The present invention will be further described below with reference to embodiments.

[0017] Example 1 (see Figure 1 A quantitative analytical method for aldose reductase activity and its inhibitory effect, comprising at least: Step 1: Divide the microfluidic reaction channel into multiple detection segments. Set up an independent bypass sampling channel in each detection segment (directly placing the substrate sensing structure within the reaction channel could easily alter the local flow field distribution, residence time, and enzymatic kinetics). This channel draws a very small volume of local fluid from the reaction channel into an independent electrochemical detection chamber. The local substrate concentration in the corresponding detection segment is calculated in real-time based on the current response. The results are then analyzed based on the local substrate concentrations detected in each detection segment. Local substrate flow rate for the corresponding detection segment Adjustments are made to maintain a relatively consistent substrate concentration environment across each detection segment. Specifically: 1) A bypass sampling channel is provided at the corresponding position of each detection segment (preferably at the center of the corresponding detection segment, from which the bypass sampling channel is led out from the side wall of the microfluidic reaction channel). Its inlet is connected to the reaction channel of the corresponding detection segment, and its outlet is connected to an independent electrochemical detection chamber. The bypass sampling channel must meet the following requirements:

[0018] in, The bypass sampling flow rate corresponding to the j-th detection segment refers to the volume of liquid drawn from the main reaction channel into the electrochemical detection chamber per unit time by the corresponding bypass sampling channel. The flow rate of the reaction channel corresponding to the j-th detection segment is the total liquid volume passing through the detection segment per unit time.

[0019] 2) Due to the extremely small bypass sampling flow rate, the sample may experience dead volume effect, diffusion distortion and time delay during transmission. Therefore, a constant-volume transport section is set after each bypass sampling channel (by setting a rectangular channel with fixed length, width, and height, the bypass sample extracted from the reaction channel is transported to the corresponding electrochemical detection chamber with a preset and constant liquid volume, reducing the impact of detection volume fluctuations on local substrate concentration determination). A capillary flow-limiting structure (in microfluidics, liquid flow is affected by capillary forces; the narrower the channel, the more pronounced the capillary effect; narrowing the channel can be achieved, such as setting a bypass sampling channel width of 100 μm followed by 20 μm. Alternatively, the channel can be designed with many bends to increase flow resistance. By reducing the channel cross-sectional area, increasing the channel length, or adjusting the channel wall wettability, the capillary resistance of the fluid is increased, stabilizing the bypass sampling flow rate) controls the sample to enter the corresponding electrochemical detection chamber at an approximately constant volume (the volume fluctuation of the sample entering the electrochemical detection chamber is controlled within an allowable range). The transmission delay time corresponding to each detection segment is estimated based on the channel length and flow rate (the ratio of length to flow rate) to stabilize the bypass sample volume entering the electrochemical detection chamber and reduce detection delay.

[0020] in, This indicates the local substrate concentration reading time corresponding to the j-th detection segment. The value represents the transmission delay time experienced by the bypass sample corresponding to the j-th detection segment from the entrance of the bypass sampling channel to the electrochemical detection chamber, and t represents the analysis time of the reaction channel.

[0021] 3) Determining local substrate concentration in the electrochemical detection chamber:

[0022] in, Indicates the spatial location of the j-th detection segment. Electrochemical detection current under the following conditions The electrode sensitivity coefficient represents the change in current caused by a unit change in substrate concentration in the electrochemical detection chamber. The unit is [unit missing]. (This coefficient is pre-calibrated. Multiple substrate standard solutions of known concentrations can be pre-prepared, injected into the electrochemical detection chamber to record the current under steady-state conditions, and a linear calibration model can be established. The slope calculated by subtracting the background current from the recorded current and then dividing the difference by the concentration is the sensitivity.) Indicates the spatial location of the j-th detection segment. The background current is the fundamental current signal that the detection system itself will still generate even when the substrate is absent or no effective detection reaction has occurred. This includes the baseline current generated by electrode zero drift and non-specific response (usually, a blank solution without substrate is injected into the electrochemical detection chamber before detection, and the measured current is the background current). Indicates the spatial location of the j-th detection segment. The detected local substrate concentration.

