Liquid chromatography combined molecular fluorography detection system and method

Abstract

The invention relates to a molecular fluorography detection method combined with liquid chromatography, and belongs to the technical field of analysis and detection. The invention aims to solve the technical problems that the high sensitivity is difficult to maintain while the miniaturization of the existing liquid chromatogram fluorescence detector is realized, and a flexible and low-cost detection scheme is lacked. According to the technical scheme, the method is characterized in that after being subjected to liquid chromatography separation, a sample is conveyed to a silica gel coating preparation plate through a polyether-ether-ketone tube to form a detection area; exciting fluorescence by adopting a laser with a first narrow-pass optical filter; acquiring a series of fluorescence images by using an image acquisition unit with a second narrow-pass optical filter; and finally, extracting RGB (Red, Green and Blue) numerical values of a fixed pixel region through computer image processing software, and generating a chromatogram of which the fluorescence intensity changes along with time. The method is mainly used for rapid and high-sensitivity fluorescence detection in the fields of biomedical analysis, environmental monitoring, food safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a molecular fluorescence imaging detection system and method using liquid chromatography coupled with liquid fluorescence imaging. Background Technology

[0002] Liquid chromatography (LC) is an important separation and analysis technique, and the performance of its detector directly affects the analytical capability of the entire system. In many application scenarios, especially in rapid on-site detection and resource-constrained environments, there is an urgent need for miniaturized and portable detectors. However, attempts at detector miniaturization are often accompanied by a significant decrease in sensitivity. This is mainly because, in order to reduce size, many miniaturized detectors use light-emitting diodes (LEDs) as the excitation source. LEDs typically emit a wider spectral width, several orders of magnitude wider than lasers, making it difficult to effectively separate the emitted fluorescence signal from the stray light of the excitation source itself. This results in a lower signal-to-noise ratio, a higher detection limit, and difficulty in accurately detecting low-concentration or trace components. Furthermore, detection schemes based on traditional photomultiplier tubes and other core components, while achieving miniaturization, usually increase hardware costs, hindering widespread application. Therefore, under current technological conditions, simultaneously achieving miniaturization, high sensitivity, and low cost in liquid chromatography detectors presents significant technical challenges, urgently requiring new technological approaches to overcome these mutually restrictive bottlenecks. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a molecular fluorescence imaging detection system and method coupled with liquid chromatography, which can maintain high detection sensitivity while achieving miniaturization and portability of the detection system.

[0005] This invention is based on the capillary diffusion behavior of the sample solution after liquid chromatography on the surface of a silica gel coating, forming a ring-shaped sample spot structure with a concentration gradient. Due to diffusion, this ring-shaped sample spot exhibits a distribution characteristic of low concentration at the center and high concentration at the edges. Under laser excitation, its fluorescence signal intensity also shows a gradient change from the center to the edge, gradually increasing and then decreasing. By controlling a conveyor belt to drive the preparation plate in intermittent motion, different regions of the ring-shaped sample spot (including the outer ring, middle region, inner ring, and center) are sequentially scanned. Fluorescence images of the blank areas on both sides are acquired before and after scanning the sample spot as background references. After image processing to extract the fluorescence intensity of each region, the signal from the blank areas is used to determine the baseline level, obtaining a high signal-to-noise ratio chromatographic peak signal, achieving high sensitivity and high accuracy fluorescence detection.

[0006] To achieve these objectives and other advantages of the present invention, a molecular fluorescence imaging detection method coupled with liquid chromatography is provided, comprising the following steps: S1: After separation by the liquid chromatography system, the sample is transported to the sample collection unit through a polyetheretherketone tube. The sample collection unit is a preparation plate with a silicone coating. The lower end of the polyetheretherketone tube is attached to the silicone coating surface of the preparation plate, and the sample solution spreads on the silicone coating surface to form a detection area. S2: The test area of ​​the preparation plate is irradiated by a laser excitation unit to excite fluorescence. The laser wavelength range emitted by the laser excitation unit is 430-470 nm, and a first narrow pass filter is set in the laser output path in front of the laser excitation unit. S3: The excited fluorescence image is acquired using an image acquisition unit, wherein a second narrow pass filter is provided in front of the lens of the image acquisition unit, and the transmission wavelength range of the second narrow pass filter is 530-570 nm. S4: Transmit a series of fluorescence images acquired by the image acquisition unit in chronological order to the computer; S5: Using image processing software on a computer, extract the intensity value of at least one channel in the RGB channels of a fixed pixel region in a series of fluorescence images, and correlate the intensity value of the RGB channel with the image acquisition time to generate a chromatogram of fluorescence intensity changing over time.

