Low-cost portable high-sensitivity fluorescence detection system and method

Abstract

The invention discloses a low-cost portable high-sensitivity fluorescence detection system and method, and belongs to the field of fluorescence detection.The low-cost portable high-sensitivity fluorescence detection system is of a compact structure comprising a wide-field excitation light source, an optical separation module, a sample container, a macro-imaging fluorescence signal acquisition module and a pixel area average signal processing module. And a large-area pixel averaging technology is utilized to effectively suppress noise, and high-sensitivity and high-interference-resistance fluorescent quantitative analysis is realized under the condition that a complex optical scanning mechanism and sample pretreatment are not needed. The cost of the system is remarkably lower than that of traditional laboratory equipment, and the system is small in size, convenient to carry and particularly suitable for direct detection of complex biological samples, for example, circulating free DNA in urine can be rapidly and accurately quantified under the condition that an extraction step is not needed, the system is highly related to a standard qPCR method result, and the detection accuracy is high. And an efficient solution is provided for bedside detection and on-site instant analysis.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection, and particularly relates to a low-cost, portable, highly sensitive fluorescence detection system and method. Background Technology

[0002] Fluorescence detection technology, due to its high sensitivity and specificity, has been widely used in biomedical analysis, environmental monitoring, and chemical detection. Especially in the quantitative analysis of trace biomarkers such as circulating cell-free DNA (cfDNA), fluorescence detection has become a key method. Currently, in laboratory environments, high-sensitivity fluorescence detection is mainly achieved using equipment such as microplate readers or benchtop fluorometers. These devices typically employ sophisticated optical components, scanning mechanisms, or point detection modes to achieve excellent detection performance.

[0003] However, these laboratory-grade devices have significant limitations: First, their complex structure, large size, and high cost (typically tens of thousands of US dollars) make them difficult to deploy and apply at the bedside, in the field, or in resource-constrained environments. Second, their high sensitivity often depends on an ideal sample matrix; when faced with biological samples with complex compositions, such as urine, background fluorescence and scattered light interference can significantly reduce the signal-to-noise ratio, affecting detection accuracy. Furthermore, existing detection procedures typically require complex extraction and purification pretreatment of samples (such as DNA extraction), which is cumbersome and time-consuming, failing to meet the needs of rapid on-site detection. Therefore, there is an urgent need to develop a fluorescence detection technology and system that combines high sensitivity, strong anti-interference capabilities, low cost, portability, and simplified pretreatment procedures. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a low-cost, portable, high-sensitivity fluorescence detection system, comprising:

[0005] The excitation light source module is used to provide wide-field excitation light to the sample to be tested in the sample container;

[0006] An optical separation module, disposed on the excitation light source module, is used to suppress the excitation light and transmit the fluorescence emission signal generated by the sample to be tested;

[0007] A sample container for receiving and holding the sample to be tested after being irradiated by the wide-field excitation light;

[0008] A macro-imaging fluorescence signal acquisition module is used to perform macro-scale imaging of the fluorescence emission signal from the sample container to obtain a fluorescence image;

[0009] The pixel region averaging signal processing module is connected to the macro-imaging fluorescence signal acquisition module. It is used to perform signal averaging processing on the predefined pixel regions in the fluorescence image to obtain the fluorescence signal intensity after noise reduction and to perform quantitative analysis.

[0010] Optionally, the excitation light source module includes a light source for generating collimated excitation light and an optical homogenizing element for converting the collimated excitation light into a uniform surface light source.

[0011] Optionally, the optical homogenizing element is a Powell prism, used to achieve wide-field uniform illumination.

[0012] Optionally, the optical separation module includes at least one filter assembly for separating the optical signal based on the difference between the excitation wavelength and the emission wavelength.

[0013] Optionally, the macro-imaging fluorescence signal acquisition module includes an imaging lens and an image sensor, wherein the imaging lens is used to image the fluorescence signal from the sample container onto the image sensor.

[0014] Optionally, the image sensor is a CMOS sensor used to convert optical signals into the form of an electrical signal for the fluorescence image.