[0023] In the above, each detection segment has an independent compensation injection point (located upstream of or at the inlet of the detection segment, replenishing the substrate within the reaction channel containing that detection segment). When the local substrate concentration of the detection segment... Below the target value When the substrate is in a certain quantity, the system increases to replenish more substrate; conversely, it decreases.

[0024] By maintaining a stable and consistent local substrate concentration, steps 2-6 are performed under this stable substrate concentration environment. In this way, the resulting local dynamic enzyme reaction rate primarily reflects the effect of the inhibitor on enzyme activity, rather than spurious changes caused by substrate concentration variations. Simultaneously, this ensures that each detection segment remains under relatively constant reaction kinetics, avoiding abnormal fluctuations in enzyme reaction rate due to insufficient substrate or local concentration fluctuations, thus improving the accuracy of subsequent local half-response time identification and overall inhibition intensity calculation.

[0025] In this embodiment, the substrate refers to the chemical substance acted upon by aldose reductase, typically an aldose, preferably D-glucose. D-galactose or DL-glyceraldehyde can also be used. Aldose reductase catalyzes the reduction of the substrate while consuming the coenzyme NADPH. Enzyme activity is reflected by measuring the decrease in NADPH absorbance during detection; therefore, substrate concentration directly affects the reaction rate.

[0026] For supplementary injection points: In microfluidic chips, compensation injection points inject additional substrate solution into the main reaction channel. Since fluids at the microscale are typically in laminar flow (low Reynolds number), the injected substrate solution does not immediately mix uniformly with the main solution, but may form parallel laminar bands or localized high-concentration clumps. Immediately detecting the concentration after injection will result in a value highly dependent on the specific location of the detection point on the channel cross-section, failing to represent the average concentration of that segment and affecting the accuracy of feedback control. Therefore, a channel length for mixing is pre-set downstream of each compensation injection point, called a mixing section. As the fluid flows through the mixing section, molecular diffusion and chaotic convection gradually achieve a spatially uniform distribution of the injected substrate and the main solution. Generally, the longer the mixing section, the more uniform the concentration distribution at the outlet.

[0027] A mixing section is set after each compensation injection point. By pre-determining the mixing section length (e.g., 2mm, 5mm, 10mm), the coefficient of variation (i.e., standard deviation / mean) of the local substrate concentration is measured at the outlet of each candidate mixing section length. As the mixing section length increases, the coefficient of variation gradually decreases. The shortest length that meets the preset threshold, such as a coefficient of variation ≤ 5%, is selected as the design length of the microfluidic chip. This ensures uniform mixing and avoids unnecessary reaction delays. The concentration variation coefficient at the mixing section outlet meets the preset uniform mixing threshold.

[0028] satisfy:

[0029] in, This represents the concentration variation coefficient at the outlet of the j-th detection segment mixing section. The standard deviation of the substrate concentration at the outlet of the mixing section (where there are multiple sampling points) represents the standard deviation of the substrate concentration. This represents the average substrate concentration at the outlet of the mixing section. This indicates the preset threshold for uniform mixing.

[0030] It is important to note that in microfluidic reaction channels, substrate transport and reaction consumption occur simultaneously. If corresponding bypass sampling channels and compensation injection points are not set up in each detection segment, a significant substrate concentration gradient will form between upstream and downstream locations, causing inconsistent reaction conditions at subsequent detection sites and resulting in incomparable enzymatic responses at different locations at the same time. Furthermore, the compensated fluid is thoroughly mixed after injection, reducing potential concentration fluctuations and local aggregation within each detection segment. This helps prevent increased dispersion in detection values ​​and maintains a relatively stable substrate environment.

[0031] The bypass sampling channel samples are discharged after testing and do not flow back to the reaction channel. This results in fluid loss in the reaction channel. To compensate for this, the bypass sampling flow rate can be estimated in advance and the total input flow rate at the reaction channel inlet can be increased.

