An automatic sample injection puerarin fluorescence detection analysis device

Abstract

The application discloses a kind of automatic sample introduction puerarin fluorescence detection analysis device, belong to traditional Chinese medicine active ingredient detection equipment field. Including cell pretreatment module, automatic sample introduction module, fluorescence spectrophotometric detection module, data processing module and time sequence linkage controller, can complete in situ cell crushing, centrifugation, preparation high-purity puerarin extract.The automatic sample introduction module integrates filtration and negative pressure quantitative structure, can prevent blockage, precise continuous sampling;Fluorescence module uses orthogonal light path layout, accurately collects characteristic fluorescence signal.Data module carries double correction model to eliminate detection deviation, and time sequence controller realizes equipment whole process unattended batch detection.The application improves the automation level and precision of puerarin detection, data is reliable, applicable to cell uptake kinetics batch detection scene.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing equipment, specifically to an automatic sample introduction device for puerarin fluorescence detection and analysis. Background Technology

[0002] Puerarin is the core active substance in the traditional Chinese medicine kudzu root. Existing puerarin fluorescence detection equipment has many shortcomings in cell sample detection scenarios. Conventional detection equipment requires manual completion of the entire process of cell washing, digestion, disruption, and centrifugation. Multiple sample transfers can easily lead to puerarin loss, resulting in low detection recovery rates and time-consuming batch sample testing.

[0003] Existing automated sample-injection fluorescence equipment is only suitable for clear, pure solutions. For cell lysate extracts, the tubing is easily clogged by proteins and cell debris. During continuous sample injection, puerarin is adsorbed onto the inner wall of the tubing, resulting in consistently low detection concentrations and poor precision in batch sample detection. Furthermore, cell extracts contain a large amount of protein and nucleic acid impurities, which can cause matrix filtration. Traditional quantitative fluorescence formulas do not consider the effect of matrix attenuation, leading to significant deviations in the detection results for low-concentration puerarin.

[0004] Traditional fluorescence detection optical paths lack a standardized orthogonal layout, allowing stray excitation light to directly enter the photoelectric acquisition unit, resulting in high baseline noise. Furthermore, the equipment cannot automatically complete the entire process of cell sampling, pretreatment, sample introduction, and detection according to preset time nodes. When conducting drug cell time-series distribution experiments, personnel are required to monitor sampling at regular intervals, introducing human error and compromising the reliability of experimental data. Therefore, there is an urgent need for an automated sample introduction device for puerarin fluorescence detection and analysis that can perform fully automated time-series detection, possesses dual correction for matrix and tubing adsorption, and is compatible with cell extract solutions. Summary of the Invention

[0005] The present invention aims to solve the problems of large amount of manual operation, low quantitative accuracy caused by tube adsorption and matrix interference, and inability to perform time-series automatic detection of puerarin fluorescence detection in the prior art, and provides an automatic sample introduction device for puerarin fluorescence detection and analysis.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An automated sample introduction device for puerarin fluorescence detection and analysis includes a cell pretreatment module, an automated sample introduction module, a fluorescence spectrophotometer, and a data processing module. The cell pretreatment module has multiple isothermal cell placement stations, and its output station connects to the sampling end of the automated sample introduction module to perform cell sample disruption and centrifugation to prepare puerarin cell extract. The automated sample introduction module is positioned between the cell pretreatment module and the fluorescence spectrophotometer, and its output end is connected to the fluorescence detection cell of the fluorescence spectrophotometer via a transport path. The automated sample introduction module can perform sample transport, quantitative delivery, and online impurity removal. The system features filtration and flow path cleaning, coupled with a constant-temperature cell placement station to achieve continuous automatic sample injection at preset time points. The fluorescence spectrophotometer is electrically connected to the data processing module. The fluorescence spectrophotometer can output a fixed wavelength excitation light to irradiate the extract in the fluorescence detection cell. After filtering out stray light, the fluorescence signal generated by puerarin is converted into an electrical signal and transmitted to the data processing module. The data processing module has a built-in main control unit and quantitative analysis program. It can call the corrected internal filtration effect model and the tubing adsorption correction model to perform correction calculations on the detection electrical signal, eliminating the quantitative deviation caused by the cell matrix internal filtration effect and the adsorption of the sample injection tubing, and outputting the true concentration result of puerarin.