[0015] Optionally, the pixel region averaging signal processing module is configured to: acquire the fluorescence image from the macro-imaging fluorescence signal acquisition module, and select a region containing at least 40,000 pixels for signal intensity averaging calculation based on the corresponding position of the sample container in the image.

[0016] Optionally, the pixel region average signal processing module is further configured to: perform quantitative analysis of the target substance in the sample to be tested by means of the standard addition method or the external standard curve method based on the fluorescence signal intensity calculated by the average.

[0017] To address the aforementioned technical problems, this invention also provides a low-cost, portable, and highly sensitive fluorescence detection method, comprising:

[0018] Provides wide-field excitation light to illuminate the container holding the sample to be tested;

[0019] Receive the fluorescence emission signal generated by irradiation from the container, and optically separate the fluorescence emission signal from the wide-field excitation light;

[0020] Macro-imaging is performed on the separated fluorescence emission signal to obtain a fluorescence image containing the container region;

[0021] Pixel signals corresponding to the container region are extracted from the fluorescence image, and the pixel signals are averaged to obtain an average signal value representing the fluorescence intensity of the sample to be tested.

[0022] Based on the average signal value, the target analyte in the sample to be tested is quantitatively analyzed.

[0023] Furthermore, before providing wide-field excitation light irradiation, the method further includes a pretreatment step of the test sample, the pretreatment including diluting the test sample and adding a fluorescent dye that specifically binds to the target analyte thereto;

[0024] The sample to be tested is a biological liquid sample, the target analyte is circulating cell-free DNA, and the pretreatment includes high-multiplier dilution of the urine sample and addition of PicoGreen dye.

[0025] On the other hand, the present invention also provides an electronic device including a memory, a processor, and a computing program stored in the memory and executable on the processor, wherein the processor implements the method when executing the computing program.

[0026] On the other hand, the present invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the method.

[0027] Compared with the prior art, the present invention has the following advantages and technical effects:

[0028] 1) The equipment has a compact structure and low cost, making it suitable for on-site testing;

[0029] 2) The optical structure is simplified by using a simple orthogonal optical path arrangement.

[0030] 3) High-sensitivity fluorescence detection is achieved using wide-field excitation and micro-imaging techniques;

[0031] 3) Suitable for complex sample matrices, with strong anti-interference ability;

[0032] 4) It has good versatility and can be adapted to a variety of fluorescent dyes and detection objects. Attached Figure Description

[0033] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0034] Figure 1 This is a schematic diagram of the system structure according to an embodiment of the present invention;

[0035] Figure 2 This is a flowchart of a method according to an embodiment of the present invention. Detailed Implementation

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0037] It should be noted that the steps shown in the flowchart in the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although a logical order is shown in the flowchart, in some cases the steps shown or described may be executed in a different order than that shown here.

[0038] Example 1

[0039] like Figure 1 As shown, this embodiment discloses a low-cost, portable, high-sensitivity fluorescence detection system, comprising:

[0040] I. Core Components of the System

[0041] Excitation source module: A 25mW 488nm diode laser is used as the excitation source. The laser spot is shaped into a rectangle with a length of 12mm and a width of 1mm by diffraction optical elements, providing wide-field excitation light for the sample to be tested (200 μL, with a height of about 12.5mm in a sample cell with an inner diameter of 4.5mm) in the sample container. With the help of a Powell prism, uniform wide-field illumination of the detection cell area is achieved, and the coefficient of variation of the excitation light intensity uniformity is <5%.

[0042] Optical separation module: Located on the excitation light source module, it consists of a 540±20nm bandpass filter. Its core function is to suppress the excitation light while allowing the fluorescence emission signal generated by the sample to pass through, thus avoiding interference from the excitation light with the detection results.

[0043] Sample container: Used to receive and hold the test sample irradiated by wide field excitation light. Unlike commonly used PCR reaction tubes, it uses a low background glass capillary tube (specifically a Durham fermentation tube with an outer diameter of 6 mm, an inner diameter of 4.5 mm, and a height of 3 cm) to reduce the influence of the container's own background on the detection.