[0032] Furthermore, since step one is established in a stable substrate environment with real-time substrate flow maintained, although substrate concentration fluctuations have been minimized, non-enzymatic factors such as coenzyme attenuation, channel optical drift, and baseline drift will still be superimposed on the background signal. In particular, within microfluidic enzyme reaction channels, the optical path length, fluid state, and local mixing degree at different locations may not be entirely consistent, and background attenuation may also exhibit slight spatial differences. Therefore, the following is proposed: Step 2: Set up an enzyme-free reference channel in each detection segment, and simultaneously collect the total absorbance change and the background natural attenuation absorbance change under a stable substrate concentration maintained by real-time substrate flow rate. After subtracting the background, obtain the true enzymatic absorbance change as the input for local dynamic enzyme activity, using the reference channel background separation method: , This represents the actual change in enzymatic absorbance at time t for the j-th detection segment. This represents the change in total absorbance of the j-th detection segment at time t. This represents the change in background natural attenuation absorbance of the j-th detection segment at time t.

[0033] It should be noted that in the microfluidic chip, each detection segment is designed with two parallel optical detection channels: The reaction pathway (containing aldose reductase, substrate, NADPH, and the inhibitor to be tested).

[0034] Enzyme-free reference channel (except for the absence of aldose reductase, the other components are exactly the same as the reaction channel).

[0035] The ultraviolet absorbance detection module (composed of an ultraviolet light source, such as a deuterium lamp, LED, or laser diode; optical transmission elements, such as optical fibers, lenses, or waveguides integrated on a microfluidic chip; sample detection: a window passing through the microfluidic channel; and a photodetector, such as a photodiode or photomultiplier tube; its working principle follows the Lambert-Beer law: when ultraviolet light passes through a solution containing NADPH, NADPH has characteristic absorption at 340 nm, and the absorbance is proportional to the NADPH concentration) continuously acquires absorbance signals from two channels. The total absorbance change and the background natural attenuation absorbance change are calculated with the initial absorbance of the same segment before the addition of the inhibitor as the reference zero point.

[0036] for Based on the spatial location of the reaction channels The total change in absorbance is obtained by subtracting the initial absorbance before the addition of the inhibitor (usually the average value before the inhibitor addition time) from the absolute absorbance measured at time t. By taking the average value before the inhibitor was added as the baseline, the absorbance at the start of the enzyme-catalyzed reaction in the uninhibited state can be obtained, and the change in absorbance relative to the baseline after the addition of the inhibitor can be accurately calculated.

[0037] for Based on the spatial location of the enzyme-free reference channel The absolute absorbance measured at time t is subtracted from the initial absorbance of the uniform reference channel (similar to the reaction channel).

[0038] Therefore, by using real-time differential processing of the parallel reference channels to eliminate common-mode background attenuation interference, the remaining... This is caused solely by the consumption of NADPH catalyzed by aldose reductase.

[0039] Step 3: Calculate the actual change in enzymatic absorbance. Savitzky-Golay smoothing, a well-known technique, is used to extract local dynamic enzyme reaction rates. This smoothing reduces the influence of random fluctuations on rate changes between different segments, truly reflecting the continuous trend of the enzyme reaction process. Furthermore, based on Lambert-Beer's law, the relationship between absorbance and the concentration of the coenzyme NADPH is as follows: A represents absolute absorbance. is the molar extinction coefficient of NADPH at 340 nm. is the optical path length of the microfluidic reaction channel (the effective thickness of the solution through which ultraviolet light passes, identical across all detection segments on the same chip), and c is the NADPH concentration. For the reaction channel, equal This includes enzymatic consumption and background decay. Indicates the spatial location of the reaction channel The absolute concentration of NADPH at time t. This indicates the initial absolute concentration of NADPH in the reaction channel at the moment the inhibitor is added; equal Includes only background attenuation. Indicates the spatial location of the enzyme-free reference channel. The absolute concentration of NADPH at time t. This indicates the initial absolute concentration of NADPH in the enzyme-free reference channel at the moment of inhibitor addition. Due to the differences between the two channels... The conditions for degradation of enzymes c and non-enzymatic enzymes are the same. Subtracting them, we get: Therefore, the difference is exactly equal to the change in NADPH concentration caused by the enzyme-catalyzed reaction alone multiplied by [the value of the value]. Therefore, the actual enzyme-catalyzed reaction rate can be calculated from the rate of change in absorbance:

[0040] in, This represents the actual change in enzymatic absorbance after smoothing. Indicates the local dynamic enzyme reaction rate. This represents the length of the sliding time window, which is the time interval extending forward by a fixed time length from the current moment. Since all detection segments on the same microfluidic chip have the same optical path length and a fixed detection wavelength, and the molar extinction coefficient is the same constant, the actual calculation of the suppression ratio... They will cancel each other out, and the formula can be simplified to defining the relative enzyme reaction rate, that is... The negative sign indicates that the decrease in absorbance corresponds to the consumption of substrate.

[0041] The sliding time window is set to calculate the local dynamic enzyme reaction rate near each time t based on the real change in enzyme absorbance over the most recent period. This allows for continuous dynamic tracking of the local reaction process, reduces the high-frequency noise amplification problem caused by instantaneous differences, preserves the dynamic change characteristics of the enzyme reaction process, and ensures that subsequent stable interval identification, half-response time calculation, and comprehensive inhibition intensity integration are all based on continuous dynamic response.

[0042] In this embodiment, aldose reductase may be adsorbed and retained on the wall of the microfluidic reaction channel, leading to a gradual decrease in the effective enzyme concentration in different detection segments. Since the wall adsorption effect gradually accumulates along the flow direction, the local dynamic enzyme reaction rate in different detection segments is affected not only by the actual inhibition but also by changes in the effective enzyme concentration. Therefore, a wall adsorption correction is established to correct the local dynamic enzyme reaction rate, resulting in the adsorption-corrected local dynamic enzyme reaction rate:

[0043] in, This represents the local dynamic enzyme reaction rate after adsorption correction. This represents the wall adsorption correction factor. This represents the wall adsorption coefficient, a preset value, ranging from 0 to 1. (In calculation...) The time used Dimensionless normalization is required (the ratio of the path length along the main flow direction from the inlet of the microfluidic reaction channel to the center of the j-th detection segment to the total geometric length of the microfluidic reaction channel along the main flow direction. The total geometric length can also be replaced by the reaction path length for dimensionless normalization).

[0044] In the initial stage of the reaction, after the substrate enters the microfluidic reaction channel, there is still a mixing and transition phase. At this time, the local concentration distribution in each detection segment is not yet stable, and the interaction relationship between the inhibitor and the enzyme is not yet fully established, leading to significant fluctuations in the local dynamic enzyme reaction rate after adsorption correction. In the later stage of the reaction, as the substrate is gradually consumed and the reaction products accumulate, the local reaction rate may drift or exhibit non-true decay, affecting the stability of subsequent inhibition analysis results. Therefore, it is necessary to identify the unstable reaction regions in the local dynamic enzyme reaction rate after adsorption correction to avoid these fluctuating regions from entering the subsequent integration and statistical process. Step 4: Calculate the coefficient of variation for the local dynamic enzyme reaction rate:

[0045] in, The coefficient of variation represents the local fluctuation of the j-th detection segment at time t, characterizing the relative fluctuation of the local dynamic enzyme reaction rate after adsorption correction within the sliding time window. This represents the standard deviation of the adsorption-corrected local dynamic enzyme reaction rate within the sliding time window ending at time t for the j-th detection segment, characterizing the degree of dispersion of the local dynamic enzyme reaction rate within this time window. The average local dynamic enzyme reaction rate after adsorption correction within the sliding time window ending at time t for the j-th detection segment.

[0046] When the local fluctuation variation coefficient within the window is lower than the stability threshold When the time interval (usually set to 0.1-0.2) is considered a stable interval, the corresponding local stability indicator is output. ,Right now =1; otherwise Above the stability threshold When this occurs, it indicates that the corresponding time period is not within a stable range, that is... It is 0.