[0007] Preferably, the liquid chromatography system includes an injector, a chromatographic column, a delivery pump, and a container for holding the mobile phase, and the polyetheretherketone tube is connected between the column outlet and the sample collection unit.

[0008] Preferably, the preparation plate is fixed horizontally, and the polyether ether ketone tube is vertically positioned directly above the preparation plate.

[0009] Preferably, the preparation plate with the detection area is placed on a conveyor belt, and the control unit drives the conveyor belt to move intermittently. The laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation plate moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires the fluorescence image of the preparation plate on the positioning area. In the period of the intermittent movement, the movement time ranges from 0.8 to 1.2 seconds, and the static time ranges from 0.8 to 1.2 seconds.

[0010] Preferably, the control unit includes an Arduino controller, a stepper motor driver, and a stepper motor. The Arduino controller outputs pulse signals to the stepper motor driver to drive the stepper motor to drive the conveyor belt to perform intermittent motion.

[0011] Preferably, the image acquisition unit is fixed on the bracket and oriented vertically toward the detection area; the shooting parameters of the image acquisition unit are set to manual mode, the ISO value is set to 50-400, and the exposure time is set to 1 / 10-1 / 30 seconds.

[0012] Preferably, the specific steps for extracting the RGB channel values ​​of a fixed pixel region from a series of fluorescence images are as follows: identify the image with the strongest fluorescence signal from the series of images, and determine the pixel coordinates of the point with the strongest signal in the image; delineate a square region with a side length of 3-7 pixels centered on the pixel coordinates; calculate the average value of the R, G, and B channel values ​​of all pixels within the square region, and extract the average value of the RGB channel values ​​for each image in the series based on the determined pixel coordinates and region size.

[0013] A molecular fluorescence radiography detection system coupled with liquid chromatography, comprising: A liquid chromatography system, comprising a chromatographic column and a polyetheretherketone delivery tube connected to the column outlet; The sample collection unit is a preparation plate with a silicone coating on its surface; the outlet end of the polyetheretherketone delivery tube is configured to adhere to the silicone coating surface of the preparation plate to deliver the sample solution separated by the chromatographic column to the surface and form a detection area. A laser excitation unit is configured to emit excitation light with a wavelength of 430-470 nm into the detection area, and a first narrow-pass filter is provided in the light output path of the laser excitation unit. The image acquisition unit is a mobile phone or camera with a camera. The image acquisition unit is fixedly set above the detection area, and a second narrow pass filter with a transmission wavelength of 530-570 nm is set in front of its lens. The conveying control unit includes a conveyor belt, a stepper motor driving the conveyor belt, a driver controlling the stepper motor, and a programmable controller signal-connected to the driver. A preparation board with a detection area is placed on the conveyor belt. The programmable controller is configured to control the conveyor belt to move intermittently. A laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation board moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires the fluorescence image of the preparation board on the positioning area. The data processing unit is a computer that is communicatively connected to the image acquisition unit. The computer is equipped with image processing software, which is configured to extract the RGB channel values ​​of a fixed pixel region from a series of fluorescence images captured by the image acquisition unit in chronological order, and generate a chromatogram based on the correspondence between the values ​​and the acquisition time.

[0014] The present invention has at least the following beneficial effects: First, by using a laser as the excitation source and in conjunction with a narrow-pass filter of a specific wavelength, this invention effectively overcomes the problem of fluorescence and stray light separation caused by the excessively broad spectrum of LED light sources in traditional miniaturized detectors. Thus, while miniaturizing the detection system, it significantly improves the signal-to-noise ratio and detection sensitivity.

[0015] Secondly, this invention uses a commonly available mobile phone or camera camera as the core image acquisition component, replacing the expensive photomultiplier tube in the traditional fluorescence detector. At the same time, it uses a conventional preparation plate as the sample carrier, which greatly reduces the hardware cost and complexity of the entire detection system and provides an economical and feasible solution for rapid on-site detection.

[0016] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a liquid phase analysis system based on a molecular fluorescence radiography detector, according to one technical solution of the present invention. Figure 2 This is a schematic diagram of a preparation plate collection device according to one technical solution of the present invention; Figure 3 This is a schematic diagram of a detection system based on a fabrication plate detector, according to one technical solution of the present invention. Figure 4 This is a diagram illustrating the data processing steps of one technical solution of the present invention; Figure 5 The chromatogram of an analyte sample is shown as an example of the present invention. Figure 6 A comparison of fluorescence intensity responses of target analytes at different concentrations; Figure 7 The standard curve and limit of detection (LOD) analysis chart for the target analyte; Figure 8 This is a schematic diagram of a device for collecting mixed samples according to one technical solution of the present invention; Figure 9 The chromatogram for detecting a mixture sample is an example of the present invention. Figure 10 This is a chromatogram of a mixture sample detected by a liquid chromatography-ultraviolet detector.