[0044] Macro-imaging fluorescence signal acquisition module: Responsible for macro-scale imaging of the fluorescence emission signal generated by the sample container to obtain fluorescence images. The hardware configuration is a 12-bit 1 / 1.8-inch, 2.3MP monochrome CMOS camera, paired with an f / 1.4 lens with a focal length of 16mm to collect fluorescence signals. By increasing the distance between the lens and the camera, macro imaging is achieved, maximizing the signal imaging area.

[0045] Pixel region average signal processing module: used for signal averaging, noise reduction, and quantitative analysis to ensure detection accuracy.

[0046] II. Key Technical Details:

[0047] (a) Signal processing flow;

[0048] In the fluorescence image, a pixel region (denoted as A1) corresponding to the detection cell is predefined.

[0049] The average grayscale values ​​of all pixels in this region are calculated (formula: Where N is the total number of pixels in the pixel region, and Ij is the grayscale value of the j-th pixel in the region).

[0050] The average gray value S is converted into fluorescence signal intensity for subsequent quantitative analysis.

[0051] (ii) Sensitivity adjustment method;

[0052] Different detection sensitivity requirements can be adapted by adjusting the camera's exposure time (adjustable range 10–1000ms) and gain (adjustable range 1–300x). For example, in high-sensitivity detection scenarios, a parameter combination of 300x gain and 100ms exposure time can be used.

[0053] III. Core Advantages of the System;

[0054] Structure adapted for on-site testing: The whole system consists of a wide field excitation light source module, an optical separation module, a sample container, a macro imaging fluorescence signal acquisition module, and a pixel area averaging signal processing module. Its structure is different from that of traditional scanning microplate readers, making it highly portable.

[0055] Significant low-cost advantage: It adopts a wide-field excitation + area array imaging optical path design and pixel area average noise reduction algorithm, eliminating the need for expensive photomultiplier tubes and keeping material costs below $500.

[0056] Excellent portability: The overall size is only 30cm×18cm×12cm, which makes it easy to carry and transport, and can meet the needs of on-site rapid testing scenarios.

[0057] Furthermore, the wide-field excitation module uses a 488nm laser light source paired with a Powell prism to achieve uniform wide-field illumination in the detection cell area, with an excitation light intensity uniformity variation coefficient of <5%.

[0058] Signal processing module

[0059] Furthermore, the signal processing module employs a pixel region averaging algorithm to perform average noise reduction on the pixel region corresponding to the detection pool, with the number of pixels participating in the averaging being ≥40,000, in order to improve the signal-to-noise ratio.

[0060] Sample processing and testing procedures

[0061] Furthermore, the testing procedure for urine samples is as follows:

[0062] Sample pretreatment: Urine samples were pretreated using an HLB solid-phase extraction column. The eluent was diluted 10 times to obtain the sample to be tested.

[0063] Fluorescent staining: Add SYBR Green I fluorescent dye to the sample to be tested to a final concentration of 1 μM, mix thoroughly, and incubate at room temperature for 10 minutes;

[0064] Detection and analysis: The stained sample is added to the detection cell, and detection is completed through wide-field excitation, macro-imaging acquisition, and pixel-average quantitative analysis.

[0065] Technical Comparison and Structural Explanation

[0066] The core technological differences between this system and the scanning microplate reader are as follows:

[0067] Example 2

[0068] like Figure 2 As shown, this embodiment provides a low-cost, portable, and highly sensitive fluorescence detection method, including:

[0069] I. Core Principles of the Detection Method;

[0070] This method is based on the technical chain of "wide field excitation-optical separation-macro imaging acquisition-regional averaging noise reduction-quantitative analysis". Relying on the hardware system of Example 1, it achieves high-sensitivity detection of target analytes through a standardized process. Its core advantage lies in balancing detection accuracy and on-site applicability, and it can be operated without a professional laboratory environment.

[0071] II. Detailed steps of the testing method;

[0072] (a) Sample pretreatment and preparation;

[0073] Sample selection and preprocessing:

[0074] Applicable sample types: biological samples (blood, plasma, serum, urine, etc.), chemical samples, or environmental samples; priority is given to the detection of low-concentration target analytes such as circulating cell-free DNA (cfDNA).