[0047] Furthermore, within the microfluidic reaction channel, the inhibitor diffusion paths and transport times differ between different detection segments, leading to asynchronous inhibitory onset processes at various spatial locations. The inhibitor is also affected by diffusion attenuation and wall adsorption during transport, causing variations in the effective inhibitor concentration at different locations. If the inhibitor addition time is uniformly used as the starting point for subsequent integration analysis, time periods before true inhibition will be incorrectly included in the statistical process. Therefore: Step 5: Within the stable range indicated by the local stability marker, identify the local half-response time of each detection segment based on the adsorption-corrected local dynamic enzyme reaction rate, and determine the true local onset time based on the local half-response time. .

[0048] in, This represents the average enzyme reaction rate of the j-th detection segment (taken before the inhibitor was added, with a length of...). (average value within the time window) This represents the number of sampling points in the baseline enzyme activity statistics window, where i represents the sampling point summation index variable. This indicates that during the local dynamic decrease in enzyme activity, the last one is not lower than the threshold. The sampling time, that is, the last sampling point before the threshold crossover, This represents the effectiveness determination coefficient (a preset constant, typically 0.5). This indicates the time interval between adjacent sampling times. This represents the time point in the j-th detection segment where the adsorption-corrected local dynamic enzyme reaction rate decreases to 50% of the baseline enzyme activity, which is also the local half-response time. The j-th detection segment is in The local dynamic enzyme reaction rate. Using The threshold for determining the local dynamic enzyme reaction rate is used because the local dynamic enzyme reaction rate is usually in the stage where the inhibitory response is most sensitive when it drops to about 50% of the baseline enzyme activity. At this time, it can avoid the instantaneous errors caused by local mixing fluctuations, diffusion instability and flow disturbances in the early stage of the reaction, and avoid the influence of non-true decay caused by substrate depletion, natural decay of coenzyme and wall adsorption accumulation in the later stage of the reaction. It can more stably characterize the true effective process of the inhibitor in the corresponding detection segment.

[0049] It should be noted that, Not less than . Less than Both together pinpoint the specific time period in which the threshold crossover occurs, providing a boundary for subsequent calculations of the local onset time.

[0050] Establish a local inhibition hysteresis index:

[0051] Inhibitor transport attenuation model:

[0052] in, This represents the local inhibition hysteresis index of the j-th detection segment, characterizing the effect of inhibitor addition from the time of detection. (The initial moment when the inhibitor is added to the reaction system) until the half-inhibition state is reached ( The time delay between ( ). Indicates the initial injection concentration of the inhibitor. This represents the time decay coefficient of the inhibitor in the microfluidic channel, where the effective concentration of the inhibitor decays exponentially with the onset delay time. This indicates the exponential decay effect of the inhibitor as the lag time increases. This represents the effective inhibitor concentration corresponding to the j-th detection segment (which needs to be divided by a concentration reference constant for dimensionless normalization during calculation), characterizing the actual effective concentration of the inhibitor after diffusion attenuation and local transport loss during microfluidic channel transport. This represents the local onset time after correction for the j-th detection segment. This time reflects the true onset boundary after deducting the effects of inhibitor transport delay and concentration decay.

[0053] The time decay coefficient is determined based on the flow rate conditions within the microfluidic reaction channel. Typically, it is smaller under low flow rate diffusion conditions and larger under high flow rate diffusion conditions, with a common range of 0.001-1 s⁻¹.

[0054] The local inhibition hysteresis index is defined to quantify the total delay experienced by an inhibitor from its addition to the actual achievement of half-inhibition. This delay includes the time cost of inhibitor transport, diffusion, and binding along the pathway. Introducing an inhibitor transport attenuation model separates the passive transport component of the total delay from the concentration-dependent onset rate, yielding a more accurate local onset time. When the effective concentration of inhibitors in different detection segments differs due to degradation or adsorption, the original half-inhibition time alone truly approximates the moment when inhibition begins after contact with the enzyme. Subsequent integrals of inhibition intensity can be aligned to the same physiological starting point, improving spatial comparability.