[0018] 101. Sample to be tested; 102. Liquid chromatograph; 103. Sample collection module; 104. Detection module; 105. Data analysis; 106. Chromatogram; 2. Preparation plate; 201. Circular sample spot; 202. Silica gel coating surface; 204. Glass layer; 301. Power supply; 302. Conveyor structure; 303. Smartphone; 304. 550 nm narrow-pass filter; 305. Laser; 306. 450 nm narrow-pass filter; 801. Theoretical peak; 802. Sample spot. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0022] A molecular fluorescence imaging method coupled with liquid chromatography, comprising the following steps: S1: After the sample is separated by the liquid chromatography system, it is transported to the sample collection unit through a polyether ether ketone tube. The sample collection unit is a preparation plate with a silicone coating, so that the lower end of the polyether ether ketone tube is attached to the silicone coating surface of the preparation plate, and the sample solution spreads on the silicone coating surface to form a detection area. S2: The test area of ​​the preparation plate is irradiated by a laser excitation unit to excite fluorescence. The laser wavelength range emitted by the laser excitation unit is 430-470 nm, and a first narrow pass filter is set in the laser output path in front of the laser excitation unit. S3: The excited fluorescence image is acquired using an image acquisition unit, wherein a second narrow pass filter is provided in front of the lens of the image acquisition unit, and the transmission wavelength range of the second narrow pass filter is 530-570 nm. S4: Transmit a series of fluorescence images acquired by the image acquisition unit in chronological order to the computer; S5: Using image processing software on a computer, extract the intensity value of at least one channel in the RGB channels of a fixed pixel region in a series of fluorescence images, and correlate the intensity value of the RGB channel with the image acquisition time to generate a chromatogram of fluorescence intensity changing over time.

[0023] This invention innovatively combines liquid chromatography separation technology with fluorescence excitation imaging technology based on a preparative plate. It employs a laser with a specific wavelength range as the excitation source and uses a mobile phone or camera equipped with a narrow-pass filter to acquire fluorescence images. Finally, computer image processing extracts the RGB values ​​of the time series to generate a chromatogram. This invention resolves the contradiction between detector miniaturization and high sensitivity in existing technologies. Because the narrow spectral characteristics of the laser, combined with the filter, significantly reduce stray light interference, it simplifies the system structure, greatly reduces hardware costs, and ensures high detection sensitivity and signal-to-noise ratio, providing a flexible and reliable solution for rapid on-site detection.

[0024] In another embodiment, the liquid chromatography system includes an injector, a column, a delivery pump, and a container for holding the mobile phase, with the polyetheretherketone (PEEK) tube connected between the column outlet and the sample collection unit.

[0025] This technical solution constructs a complete liquid chromatography separation unit including an injector, chromatographic column, delivery pump, and mobile phase container, and uses polyetheretherketone tubing to directly connect the column outlet to the subsequent sample collection unit, establishing a stable and closed sample transport path. This design ensures that each component after fine separation by the chromatographic column can be guided intact and undamaged to the designated area of ​​the preparation plate, thus providing a reliable and consistent sample basis for subsequent fluorescence excitation and imaging detection. It effectively avoids sample diffusion, adsorption loss, or cross-contamination during the transport process, improving the reproducibility and accuracy of the entire analytical process. At the same time, the standardized interface and tubing design also facilitates system integration and miniaturization.

[0026] In another embodiment, the preparation plate is fixed horizontally, and the polyether ether ketone tube is vertically positioned directly above it.

[0027] In the sample collection step, this technical solution ensures that the sample solution can be accurately delivered to the collection surface in a vertical and centered manner by keeping the preparation plate horizontally fixed and setting the polyetheretherketone tube vertically above it. This precise spatial positioning and orientation allows the solution to achieve controllable and uniform radial spreading on the silicone coating or optical surface, thereby forming a detection area with a regular shape and relatively consistent thickness. This provides a stable and repeatable sample substrate for subsequent laser excitation, effectively reducing fluorescence signal fluctuations caused by droplet position deviation or uneven spreading, and improving the reliability of quantitative analysis.