[0007] Furthermore, the modified internal filtration effect model employs a modified fluorescence quantitative equation, with fluorescence intensity... With puerarin concentration The following relationship must be satisfied: , in, The overall efficiency coefficient of the instrument; The initial intensity of the incident excitation light; The molar absorptivity of puerarin at 342 nm; The effective optical path length of the fluorescence detection cell; This represents the filtration attenuation coefficient within the cell matrix.

[0008] Furthermore, the pipeline adsorption correction model employs a dynamic correction relationship, based on the true concentration of puerarin. Compared with apparent concentration The following relationship must be satisfied: , in, The saturated adsorption rate of the inner wall of the sample inlet tube; This represents the cumulative number of consecutive injections. The adsorption saturation characteristic number is denoted as .

[0009] Furthermore, the quantitative analysis program of the data processing module includes a light intensity quantification unit, a matrix background subtraction unit, and a data storage unit; the light intensity quantification unit acquires and quantifies the original fluorescence electrical signal, the matrix background subtraction unit subtracts the fluorescence background of the blank cell matrix, and the data storage unit stores the sample information and detection results together.

[0010] Furthermore, the fluorescence spectrophotometer includes an excitation optical path unit, an emission filtering optical path unit, and a photoelectric acquisition unit. The excitation optical path unit is equipped with a xenon lamp light source and a monochromator, with a fixed output wavelength of 342nm and an excitation slit width of 10nm. The output optical path of the excitation optical path unit is aligned with the fluorescence detection cell. The emission filtering optical path unit is located between the fluorescence detection cell and the photoelectric acquisition unit, and has a built-in 461nm narrowband bandpass filter with a half-width of 10nm. The photoelectric acquisition unit receives the filtered fluorescence signal and converts it into an electrical signal.

[0011] Furthermore, the photoelectric acquisition unit of the fluorescence spectrophotometer is equipped with a photomultiplier tube detector, which has a built-in temperature compensation unit to suppress dark current noise; the spectral response range of the photomultiplier tube detector covers 380nm~600nm.

[0012] Furthermore, the automatic sample introduction module includes a negative pressure quantitative loop and a filter unit; the filter unit and the negative pressure quantitative loop are connected in series through a flow path, the filter unit is connected to the output station of the cell pretreatment module, and the negative pressure quantitative output terminal is connected to the fluorescence detection cell.

[0013] Furthermore, the negative pressure quantitative loop is a fixed-volume pipeline, which is filled with extract by negative pressure suction to achieve fixed-volume injection.

[0014] Furthermore, the cell pretreatment module includes a processing chamber, which contains a sample carrying station. An ultrasonic disruptor is installed at the bottom of the sample carrying station, and a miniature high-speed centrifugal rotor is coaxially connected to the sample carrying station.

[0015] Furthermore, it also includes a timing linkage controller, which is electrically connected to the cell pretreatment module, the automatic sample injection module, the fluorescence spectrophotometer detection module, and the data processing module, respectively.

[0016] Compared with the prior art, the present invention has the following significant advantages:

[0017] 1. The whole machine integrates cell pretreatment, automatic sample introduction, fluorescence detection and data correction modules, and is equipped with a timing linkage controller to realize unattended sequential batch detection, which greatly reduces the amount of manual operation and avoids experimental errors caused by manual sampling and transfer.

[0018] 2. A dual quantitative correction mathematical model was set up to eliminate the concentration deviation caused by the intracellular matrix filtration effect and the dynamic adsorption of the tubing, which significantly improved the accuracy and precision of the detection of low-concentration puerarin samples.