[0055] Step Six: Based on the local onset time corresponding to each detection segment as the starting boundary for integration, and under the constraint of local stability markers, weighted integrals are performed on the local dynamic enzyme reaction rates corresponding to the inhibited state and the reference state to obtain the comprehensive inhibition intensity corresponding to each detection segment:

[0056] in, This represents the overall suppression intensity corresponding to the j-th detection segment. This represents the local dynamic enzyme reaction rate of the j-th detection segment under the inhibition state at time t (inhibition state: the state of the enzyme reaction after the addition of the inhibitor to be tested. At this time, due to the effect of the inhibitor, the enzyme activity is partially or completely inhibited, which is manifested as the local dynamic enzyme reaction rate being lower than the local dynamic enzyme reaction rate under the reference state without inhibition). The value represents the adsorption-corrected local dynamic enzyme reaction rate of the j-th detection segment under the reference state at time t (obtained by conducting a single control experiment without adding inhibitors, using the same microfluidic chip and the same segment substrate compensation conditions, and recording the local dynamic enzyme reaction rate of each detection segment at each time point; if multiple independent control experiments are conducted, the arithmetic mean can be taken), and T represents the overall analysis termination time. This represents extremely small positive terms, avoiding a denominator of 0.

[0057] Finally, the analysis results set can be output. , m represents the number of detection segments. Each element records the spatial location, local onset time, and overall inhibitory intensity of the j-th detection segment, providing a clear indication of the inhibitor's onset sequence along the reaction pathway and the uniformity of its inhibitory intensity.

[0058] Quantitative analysis system, including: The concentration environment adjustment module divides the microfluidic reaction channel into multiple detection segments and sets up a bypass sampling channel and an electrochemical detection chamber; fluid is led out through the bypass sampling channel to the electrochemical detection chamber to detect the local substrate concentration; the local substrate concentration is compared with the preset target substrate concentration to determine the substrate injection amount at the corresponding compensation injection point; The absorbance change analysis module sets an enzyme-free reference channel for each detection segment, and collects the total absorbance change and the background natural attenuation absorbance change respectively in combination with the reaction channel to calculate the true enzymatic absorbance change for the corresponding detection segment. The enzyme reaction analysis module calculates the actual absorbance change between consecutive moments based on a sliding time window, and obtains the local dynamic enzyme reaction rate of each detection segment by combining the Lambert-Beer law. The local dynamic enzyme reaction rate calculates the local fluctuation variation coefficient of each detection segment within the sliding time window, and determines the corresponding local stability indicator when the local fluctuation variation coefficient is lower than a preset stability threshold. The onset time determination module identifies the local half-response time of each detection segment within the stable range indicated by the local stability indicator and determines the true local onset time. The inhibition state analysis module integrates the local dynamic enzyme reaction rate corresponding to the inhibition state and the reference state with the local onset time as the starting boundary, and obtains the comprehensive inhibition intensity corresponding to each detection segment.

[0059] Furthermore, if the aforementioned function is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0060] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequenced list of executable instructions for implementing logical functions, and can be embodied in any computer-readable medium for use by, or in conjunction with, an instruction execution system, apparatus, or device (such as a computer-based system, a processor-including system, or other system that can fetch and execute instructions from, an instruction execution system, apparatus, or device). For the purposes of this specification, "computer-readable medium" can be any means that can contain, store, communicate, propagate, or transmit programs for use by, or in conjunction with, an instruction execution system, apparatus, or device.

[0061] More specific examples of computer-readable media (a non-exhaustive list) include: electrical connections (electronic devices) having one or more wires, portable computer disk drives (magnetic devices), random access memory (RAM), read-only memory (ROM), erasable and editable read-only memory (EPROM or flash memory), fiber optic devices, and portable optical disc read-only memory (CDROM). Furthermore, computer-readable media can even be paper or other suitable media on which the program can be printed, because the program can be obtained electronically, for example, by optically scanning the paper or other medium, followed by editing, interpreting, or otherwise processing as necessary, and then stored in computer memory.