[0028] In another approach, the sample solution flows out from the lower end of the polyetheretherketone (PEEK) tube, spreads on the silicone coating surface, and diffuses due to capillary action, eventually forming a clear annular sample spot. Once the solution delivery is complete, collection is finished. The annular sample spot is the detection area; the right side of the annular sample spot is the blank detection area A, and the left side is the blank detection area B. The preparation plate with the detection area is placed on a conveyor belt, which is driven by a control unit to move intermittently. A laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation plate moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires a fluorescence image of the preparation plate on the positioning area. The period of the intermittent movement ranges from 0.8 to 1.2 seconds, and the static time ranges from 0.8 to 1.2 seconds.

[0029] Specifically, the laser excitation unit continuously irradiates the positioning area on the conveyor belt. Initially, the preparation plate is placed on the conveyor belt, aligning its blank detection area A with the left side of the positioning area. The programmable controller controls the conveyor belt to move horizontally from left to right. When the conveyor belt moves the preparation plate for 0.8-1.2 seconds to bring the test area of ​​the preparation plate into the positioning area, it pauses for 0.8-1.2 seconds. During this static period, the laser excitation unit excites the current test area with a laser, while the image acquisition unit simultaneously captures a fluorescence image. After a single image acquisition is completed, the conveyor belt resumes movement, repeating the above steps of moving-pausing-image acquisition, sequentially completing the laser excitation and fluorescence image acquisition of the blank detection area A, different areas of the detection area, and the blank detection area B on the preparation plate.

[0030] This technical solution places the sample-carrying preparation plate on a conveyor belt and uses a control unit to drive the conveyor belt to perform intermittent motion in specific cycles, completing the acquisition of fluorescence images within each brief static window. This intermittent motion mode allows the sample detection area to pass through the excitation and imaging positions sequentially and stably, effectively avoiding image blurring caused by continuous sample movement. It also prevents fluorescence quenching or sample damage that may be caused by prolonged laser irradiation of fixed points, thereby ensuring that the fluorescence intensity signal extracted from the series of images has higher temporal resolution and accuracy. This provides a reliable data foundation for generating clear time-series chromatograms and enhances the system's adaptability to detecting unstable fluorescent samples.

[0031] In another embodiment, the control unit includes an Arduino controller, a stepper motor driver, and a stepper motor. The Arduino controller outputs pulse signals to the stepper motor driver to drive the stepper motor to drive the conveyor belt to perform intermittent motion.

[0032] This technical solution employs a control unit comprising a programmable logic controller (PLC), a stepper motor driver, and a stepper motor. The PLC generates and outputs a specific pulse signal sequence to the driver, thereby precisely driving the stepper motor to drive the conveyor belt to execute a preset intermittent motion mode. This digital pulse-based motor control method achieves millisecond-level precise management of the conveyor belt's start and stop positions and motion duration, ensuring that the sample's dwell time at each detection site is strictly consistent. This provides a stable and reliable imaging window for the image acquisition unit, effectively overcoming image focusing deviations or timing errors caused by transmission instability, and ensuring the repeatability and comparability of subsequent chromatogram data.

[0033] In another embodiment, the image acquisition unit is fixed on a bracket and oriented vertically toward the detection area; the shooting parameters of the image acquisition unit are set to manual mode, with the ISO value set to 50-400 and the exposure time set to 1 / 10-1 / 30 seconds.

[0034] This technical solution constructs a stable and controllable light signal capture environment by fixing the image acquisition unit, such as a mobile phone or camera, to a bracket and vertically aligning it with the detection area, while setting the shooting parameters to manual mode and limiting the ISO sensitivity and exposure time to specific ranges. This setting eliminates image position shifts and brightness fluctuations caused by hand shake or changes in automatic metering, ensuring that each frame of the acquired fluorescence image has a consistent spatial reference and exposure level during intermittent motion detection. This lays a solid foundation for extracting accurate and comparable RGB values ​​from time-series images, significantly improving the consistency of fluorescence intensity data and the reproducibility of chromatograms.

[0035] In another approach, the specific steps for extracting the RGB channel values ​​of a fixed pixel region from a series of fluorescence images are as follows: identify the image with the strongest fluorescence signal from the series of images and determine the pixel coordinates of the point with the strongest signal in that image; delineate a square region with a side length of 3-7 pixels centered on the pixel coordinates; calculate the average value of the R, G, and B channel values ​​of all pixels within the square region; for each image in the series, extract the average value of the RGB channel values ​​according to the determined pixel coordinates and region size.