[0019] 3. The automatic sample injection module is equipped with a series filter unit and a fixed-volume negative pressure quantitative loop, which is suitable for complex matrix of cell extracts, effectively prevents pipeline blockage, and ensures the accuracy of continuous sample injection volume; the fluorescence optical path adopts an orthogonal layout with a dedicated filter to reduce baseline noise and improve the fluorescence signal-to-noise ratio. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall structural framework in an embodiment of the present invention; Figure 2 This is a schematic diagram of the processing chamber structure in an embodiment of the present invention; Figure 3 This is a schematic diagram of the fluorescence spectrophotometer detection module in an embodiment of the present invention; Figure 4 This is a schematic diagram of the flow path connection of the automatic sample delivery module in an embodiment of the present invention; Figure 5 This is a flowchart illustrating the overall automated detection timing process of the device of the present invention. Figure 6 This is a flowchart of the dual-model data correction operation of the present invention.

[0021] The components include: 1. Cell pretreatment module; 11. Constant temperature cell placement station; 12. Processing chamber; 121. Sample carrying station; 122. Ultrasonic disruptor; 123. Miniature high-speed centrifuge rotor; 2. Automatic sample loading module; 21. Filtering unit; 22. Negative pressure quantitative loop; 3. Fluorescence spectrophotometry detection module; 31. Excitation optical path unit; 32. Fluorescence detection cell; 33. Emission filtering optical path unit; 34. Photoelectric acquisition unit. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0023] See Figures 1 to 6This invention discloses an automated sample-injection puerarin fluorescence detection and analysis device, in a first embodiment, comprising a cell pretreatment module 1, an automated sample-injection module 2, a fluorescence spectrophotometer detection module 3, and a data processing module. The cell pretreatment module 1 is equipped with multiple sets of isothermal cell placement stations 11. The output station of the cell pretreatment module 1 is connected to the sampling end of the automated sample-injection module 2, enabling the disruption and centrifugation of cell samples to prepare puerarin cell extract. The automated sample-injection module 2 is positioned between the cell pretreatment module 1 and the fluorescence spectrophotometer detection module 3. The output end of the automated sample-injection module 2 is connected to the fluorescence detection cell 32 of the fluorescence spectrophotometer detection module 3 via a transport flow path. The automated sample-injection module 2 can complete sample transport, ... Quantitative delivery, online impurity removal filtration, and flow path cleaning, combined with the constant temperature cell placement station 11, enable continuous automatic sample injection according to preset time nodes; the fluorescence spectrophotometer detection module 3 is electrically connected to the data processing module. The fluorescence spectrophotometer detection module 3 can output a fixed wavelength excitation light to irradiate the extract to be tested in the fluorescence detection cell 32. After filtering out stray light, the fluorescence signal generated by puerarin is converted into an electrical signal and transmitted to the data processing module; the data processing module has a built-in main control unit and quantitative analysis program, which can call the corrected internal filtration effect model and the pipeline adsorption correction model to perform correction calculations on the detection electrical signal, eliminate the quantitative deviation caused by the cell matrix internal filtration effect and the adsorption of the sample injection pipeline, and output the true concentration result of puerarin.

[0024] Specifically, the cell pretreatment module 1 carries cell samples and integrates disruption and centrifugation functions, eliminating the need for manual sample transfer, reducing puerarin loss, and serving as the basic carrier for achieving automated pretreatment.

[0025] The isothermal cell placement station 11 is used to place cell culture dishes and maintain the isothermal environment required for cell survival. It works in conjunction with the timing controller to complete fixed-point and timed sampling.

[0026] The automatic sample injection module 2 receives the pre-treated extract and automatically completes filtration, quantitative delivery, and pipeline cleaning, achieving continuous sample injection without manual intervention and solving the problems of large errors and easy cross-contamination in traditional manual sample injection equipment.

[0027] The fluorescence detection cell 32 serves as a fluorescent reaction carrier, receiving the quantitatively delivered extract. It is located at the intersection of the excitation and emission light paths, providing space for the generation of fluorescence signals.

[0028] The fluorescence spectrophotometer module 3 outputs characteristic excitation light and collects characteristic fluorescence signals of puerarin, converting the optical signal into a calculable electrical signal to realize the digitization of the optical signal.

[0029] The data processing module is equipped with a dual-correction mathematical model to perform correction calculations on the original detection signal, eliminate two types of systematic errors: matrix and pipeline adsorption, and output accurate puerarin concentration values.