[0062] Furthermore, in order to provide a concise description of exemplary embodiments, not all features of actual embodiments (i.e., those features that are not relevant to the best mode of carrying out the invention as currently considered, or those features that are not relevant to implementing the invention) may be omitted.

[0063] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. A quantitative analytical method for aldose reductase activity and its inhibitory effect, characterized in that, include: The microfluidic reaction channel is divided into multiple detection segments, and a bypass sampling channel and an electrochemical detection chamber are set up. Fluid is drawn out through a bypass sampling channel to the electrochemical detection chamber to detect local substrate concentration; The difference between the local substrate concentration and the preset target substrate concentration is calculated to determine the substrate injection amount at the corresponding compensation injection point. Each detection segment is equipped with an enzyme-free reference channel. The total absorbance change and the background natural attenuation absorbance change are collected in combination with the reaction channel to calculate the true enzymatic absorbance change of the corresponding detection segment. Based on the sliding time window, the actual change in absorbance between consecutive time points is calculated, and the local dynamic enzyme reaction rate of each detection segment is obtained by combining the Lambert-Beer law. The local dynamic enzyme reaction rate is calculated by the local fluctuation variation coefficient of each detection segment within the sliding time window, and the corresponding local stability indicator is determined when the local fluctuation variation coefficient is lower than the preset stability threshold. Within the stable interval indicated by the local stability marker, the local half-response time of each detection segment is identified, and the true local onset time is determined as the integration starting boundary. The local dynamic enzyme reaction rate corresponding to the inhibition state and the reference state is integrated to obtain the comprehensive inhibition intensity corresponding to each detection segment. When determining the substrate injection volume at the compensation injection point, it is also necessary to ensure that the substrate is mixed evenly at the compensation injection point. A mixing section is set after each compensation injection point; Multiple sampling locations are set at the outlet of the mixing section, and the local substrate concentrations at the multiple sampling locations are measured to calculate the average substrate concentration. Determine the difference between the local substrate concentration at each sampling location and the average substrate concentration at the outlet of the mixing section; Establish the standard deviation of the substrate concentration at the outlet of the mixing section; The concentration variation coefficient at the outlet of the mixing section is obtained by using the standard deviation and the average substrate concentration at the outlet of the mixing section; When the concentration variation coefficient at the outlet of the mixing section is less than or equal to the threshold, it is determined that the current mixing section meets the mixing conditions. To obtain the true changes in enzymatic absorbance, including: The absolute absorbance of the reaction channel and the absolute absorbance of the corresponding enzyme-free reference channel were collected. The total absorbance change is obtained by comparing the absolute absorbance of the reaction channel at the current moment with the initial absorbance before the inhibitor was added. The difference between the absolute absorbance of the enzyme-free reference channel at the current moment and the initial absorbance before the addition of the inhibitor is used to obtain the change in background natural attenuation absorbance. The difference between the total absorbance change and the background natural attenuation absorbance change is used to obtain the actual enzymatic absorbance change produced by the aldose reductase catalytic reaction. After obtaining the local dynamic enzyme reaction rate of each detection segment based on the Lambert-Beer law, the method also includes wall adsorption correction, specifically: Calculate the path length from the center of each detection segment to the inlet of the microfluidic reaction channel; Divide the path length by the total geometric length of the microfluidic reaction channel to obtain the normalized path position; The normalized path position and the preset wall adsorption coefficient are used to obtain the corresponding wall adsorption correction coefficient. The adsorption-corrected local dynamic enzyme reaction rate is obtained by comparing the local dynamic enzyme reaction rate with the wall adsorption correction coefficient. The method for determining the local onset time is as follows: Within a sliding time window, the local dynamic enzyme reaction rates after adsorption correction at multiple time points are summed, and the average value of the window is obtained by the ratio of the summation value to the number of sampling points. Calculate the difference between the local dynamic enzyme reaction rate after each adsorption correction and the window average value. Sum the squares of each difference, divide by the number of sampling points, and take the square root to obtain the window standard deviation. Based on the aforementioned standard deviation and mean, a local fluctuation variation coefficient is constructed; When the local fluctuation variation coefficient is lower than the preset stability threshold, the corresponding time period is marked as a local stability indicator; The adsorption-corrected local dynamic enzyme reaction rates at multiple moments within a preset time window before the addition of the inhibitor are summed and divided by the number of sampling points to obtain the average enzyme reaction rate. A half-response threshold is established by comparing the average enzyme reaction rate with a preset efficacy determination coefficient. The time point at which the local dynamic enzyme reaction rate, after adsorption correction, first drops to the half-response threshold is identified and determined as the local half-response time. Subtracting the inhibitor addition time from the local half-response time yields the local inhibition hysteresis index; The time decay coefficient is combined with the local inhibition hysteresis exponent for exponential decay. The effective inhibitor concentration is then established by multiplying the initial inhibitor injection concentration with the exponential decay result. The local onset time is obtained by using the local inhibition hysteresis index, the effective inhibitor concentration, and the inhibitor addition time; The method for obtaining the overall suppression strength includes: The local onset time is used as the starting boundary for integration; Within the stable range defined by the local stability marker, the local dynamic enzyme reaction rate after adsorption correction under the inhibited state and the local dynamic enzyme reaction rate after adsorption correction under the reference state are obtained, a ratio is established, and the instantaneous degree of inhibition corresponding to each time point is calculated. The instantaneous inhibition level at each moment is combined with the corresponding time interval and then summed to obtain the integral value of the inhibition effect. By combining the local stability markers at each moment with the corresponding time intervals, a normalized reference integral value is established. The ratio of the integral value of the inhibition effect to the integral value of the normalized reference value constitutes the overall inhibition strength.