[0036] In the image processing stage, this technical solution identifies and locates the pixel with the strongest fluorescence signal within the time-series image. A fixed-size square region is then defined centered on the coordinates of this point, and the average RGB channel values ​​of all pixels within this region are calculated. This establishes a unified and objective signal extraction standard for the entire image sequence. This method effectively eliminates signal fluctuations caused by random noise or slight positional shifts in single pixels, making the fluorescence intensity data obtained from each image more representative and stable. It significantly improves the comparability of data collected at different time points and provides a reliable and consistent data processing foundation for constructing chromatograms that accurately reflect the changes in fluorescence intensity over time.

[0037] A molecular fluorescence radiography detection system coupled with liquid chromatography, comprising: A liquid chromatography system, comprising a chromatographic column and a polyetheretherketone delivery tube connected to the column outlet; The sample collection unit is a preparation plate with a silicone coating on its surface; the outlet end of the polyetheretherketone delivery tube is configured to adhere to the silicone coating surface of the preparation plate to deliver the sample solution separated by the chromatographic column to the surface and form a detection area. A laser excitation unit is configured to emit excitation light with a wavelength of 430-470 nm into the detection area, and a first narrow-pass filter is provided in the light output path of the laser excitation unit. The image acquisition unit is a mobile phone or camera with a camera. The image acquisition unit is fixedly set above the detection area, and a second narrow pass filter with a transmission wavelength of 530-570 nm is set in front of its lens. The conveying control unit includes a conveyor belt, a stepper motor driving the conveyor belt, a driver controlling the stepper motor, and a programmable controller signal-connected to the driver. A preparation board with a detection area is placed on the conveyor belt. The programmable controller is configured to control the conveyor belt to move intermittently. A laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation board moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires the fluorescence image of the preparation board on the positioning area. The data processing unit is a computer that is communicatively connected to the image acquisition unit. The computer is equipped with image processing software, which is configured to extract the RGB channel values ​​of a fixed pixel region from a series of fluorescence images captured by the image acquisition unit in chronological order, and generate a chromatogram based on the correspondence between the values ​​and the acquisition time.

[0038] The present invention will be further described in detail below with reference to the embodiments.

[0039] Example 1 1. Overall System Structure See appendix Figure 1 The detection system in this embodiment mainly includes the following subsystems: sample injection and liquid chromatography separation subsystem, sample collection subsystem, fluorescence excitation and image acquisition subsystem, and data processing subsystem.

[0040] The sample solution to be tested is injected into the liquid chromatograph via an injector. The liquid chromatograph includes a mobile phase reservoir, a liquid phase pump, a column oven, and a chromatographic column. The liquid phase pump provides the power for mobile phase delivery, and the sample is separated within the chromatographic column. The separated components elute sequentially from the column.

[0041] 2. Sample collection subsystem Once the actual sample is injected into the liquid chromatography system, the sample collection process is initiated. The eluent of the target component separated by the chromatographic column is precisely delivered to the sample collection unit through a polyetheretherketone (PEEK) tube.

[0042] See appendix Figure 2 A smooth, clean silica gel plate for thin-layer chromatography was selected as the preparation plate. It was horizontally fixed on a worktable at a suitable height. A polyetheretherketone (PEEK) tube was vertically fixed directly above the preparation plate, and its lower end was adjusted to ensure close contact with the silica gel coating on the plate surface. The liquid chromatography system was started, and the flow rate was set to 0.1 mL / min. The sample solution flowed out from the lower end of the PEEK tube, spread across the silica gel coating surface, and diffused due to capillary action, eventually forming a clear circular sample spot. Once the solution delivery was complete, the sample was collected. The right side of the circular sample spot is the blank detection area A, and the left side is the blank detection area B.

[0043] 3. Fluorescence Excitation and Image Acquisition Subsystem After sample collection, the preparation plate carrying the sample is placed on a conveyor belt and enters the testing stage. This system integrates laser excitation, filtering, motion control, and image acquisition functions.

[0044] 3.1 Transmission and Motion Control The conveyor belt is driven by a control unit to perform intermittent motion. This control unit includes an Arduino controller, a stepper motor driver, and stepper motors. The Arduino controller outputs a preset pulse sequence, which precisely controls the stepper motors via the driver, thereby driving the conveyor belt to perform motion-stationary cycles. The motion time is approximately 1 second, and the stationary time is approximately 1 second.