[0030] A second embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention uses a modified fluorescence quantitative equation to correct the internal filtration effect model, and the fluorescence intensity... With puerarin concentration The following relationship must be satisfied: , in, The overall efficiency coefficient of the instrument; The initial intensity of the incident excitation light; The molar absorptivity of puerarin at 342 nm; The effective optical path of the fluorescence detection cell is 32. This represents the filtration attenuation coefficient within the cell matrix.

[0031] In this embodiment, the equation is derived based on the classic Beer-Lambert fluorescence quantitative theory combined with biological matrix absorption correction. Traditional fluorescence formulas are only applicable to pure solutions without impurities. Cell extracts contain a large amount of protein and nucleic acid, which will absorb excitation light and emit fluorescence, resulting in an internal filtration effect. This equation adds a matrix attenuation correction term to adapt to cell sample detection scenarios.

[0032] The effective fluorescence intensity represents the final collected data and is the basis for subsequent calculations of puerarin concentration.

[0033] The comprehensive coefficient integrates fluorescence quantum yield, optical path transmission loss, and detector response efficiency, and uniformly characterizes the inherent parameters of the entire instrument's optical system.

[0034] The initial output light intensity of the light source remains constant during the detection process using the same device.

[0035] The molar absorptivity of puerarin at an excitation wavelength of 342 nm is a fixed constant obtained by calibration with a standard puerarin solution.

[0036] The length of the liquid inside the fluorescence detection cell 32 that allows light to pass through is a fixed structural parameter of the equipment.

[0037] This is a cell matrix-specific attenuation coefficient, pre-calibrated using a blank puerarin-free cell extract to compensate for fluorescence attenuation caused by impurity absorption.

[0038] Furthermore, the overall efficiency coefficient of the equipment was calibrated using a standard puerarin gradient solution. Incident light intensity molar absorptivity Detection cell optical path Matrix attenuation coefficient When the concentration of puerarin to be measured Substituting into the equation, the effective fluorescence intensity is calculated. It completes matrix attenuation correction, and compared with the traditional uncorrected formula, the detection deviation of low concentration samples is reduced by more than 85%.

[0039] In a third embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention, the tubing adsorption correction model adopts a dynamic correction relationship, and the true concentration of puerarin is... Compared with apparent concentration The following relationship must be satisfied: , in, The saturated adsorption rate of the inner wall of the sample inlet tube; This represents the cumulative number of consecutive injections. The adsorption saturation characteristic number is denoted as .

[0040] In this embodiment, the relationship is derived based on the first-order adsorption saturation kinetic model of the solid-liquid interface. The organic material on the inner wall of the pipeline will continuously adsorb puerarin molecules. The adsorption loss of the previous sample will cause the subsequent detection results to be lower. As the number of injections increases, the adsorption gradually becomes saturated, and the loss decreases exponentially. This formula can compensate for the adsorption loss in real time.

[0041] This is to correct the true concentration of puerarin, and to output the final test results.

[0042] The apparent concentration is calculated solely from fluorescence intensity and is therefore uncorrected, resulting in a negative adsorption bias.

[0043] The maximum saturated adsorption ratio of the pipeline is 0 to 1, and is determined by continuous injection of gradient standard solutions.

[0044] This represents the current cumulative number of continuously injected samples, which is counted in real time by the program.

[0045] The adsorption saturation characteristic number represents the number of injections required for the pipeline to approach adsorption saturation, and is a fixed calibration parameter for the equipment.

[0046] Specifically, pipeline saturation adsorption rate Adsorption saturation characteristic number On the sixth consecutive injection, the instrument measured the apparent concentration. Substitute into the formula to calculate The concentration error caused by adsorption in the correction pipeline was reduced, and the RSD of batch continuous detection was reduced from 12.6% to 2.1%.

[0047] The fourth embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention includes a quantitative analysis program of a data processing module comprising a light intensity quantification unit, a matrix background subtraction unit, and a data storage unit; the light intensity quantification unit acquires and quantifies the original fluorescence electrical signal, the matrix background subtraction unit subtracts the fluorescence background of the blank cell matrix, and the data storage unit stores the sample information and detection results together.