2. The method according to claim 1, characterized in that, The bypass sampling channel leads fluid to the electrochemical detection chamber to detect local substrate concentration, including: A bypass sampling channel is set up in each detection segment, and the bypass sampling flow rate is controlled to be less than the flow rate of the reaction channel formed by the total liquid volume in the corresponding detection segment. A constant-volume transport section is set up. Based on the channel length and fluid flow velocity of the bypass sampling channel, the transmission delay time is determined. Combined with the analysis time of the reaction channel, the concentration reading time is determined, and the local substrate concentration is output. Based on the electrode sensitivity coefficient and background current, the local substrate concentration corresponding to the detection segment is obtained.

3. A quantitative analysis system, applied to the method of any one of claims 1-2, characterized in that, include: The concentration environment adjustment module divides the microfluidic reaction channel into multiple detection segments and sets up a bypass sampling channel and an electrochemical detection chamber; Fluid is led out through the bypass sampling channel to the electrochemical detection chamber to detect the local substrate concentration; the local substrate concentration is compared with the preset target substrate concentration to determine the substrate injection amount at the corresponding compensation injection point; The absorbance change analysis module sets an enzyme-free reference channel for each detection segment, and collects the total absorbance change and the background natural attenuation absorbance change respectively in combination with the reaction channel to calculate the true enzymatic absorbance change for the corresponding detection segment. The enzyme reaction analysis module calculates the actual absorbance change between consecutive moments based on a sliding time window, and obtains the local dynamic enzyme reaction rate of each detection segment by combining the Lambert-Beer law. The local dynamic enzyme reaction rate calculates the local fluctuation variation coefficient of each detection segment within the sliding time window, and determines the corresponding local stability indicator when the local fluctuation variation coefficient is lower than a preset stability threshold. The onset time determination module identifies the local half-response time of each detection segment within the stable range indicated by the local stability indicator and determines the true local onset time. The inhibition state analysis module integrates the local dynamic enzyme reaction rate corresponding to the inhibition state and the reference state with the local onset time as the starting boundary, and obtains the comprehensive inhibition intensity corresponding to each detection segment.

4. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that, When the processor executes the computer program, it implements the method of any one of claims 1 to 2.

5. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the method of any one of claims 1 to 2.

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