[0045] The specific process is as follows: The laser excitation unit continuously irradiates the positioning area on the conveyor belt; initially, the preparation plate is placed on the conveyor belt, with its blank detection area A aligned with the left side of the positioning area; the control unit controls the conveyor belt to move horizontally from left to right at a constant speed; each test area moves sequentially to the positioning area in the following order: blank detection area A → outer ring of the annular sample spot → middle area between the outer and inner rings → inner ring → center of the inner ring → inner ring → middle area between the outer and inner rings → outer ring → blank detection area B. Within each stationary window (approximately 1 second), the laser excitation unit excites the current area, and the image acquisition unit simultaneously captures fluorescence images. Through this sequential scanning method, a series of images reflecting the fluorescence intensity distribution of the annular sample spot can be obtained, and the signal exhibits a normal distribution over time, providing a high-quality data foundation for subsequent chromatogram construction.

[0046] 3.2 Fluorescence Excitation See appendix Figure 3 A laser with an emission wavelength of 450±5nm is fixed obliquely above the preparation plate. A first narrow-pass filter is installed before the laser's output light path to purify the excitation light. The laser angle is adjusted so that its beam accurately illuminates the sample spot area on the preparation plate on the conveyor belt.

[0047] 3.3 Image Acquisition A smartphone is vertically fixed above the sample detection area using a bracket. A second narrow-pass filter with a transmission wavelength range of 550±7.5nm is installed in front of its lens to filter out stray light from the excitation light, allowing only sample fluorescence to pass through.

[0048] The image acquisition unit's shooting parameters were set to manual mode. In one specific setting, the ISO value was set to 50, and the exposure time was set to 1 / 20 second. The image acquisition unit captured fluorescence images when the conveyor belt was stationary. With the intermittent movement of the conveyor belt, the system controlled the conveyor belt to drive the preparation plate in an intermittent motion, sequentially scanning different regions of the annular sample spot (including the outer ring, middle region, inner ring, and center), and acquiring fluorescence images of the blank areas on both sides of the sample spot before and after scanning as background references. Through this sequential scanning method, a series of image sequences reflecting the spatial distribution of fluorescence intensity on the sample spot over time can be obtained.

[0049] 4. Data Processing Subsystem The image acquisition unit transmits a series of captured fluorescence images to a computer via wireless or wired means.

[0050] Data analysis was performed using professional image processing software on a computer; the workflow is detailed in the appendix. Figure 4 : Step 401: Import the original image sequence obtained in chronological order.

[0051] Step 402: Identify the frame with the strongest fluorescence signal from the sequence. Determine the pixel coordinates of the point with the strongest signal in this image.

[0052] Step 403: Using these coordinates as the center, define a fixed-size pixel region (a square region with sides of 5 pixels). Calculate the average R, G, and B channel values ​​of all pixels within this region. This region and calculation method are used as a standard and applied to all images in this sequence.

[0053] Step 404: For all images arranged in chronological order, repeat step 403 to extract the RGB average value of the corresponding region of each frame image and correlate it with the acquisition time of that image.

[0054] Step 405: The intensity values ​​of a specific channel (Channel G) extracted are compiled and correlated with time to create a chromatogram showing the change in fluorescence intensity over time. This chromatogram visually illustrates the signal response of different components after separation by liquid chromatography and can be used for quantitative analysis.

[0055] 5. Implementation Results Using the above system and method, rhodamine 6G dissolved in methanol (concentration 0.05 μg / mL) was tested in preparative plate detection mode. Chromatographic conditions: C18 column (250 mm), mobile phase: methanol, flow rate: 0.1 mL / min, injection volume: 5 μL. The system operated stably and successfully obtained chromatograms with good signal-to-noise ratio. Figure 5 (As shown). This embodiment of the system has high-sensitivity detection capabilities, and the relevant verification data is as follows. Figure 6 , Figure 7 As shown: Among them Figure 6 The graph shows a comparison of the fluorescence intensity response of Rhodamine 6G at different concentrations (1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL). The vertical axis represents the fluorescence response value, which intuitively presents the differences in the system's signal response to samples of different concentrations and clearly reflects the positive correlation between concentration and fluorescence intensity. Figure 7 The standard curve and limit of detection (LOD) analysis of this sample were obtained through linear fitting, yielding the equation y = a + b*x. The fitting parameters showed an intercept of 149.52663 ± 13.03837, a slope of 408.73746 ± 7.09636, and a Pearson's r-correlation coefficient as high as 0.9994. 2The coefficient of determination (COD) is 0.9991, demonstrating a strong linear correlation between concentration and fluorescence intensity, providing a reliable basis for quantitative analysis. Furthermore, the limit of detection (LOD) is 0.33 ng / mL, fully validating the high sensitivity and reliability of this system for quantitative analysis. While the achieved LOD in this example is somewhat lower than that of a commercially available fluorescence detector, it still reaches 0.3 ng / mL, exhibiting a respectable level.