[0075] Pretreatment process (taking urine sample as an example): The original urine sample is purified using an HLB solid phase extraction column to remove impurities, proteins, and matrix interferences. The specific steps are: "sample loading (wash twice with 500 μL of 5% methanol aqueous solution) → elution (elution with 1 mL of urine at a flow rate of 1 mL / min and a flow rate of 200 μL)", and the eluent is collected for later use.

[0076] Sample dilution and staining:

[0077] High-level dilution: The pretreated eluent is diluted ≥10 times (using PBS buffer at pH 7.4 as the diluent) to reduce matrix residue interference and adapt to the linear response range of the detection system.

[0078] Fluorescent staining: Add PicoGreen fluorescent dye to the diluted sample to achieve a final dye concentration of 1 μM (matching the 488 nm excitation wavelength). After thorough mixing, incubate at room temperature in the dark for 15 minutes to ensure that the dye specifically binds to the target nucleic acid (such as cfDNA).

[0079] (II) Detection system debugging and sample loading;

[0080] System parameter settings: Start the detection system, set the excitation duration of the excitation source (25mW 488nm diode laser) to 20ms, and adjust the CMOS camera parameters to "exposure time 300ms, gain 300 times" (high sensitivity detection mode); confirm that the 540±20nm bandpass filter is installed in place and the optical path is aligned with the sample container area.

[0081] Sample loading: The stained sample was slowly injected into a low-background glass capillary (a Durham fermentation tube with an outer diameter of 6 mm, an inner diameter of 4.5 mm, and a height of 3 cm) at a loading volume of 200 μL (the sample height is about 12.5 mm, matching the size of the wide-field excitation spot) to avoid generating air bubbles.

[0082] (III) Excitation, Imaging and Signal Processing;

[0083] Wide-field excitation: The excitation light source is turned on, and a 12mm×1mm rectangular light spot shaped by diffractive optical elements is used to uniformly illuminate the sample area, thereby exciting the fluorescent dye to generate a specific fluorescence emission signal.

[0084] Optical separation: The 488nm excitation light is filtered out by a bandpass filter (540±20nm), allowing only the target fluorescence signal to pass through, thus ensuring signal purity.

[0085] Macro imaging acquisition: A 12-bit 1 / 1.8-inch, 2.3MP monochrome CMOS camera (with an f1.4, 16mm focal length lens) is used to acquire fluorescence images through macro imaging mode, completely covering the effective area of ​​the sample container.

[0086] Signal processing:

[0087] Region definition: The pixel region corresponding to the sample container is pre-marked in the fluorescence image (denoted as A1) to exclude interfering pixels from the container wall and background region.

[0088] Region averaging calculation: The grayscale values ​​of all pixels within region A1 are averaged using the following formula:

[0089] ;

[0090] Where N is the total number of pixels in the region, ≥40,000; Ij is the gray value of the j-th pixel, and the average gray value S is obtained.

[0091] Signal conversion: Substitute the average gray value S into the preset calibration curve to convert it into the corresponding fluorescence signal intensity.

[0092] (iv) Quantitative analysis;

[0093] Two quantitative methods are available to ensure the accuracy of the results:

[0094] External standard curve method: Prepare a series of target analyte standards with concentrations (1 pg / μL to 100 ng / μL) in advance, detect them according to the above procedure and plot the "fluorescence signal intensity-concentration" standard curve (R²≥0.99), and calculate the target analyte concentration by substituting the fluorescence signal intensity of the sample.

[0095] Standard addition method: Add a target analyte standard of known concentration to the sample to be tested, perform 3 parallel detections, and calculate the original concentration of the target analyte in the sample by signal increment to reduce the influence of matrix effect.