[0048] Specifically, the light intensity quantization unit converts the analog electrical signal output by the photoelectric acquisition unit into a standardized digital fluorescence intensity value, providing basic data for equation calculation.

[0049] The matrix background subtraction unit retrieves the fluorescence baseline of the blank cell extract and automatically cancels the background interference caused by the fluorescence of cell impurities, thus avoiding false positives and false negatives.

[0050] The data storage unit is synchronously bound to the sample number, incubation time, calibrated concentration, and detection time, enabling full traceability of experimental data without the need for manual recording.

[0051] A fifth embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention includes a fluorescence spectrophotometer detection module 3 comprising an excitation optical path unit 31, an emission filtering optical path unit 33, and a photoelectric acquisition unit 34. The excitation optical path unit 31 is equipped with a xenon lamp light source and a monochromator, with a fixed output wavelength of 342 nm and an excitation slit width of 10 nm. The output optical path of the excitation optical path unit 31 is aligned with the fluorescence detection cell 32. The emission filtering optical path unit 33 is disposed between the fluorescence detection cell 32 and the photoelectric acquisition unit 34, and has a built-in 461 nm narrow bandpass filter with a half-width of 10 nm. The photoelectric acquisition unit 34 receives the filtered fluorescence signal and converts it into an electrical signal.

[0052] The excitation optical path unit 31 outputs a dedicated 342nm excitation light for puerarin. The monochromator and slit ensure a single output wavelength and stable optical power, accurately exciting puerarin to produce characteristic fluorescence.

[0053] The fluorescence detection cell 32 is positioned at a 90° vertical position on the optical axis of the excitation optical path unit 31, and an orthogonal optical path layout is adopted to avoid high baseline noise caused by direct exposure of the excitation light to the detector.

[0054] The emission filtering optical path unit 33 has a built-in 461nm narrowband filter that only allows the characteristic emission fluorescence of puerarin to pass through, filtering out excitation stray light and autofluorescence of cellular impurities, thereby improving the signal-to-noise ratio.

[0055] The photoelectric acquisition unit 34 receives the filtered pure fluorescence signal, converts the optical signal into an analog electrical signal, and transmits it to the data processing module.

[0056] In a sixth embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention, the photoelectric acquisition unit 34 of the fluorescence spectrophotometer detection module 3 is equipped with a photomultiplier tube detector. The photomultiplier tube detector has a built-in temperature compensation unit to suppress dark current noise. The spectral response range of the photomultiplier tube detector covers 380nm~600nm.

[0057] The photomultiplier tube detector has an ultra-high amplification capability for weak light signals and is suitable for collecting weak fluorescence of low-concentration puerarin.

[0058] The temperature compensation unit compensates for dark current drift caused by temperature changes in real time, ensuring baseline stability during long-term continuous detection.

[0059] The spectral response range of 380nm~600nm completely covers the 461nm emission wavelength of puerarin, with no signal loss.

[0060] The seventh embodiment of the automatic sample introduction device for puerarin fluorescence detection and analysis of the present invention includes an automatic sample introduction module 2 comprising a negative pressure quantitative loop 22 and a filter unit 21; the filter unit 21 and the negative pressure quantitative loop 22 are connected in series through a flow path, the filter unit 21 is connected to the output station of the cell pretreatment module 1, and the negative pressure quantitative output terminal is connected to the fluorescence detection cell 32.

[0061] The filter unit 21 is a microporous filter element, positioned at the front end of the negative pressure quantitative loop 22. It traps cell debris and protein precipitates, preventing tubing blockage and adapting to complex matrices of cell extracts. The negative pressure quantitative loop 22 is connected in series at the rear end of the filter unit 21, achieving precise volumetric injection with a fixed volume, ensuring consistent injection volume in each round and improving detection repeatability.

[0062] Furthermore, the fixed-volume tubing has a constant volume. Liquid is drawn from the tubing by a negative pressure pump to completely fill it, and excess liquid is returned. Quantification is achieved by relying on physical volume and is not affected by sample viscosity, making it more stable for quantification compared to syringe pumps.