[0056] The detection mechanism of this system is based on the combination of sample diffusion on the surface of the silica gel coating and fluorescence imaging. For a single dye sample, only one spot needs to be collected on the preparation plate. The sample diffuses naturally on the silica gel coating due to capillary action, forming a distribution with low concentration at the center and high concentration at the edge. This results in an intensity gradient in the fluorescence image, which is weak at the center and strong at the edge. After extraction, the concentration information of the chromatographic effluent can be directly obtained from this gradient signal.

[0057] Example 2 For multi-component mixed samples, to accurately capture the elution profile of each chromatographic peak, time-resolved spotting is required within the elution time period of each component. This means that at least five consecutive time points must be collected for each chromatographic peak, with the spotting interval consistent with the chromatographic elution time resolution. All sample spots are arranged at equal intervals in chronological order on the same elongated preparation plate, forming a time-space correspondence array. The preparation plate is placed on a conveyor belt for sequential scanning. The fluorescence signal of each spot is acquired through laser excitation and image acquisition, and then the complete chromatogram is reconstructed through image processing. The peak morphology and elution order obtained by this method are consistent with the results of conventional liquid chromatography with UV detectors, verifying the reliability and accuracy of this system in the separation and detection of complex samples.

[0058] To further verify the system's ability to separate and detect complex real-world samples, a mixed sample detection experiment was conducted. The specific method is as follows: [See sample collection instructions]. Figure 8 The chromatographic eluent was sequentially added dropwise to the silica gel coating of the preparation plate using a polyetheretherketone (PEEK) tube, one drop every second from right to left, with each drop corresponding to a time point on the chromatographic elution curve. At least five consecutive drops were collected for each chromatographic peak until all target components were collected. Subsequently, the fluorescence of all drops was excited by laser scanning, and images were acquired. Finally, the chromatogram of the mixed sample was obtained through image processing.

[0059] To verify the system's ability to separate and detect multi-component mixed samples, a mixed sample composed of DBD-H, formaldehyde, acetaldehyde, acetone, n-propionaldehyde, and n-butyraldehyde was selected for testing. The components and their corresponding concentrations and volumes are as follows: DBD-H: 360 μL 10 mM; Formaldehyde: 100 μL 3.75 mM; Acetaldehyde: 100 μL 7.5 mM; Acetone: 100 μL 10 mM; n-Propionaldehyde: 100 μL 10 mM; n-Butyraldehyde: 100 μL 10 mM.

[0060] The test was performed in preparative plate detection mode, and the chromatographic conditions were as follows: C18 column (250 mm), mobile phase acetonitrile:water = 65:35, +0.1% formic acid, flow rate 0.5 ml / min, injection volume 20 μL.

[0061] The system runs stably, and the obtained chromatograms are as follows: Figure 9 As shown in the figure, the elution order of the components is as follows: DBD-H, formaldehyde, acetaldehyde, acetone, n-propionaldehyde, and n-butyraldehyde. This is compared with the chromatogram obtained from the same sample using a conventional liquid chromatography-UV detector. Figure 10 A comparison showed that the peak shapes and elution sequences of the two systems were basically the same, indicating that the system has reliability and consistency in the separation and detection of multiple components.

[0062] This system's detection mechanism fully utilizes the circular sample spot structure formed by the diffusion characteristics of the sample on the silicone coating surface. By sequentially scanning different regions, the obtained fluorescence intensity signals naturally exhibit a normal distribution, which highly matches the mathematical model of chromatographic elution peaks. This method not only simplifies the data reconstruction process but also significantly improves the signal-to-noise ratio and the reliability of quantitative analysis, making it particularly suitable for the rapid detection of trace components.

[0063] This invention offers operational flexibility in handling different detection scenarios: For multi-component mixed samples, since the elution time of each component is unknown, a continuous and equally spaced sampling mode (such as one sample per second) can be adopted to achieve full-process capture of the effluent without prior knowledge of the elution time. Its function is equivalent to a detector that works around the clock.

[0064] For a single target dye, targeted spotting and signal acquisition can be performed within the corresponding time period according to its expected peak time, so as to improve detection efficiency and reduce unnecessary sample and reagent consumption.