[0096] III. Performance indicators of the testing methods;

[0097] The detection performance of this method reaches the laboratory-grade fluorescence detection level, specifically as follows:

[0098] Limit of detection: The lowest limit of detection for circulating cell-free DNA (cfDNA) is ≤1 pg / μL;

[0099] Precision: For the same sample, 6 parallel tests are performed, and the relative standard deviation (RSD) is ≤5%;

[0100] Linear range: 1 pg / μL to 100 ng / μL, linear correlation coefficient R² ≥ 0.99.

[0101] IV. Related equipment and storage media;

[0102] (a) Electronic equipment;

[0103] It includes a memory, a processor, and a computer program stored in the memory and capable of running on the processor. When the processor executes the program, it realizes the full-process automated control of the above detection method, including functions such as parameter setting, image acquisition, signal calculation, and result output.

[0104] (ii) Computer-readable storage media;

[0105] It stores a computer program that, when executed by a processor, can call preset calibration curve data, signal processing algorithms, and detection process templates to automatically complete detection and quantitative analysis, and supports data export and traceability.

Claims

1. A low-cost, portable, high-sensitivity fluorescence detection system, characterized in that, include: The excitation light source module is used to provide wide-field excitation light to the sample to be tested in the sample container; An optical separation module, disposed on the excitation light source module, is used to suppress the excitation light and transmit the fluorescence emission signal generated by the sample to be tested; A sample container for receiving and holding the sample to be tested after being irradiated by the wide-field excitation light; A macro-imaging fluorescence signal acquisition module is used to perform macro-scale imaging of the fluorescence emission signal from the sample container to obtain a fluorescence image; The pixel region averaging signal processing module is connected to the macro-imaging fluorescence signal acquisition module. It is used to perform signal averaging processing on the predefined pixel regions in the fluorescence image to obtain the fluorescence signal intensity after noise reduction and to perform quantitative analysis.

2. The system according to claim 1, characterized in that, The excitation light source module includes a light source for generating collimated excitation light and an optical homogenizing element for converting the collimated excitation light into a uniform surface light source.

3. The system according to claim 2, characterized in that, The optical homogenizing element is a Powell prism, used to achieve wide-field uniform illumination.

4. The system according to claim 1, characterized in that, The optical separation module includes at least one filter assembly for separating optical signals based on the difference between the excitation wavelength and the emission wavelength.

5. The system according to claim 1, characterized in that, The macro-imaging fluorescence signal acquisition module includes an imaging lens and an image sensor. The imaging lens is used to image the fluorescence signal from the sample container onto the image sensor.

6. The system according to claim 5, characterized in that, The image sensor is a CMOS sensor, used to convert optical signals into the form of an electrical signal for the fluorescence image.

7. The system according to claim 1, characterized in that, The pixel region averaging signal processing module is configured to: acquire the fluorescence image from the macro-imaging fluorescence signal acquisition module, and select a region containing at least 40,000 pixels for signal intensity averaging calculation based on the corresponding position of the sample container in the image.

8. The system according to claim 7, characterized in that, The pixel region average signal processing module is further configured to: perform quantitative analysis of the target substance in the sample to be tested by means of the standard addition method or the external standard curve method based on the fluorescence signal intensity calculated by the average.

9. A low-cost, portable, and highly sensitive fluorescence detection method, characterized in that, include: Provides wide-field excitation light to illuminate the container holding the sample to be tested; Receive the fluorescence emission signal generated by irradiation from the container, and optically separate the fluorescence emission signal from the wide-field excitation light; Macro-imaging is performed on the separated fluorescence emission signal to obtain a fluorescence image containing the container region; Pixel signals corresponding to the container region are extracted from the fluorescence image, and the pixel signals are averaged to obtain an average signal value representing the fluorescence intensity of the sample to be tested. Based on the average signal value, the target analyte in the sample to be tested is quantitatively analyzed.

10. The method according to claim 9, characterized in that, Before providing wide-field excitation light irradiation, the method further includes a step of pretreatment of the sample to be tested, the pretreatment including diluting the sample to be tested and adding a fluorescent dye that specifically binds to the target analyte thereto; The sample to be tested is a biological liquid sample, the target analyte is circulating cell-free DNA, and the pretreatment includes high-multiplier dilution of the urine sample and addition of PicoGreen dye.

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