[0063] This invention discloses an eighth embodiment of an automated sample introduction device for puerarin fluorescence detection and analysis. The cell pretreatment module 1 includes a treatment chamber 12, within which a sample carrying station 121 is located. An ultrasonic disruptor oscillator 122 is installed at the bottom of the sample carrying station 121, which is coaxially connected to a miniature high-speed centrifugal rotor 123. The isothermal cell placement station 11 and the treatment chamber 12 are separate but linked structures. The isothermal cell placement station 11 is fixedly located in the static culture area outside the cell pretreatment module, while the treatment chamber 12 is fixedly located in the pretreatment operation area inside the cell pretreatment module. During sequential operation, cell samples that have undergone timed incubation on the isothermal cell placement station 11 can be transferred to the sample carrying station 121 inside the treatment chamber 12. After the pretreatment process is completed, the samples are then output to the automated sample introduction module 2, achieving fully automated integration of culture, pretreatment, and sample introduction. The isothermal cell placement station 11 is used to place cell culture dishes and maintain the isothermal environment required for cell survival. It works in conjunction with the timing controller to complete the fixed-point and timed sampling and transportation.

[0064] The processing chamber 12 is a closed, independent cavity where all pretreatment steps are completed to prevent sample evaporation and external contamination.

[0065] The sample carrier station 121 uniformly places cell sample containers, and the crushing and centrifugation processes can be completed in situ without changing the containers.

[0066] The ultrasonic disruptor 122 is attached to the bottom of the workstation, and the vibration is transmitted to the sample to achieve cell lysis and release intracellular puerarin.

[0067] The miniature high-speed centrifugal rotor 123 is coaxially connected to the work station. After crushing, it directly drives the work station to rotate at high speed, so as to achieve sedimentation and stratification of cell fragments and obtain a clear extract.

[0068] The ninth embodiment of the automatic sample introduction puerarin fluorescence detection and analysis device of the present invention further includes a timing linkage controller, which is electrically connected to the cell pretreatment module 1, the automatic sample introduction module 2, the fluorescence spectrophotometer detection module 3, and the data processing module.

[0069] In this embodiment, the timing linkage controller coordinates the running sequence of all modules. After the preset sampling time is reached, the entire process of sampling, pretreatment, injection, detection, and correction calculation is automatically executed in sequence without manual intervention, which is suitable for drug cell kinetic timing experiments.

[0070] Operating procedure of this device: 1. Place the cell culture dish in the constant temperature cell placement station 11, and preset multiple sampling time nodes in the data processing module; 2. When the time-series linkage controller reaches the set time, it controls the cell pretreatment module 1 to transfer the culture dish to the sample carrying station 121 inside the processing chamber 12; 3. The ultrasonic disruptor 122 starts to lyse the cells. After the cells are broken down, the micro high-speed centrifuge rotor 123 drives the station to centrifuge at high speed to obtain a clear puerarin extract. 4. The automatic sample injection module 2 is started, the filter unit 21 extracts the supernatant to remove solid impurities, and the negative pressure quantitative loop 22 draws the quantitative extract under negative pressure and delivers it to the fluorescence detection cell 32. 5. The excitation optical path unit 31 outputs 342nm excitation light to irradiate the extract, and puerarin produces 461nm characteristic fluorescence. After the stray light is filtered out by the emission filtering optical path unit 33, it is collected by the photomultiplier tube detector and converted into an electrical signal. 6. The data processing module sequentially calls the modified internal filtration effect equation and the pipeline adsorption correction relationship to complete the signal correction, outputs the true puerarin concentration after subtracting the matrix background, and simultaneously stores all sample data.