[0065] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method of detection by molecular fluorescence photography in conjunction with liquid chromatography, characterized in that, Includes the following steps: S1: After separation by the liquid chromatography system, the sample is transported to the sample collection unit through a polyetheretherketone tube. The sample collection unit is a preparation plate with a silicone coating. The lower end of the polyetheretherketone tube is attached to the silicone coating surface of the preparation plate, and the sample solution spreads on the silicone coating surface to form a detection area. S2: The test area of ​​the preparation plate is irradiated by a laser excitation unit to excite fluorescence. The laser wavelength range emitted by the laser excitation unit is 430-470 nm, and a first narrow pass filter is set in the laser output path in front of the laser excitation unit. S3: The excited fluorescence image is acquired using an image acquisition unit, wherein a second narrow pass filter is provided in front of the lens of the image acquisition unit, and the transmission wavelength range of the second narrow pass filter is 530-570 nm. S4: Transmit a series of fluorescence images acquired by the image acquisition unit in chronological order to the computer; S5: Using image processing software on a computer, extract the intensity value of at least one channel in the RGB channels of a fixed pixel region in a series of fluorescence images, and correlate the intensity value of the RGB channel with the image acquisition time to generate a chromatogram of fluorescence intensity changing over time.

2. The method of claim 1, wherein the method is used in conjunction with liquid chromatography. The liquid chromatography system includes an injector, a column, a delivery pump, and a container for holding the mobile phase. The polyether ether ketone tube is connected between the column outlet and the sample collection unit.

3. The method of claim 1, wherein the method is used in conjunction with liquid chromatography. The preparation plate is fixed horizontally, and the polyether ether ketone tube is vertically positioned directly above the preparation plate.

4. The method of claim 1, wherein the method is used in conjunction with liquid chromatography. The preparation plate with the detection area is placed on a conveyor belt, and the control unit drives the conveyor belt to move intermittently. The laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation plate moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires the fluorescence image of the test area of ​​the preparation plate on the positioning area. In the period of the intermittent movement, the movement time ranges from 0.8 to 1.2 seconds, and the static time ranges from 0.8 to 1.2 seconds.

5. The method of claim 4, wherein the liquid chromatography coupled with molecular fluorescence photography detection method is characterized by, The control unit includes an Arduino controller, a stepper motor driver, and a stepper motor. The Arduino controller outputs pulse signals to the stepper motor driver to drive the stepper motor to drive the conveyor belt to perform intermittent motion.

6. The method of claim 1, wherein the method is used in conjunction with liquid chromatography. The image acquisition unit is fixed on the bracket and oriented vertically toward the detection area; the shooting parameters of the image acquisition unit are set to manual mode, the ISO value is set to 50-400, and the exposure time is set to 1 / 10-1 / 30 seconds.

7. The method of claim 1, wherein the method is used in conjunction with liquid chromatography. The specific steps for extracting RGB channel values ​​from a fixed pixel region in a series of fluorescence images are as follows: Identify the image with the strongest fluorescence signal from the series of images and determine the pixel coordinates of the point with the strongest signal in that image; delineate a square region with a side length of 3-7 pixels centered on the pixel coordinates; calculate the average value of the R, G, and B channel values ​​of all pixels within the square region; for each image in the series, extract the average value of the RGB channel values ​​according to the determined pixel coordinates and region size.

8. A molecular fluorescence photogrammetry detection system for use with liquid chromatography, characterized by, include: A liquid chromatography system, comprising a chromatographic column and a polyetheretherketone delivery tube connected to the column outlet; The sample collection unit is a preparation plate with a silicone coating on its surface; the outlet end of the polyetheretherketone delivery tube is configured to adhere to the silicone coating surface of the preparation plate to deliver the sample solution separated by the chromatographic column to the surface and form a detection area. A laser excitation unit is configured to emit excitation light with a wavelength of 430-470 nm into the detection area, and a first narrow-pass filter is provided in the light output path of the laser excitation unit. The image acquisition unit is a mobile phone or camera with a camera. The image acquisition unit is fixedly set above the detection area, and a second narrow pass filter with a transmission wavelength of 530-570 nm is set in front of its lens. A conveying control unit includes a conveyor belt, a stepper motor that drives the conveyor belt, a driver that controls the stepper motor, and a programmable controller that is signal-connected to the driver. A preparation plate with a detection area is placed on a conveyor belt. A programmable controller is configured to control the conveyor belt to move intermittently. A laser excitation unit continuously irradiates the positioning area on the conveyor belt. When the test area of ​​the preparation plate moves to the positioning area, the conveyor belt stops moving. During the static time, the image acquisition unit acquires the fluorescence image of the preparation plate on the positioning area. The data processing unit is a computer that is communicatively connected to the image acquisition unit. The computer is equipped with image processing software, which is configured to extract the RGB channel values ​​of a fixed pixel region from a series of fluorescence images captured by the image acquisition unit in chronological order, and generate a chromatogram based on the correspondence between the values ​​and the acquisition time.

Images