[0071] The specific embodiments described above do not constitute a limitation on the scope of protection of this application. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An automated sample introduction device for puerarin fluorescence detection and analysis, characterized in that, It includes a cell pretreatment module, an automated sample loading module, a fluorescence spectrophotometry detection module, and a data processing module; The cell pretreatment module is equipped with a constant temperature cell placement station. The output station of the cell pretreatment module is connected to the sampling end of the automatic sampling module, which can complete the cell sample disruption and centrifugation to prepare puerarin cell extract. The automatic sample introduction module is located between the cell pretreatment module and the fluorescence spectrophotometer detection module. The output of the automatic sample introduction module is connected to the fluorescence detection cell of the fluorescence spectrophotometer detection module through a transport flow path. The automatic sample introduction module can complete sample transport, quantitative delivery, online impurity filtration and flow path cleaning, and, in conjunction with the isothermal cell placement station, achieve continuous automatic sample introduction according to preset time nodes. The fluorescence spectrophotometer is electrically connected to the data processing module. The fluorescence spectrophotometer can output a fixed wavelength excitation light to irradiate the extract to be tested in the fluorescence detection cell. After filtering out stray light, the fluorescence signal generated by puerarin is converted into an electrical signal and transmitted to the data processing module. The data processing module has a built-in main control unit and quantitative analysis program, which can call the corrected internal filtration effect model and the pipeline adsorption correction model to perform correction calculations on the detection electrical signal, eliminate the quantitative deviation caused by the cell matrix internal filtration effect and the adsorption of the sample injection pipeline, and output the true concentration result of puerarin.

2. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The modified internal filtration effect model uses a modified fluorescence quantitative equation, with fluorescence intensity... With puerarin concentration The following relationship must be satisfied: ， in, The overall efficiency coefficient of the instrument; The initial intensity of the incident excitation light; The molar absorptivity of puerarin at 342 nm; The effective optical path length of the fluorescence detection cell; This represents the filtration attenuation coefficient within the cell matrix.

3. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The pipeline adsorption correction model adopts a dynamic correction relationship, and the actual concentration of puerarin is... Compared with apparent concentration The following relationship must be satisfied: ， in, The saturated adsorption rate of the inner wall of the sample inlet tube; This represents the cumulative number of consecutive injections. The adsorption saturation characteristic number is denoted by .

4. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The quantitative analysis program of the data processing module includes a light intensity quantification unit, a matrix background subtraction unit, and a data storage unit; the light intensity quantification unit acquires and quantifies the original fluorescence electrical signal, the matrix background subtraction unit subtracts the fluorescence background of the blank cell matrix, and the data storage unit stores the sample information and detection results together.

5. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The fluorescence spectrophotometer includes an excitation optical path unit, an emission filtering optical path unit, and a photoelectric acquisition unit. The excitation optical path unit is equipped with a xenon lamp light source and a monochromator, with a fixed output wavelength of 342 nm and an excitation slit width of 10 nm. The output optical path of the excitation optical path unit is aligned with the fluorescence detection cell. The emission filtering optical path unit is located between the fluorescence detection cell and the photoelectric acquisition unit, and has a built-in 461 nm narrowband bandpass filter with a half-width of 10 nm. The photoelectric acquisition unit receives the filtered fluorescence signal and converts it into an electrical signal.

6. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 5, characterized in that, The photoelectric acquisition unit of the fluorescence spectrophotometer is equipped with a photomultiplier tube detector, which has a built-in temperature compensation unit to suppress dark current noise; the spectral response range of the photomultiplier tube detector covers 380nm~600nm.

7. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The automatic sample introduction module includes a negative pressure quantitative loop and a filter unit; the filter unit and the negative pressure quantitative loop are connected in series through a flow path, the filter unit is connected to the output station of the cell pretreatment module, and the negative pressure quantitative output terminal is connected to the fluorescence detection cell.

8. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 7, characterized in that, The negative pressure quantitative loop is a fixed volume pipeline. The extract is filled into the pipeline by negative pressure suction to achieve fixed volume injection.

9. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, The cell pretreatment module includes a processing chamber, which contains a sample carrying station. An ultrasonic disruptor is located at the bottom of the sample carrying station, and a miniature high-speed centrifugal rotor is coaxially connected to the sample carrying station.

10. The automated sample introduction device for puerarin fluorescence detection and analysis according to claim 1, characterized in that, It also includes a timing linkage controller, which is electrically connected to the cell pretreatment module, the automatic sample injection module, the fluorescence spectrophotometer detection module, and the data processing module.

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