Light path design method for improving sensitivity of fluorescence detector

Abstract

The invention discloses a light path design method for improving the sensitivity of a fluorescence detector, and relates to the technical field of optical detection, and the method comprises the following steps: carrying out optical bonding on a flow cell by using quartz glass, and evaporating an alloy film on the surface corresponding to the directions of exciting light and fluorescence; plating a reflecting film on one surface of the flow cell to enhance fluorescence collection; exciting light irradiates the flow cell at a specific angle and a spectroscope is additionally arranged behind the flow cell, so that transmission light is reflected back to the flow cell to form secondary excitation, the optical path is increased and the sensitivity is improved; transmission light intensity is reduced through a spectroscope to avoid detector ultralimit, and meanwhile, a photodiode detector is used for detecting light intensity of a light source to calculate a fluorescence value to improve stability and accuracy; aiming at a high-concentration sample, the light splitting sheet can be adjusted to move out of a light path and is inserted into the diffusion sheet, so that concentration adaptive detection is realized. By means of multi-light-path optimization and dynamic adjustment, the sensitivity and stability of fluorescence detection are remarkably improved, and the concentration application range of fluorescence detection is remarkably widened.

Description

Technical Field

[0001] The present invention relates to the field of optical detection technology, and specifically to a light path design method for improving the sensitivity of a fluorescence detector. This technology can be widely used in separation and analysis systems such as high-performance liquid chromatography (HPLC) and capillary electrophoresis, and is used for trace substance detection in fields such as environmental monitoring, biomedical testing, and food safety analysis. Background Art

[0002] Fluorescence detection technology is a key tool in modern analytical chemistry and biomedical testing. Fluorescence detectors measure the intensity of the fluorescent signal emitted by a sample after excitation, enabling qualitative and quantitative analysis of the substance being tested. This technology is based on the property that fluorescent substances, after absorbing excitation light of a specific wavelength, emit fluorescence at a different wavelength. According to the Lambert-Beer law, fluorescence intensity is positively correlated with excitation light intensity, sample concentration, and optical path length, resulting in fluorescence detection with high sensitivity and good specificity.

[0003] Currently, conventional fluorescence detectors primarily utilize a fixed-pathlength flow cell design. To improve detection sensitivity for low-concentration samples, the flow cell size is typically increased to extend the optical path length. However, this approach significantly increases the flow cell volume, leading to chromatographic peak broadening and decreased separation efficiency. Furthermore, existing detectors struggle to simultaneously detect both high- and low-concentration samples, and signal saturation is a common problem with high-concentration samples.

[0004] Existing fluorescence detection technology has the following major drawbacks: First, the fixed optical path design cannot automatically adjust the optimal optical path according to sample concentration, resulting in insufficient sensitivity for low-concentration detection or signal saturation for high-concentration detection. Second, the traditional method of increasing the optical path increases the volume of the flow cell, affecting chromatographic separation. Third, complex optical path design increases instrument cost and the difficulty of optical adjustment. Finally, existing systems struggle to achieve rapid concentration adaptive adjustment while maintaining high sensitivity. These issues limit the application of fluorescence detectors in trace analysis and detection over a wide concentration range. Summary of the Invention

[0005] In view of this, the present invention provides an optical path design method for improving the sensitivity of fluorescence detectors. Through innovative optical path design, the present invention can achieve dynamic adjustment of the optical path length while maintaining a small circulation pool volume, thereby significantly improving the detection sensitivity of low-concentration samples and avoiding the signal saturation problem when detecting high-concentration samples.

[0006] To this end, the present invention provides the following technical solutions: The present invention provides a light path design method for improving the sensitivity of a fluorescence detector, comprising the following steps: The flow cell is optically bonded with quartz glass; The flow cell is square in shape and includes a first side and a second side for receiving and emitting excitation light, and a third side and a fourth side for detecting fluorescence; a photodiode detector is located on one side of the second side; and a photomultiplier tube detector is located on one side of the third side; Alloy films are evaporated on the first side and the third side, and a reflective film is evaporated on the fourth side to reflect scattered fluorescence back to a photomultiplier tube detector; Excitation light illuminates the flow cell from the first side at a specific angle, wherein the angle is determined by the longitudinal dimension of the flow cell; A beam splitter is added on the fourth side, and the excitation light transmitted through the flow cell is reflected back to the flow cell by the beam splitter to form secondary excitation.

[0007] Furthermore, the transmission and reflection of the beam splitter are fixed values ​​(eg, 1:9). Furthermore, it also includes: using a photodiode detector to detect the intensity of the excitation light source, and calculating the fluorescence value according to the formula FU=PMT value / PD value, wherein FU represents the fluorescence value, PMT value represents the detection value of the photomultiplier tube detector, and PD value represents the detection value of the photodiode detector. Furthermore, the excitation light source is a xenon lamp. Furthermore, the method further includes: when analyzing a high-concentration sample, moving the beam splitter out of the light path through a transmission mechanism. Furthermore, the method further comprises: when the beam splitter is moved out of the light path, a diffuser is inserted synchronously, and a difference between the transmittance of the diffuser and the transmittance of the beam splitter does not exceed a preset threshold. Furthermore, the transmission mechanism uses motor rotation or electromagnet cooperation to achieve position adjustment of the beam splitter.

[0008] Furthermore, the beam splitter is a vapor-deposited metal film.

[0009] Advantages and positive effects of the present invention: The present invention discloses a light path design method for improving the sensitivity of a fluorescence detector, wherein the circulation cell is optically bonded with quartz glass and an alloy film is evaporated on the surface corresponding to the direction of the excitation light and the fluorescence; a reflective film is plated on one side of the circulation cell to enhance fluorescence collection; the excitation light irradiates the circulation cell at a specific angle and a spectroscope is added thereafter to reflect the transmitted light back into the circulation cell to form secondary excitation, thereby increasing the optical path and improving sensitivity; the intensity of the transmitted light is reduced by the spectroscope to avoid detector over-limit, while a photodiode detector is used to detect the light intensity of the light source to calculate the fluorescence value to improve stability and accuracy; for high-concentration samples, the spectroscope can be adjusted to move out of the light path and a diffuser can be inserted to achieve concentration-adaptive detection. The present invention significantly improves the sensitivity, stability, and concentration adaptation range of fluorescence detection through multiple light path optimization and dynamic adjustment methods. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0010] Figure 1 Schematic diagram of the structure of the fluorescence detector in an embodiment of the present invention; Figure 2 A diagram showing the implementation path of secondary excitation light in an embodiment of the present invention; Figure 3 Schematic diagram of the detection process of the fluorescence detector when analyzing high-concentration samples in an embodiment of the present invention. DETAILED DESCRIPTION

[0011] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0012] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0013] The present invention provides an optical path design method for improving the sensitivity of a fluorescence detector. The method can achieve dynamic adjustment of the optical path length while maintaining a small circulation cell volume, thereby significantly improving the detection sensitivity of low-concentration samples while avoiding the signal saturation problem when detecting high-concentration samples.

[0014] To facilitate understanding, the structure of the fluorescence detector is first explained: A fluorescence detector is a highly sensitive analytical instrument used to detect fluorescent substances in samples. Its core components vary depending on its operating principle and design, but generally include the following main components: Excitation light source: Commonly used xenon lamps, mercury lamps or LED lamps can provide excitation light of a specific wavelength to make the sample molecules transition from the ground state to the excited state.

[0015] Excitation monochromator: includes a grating or filter, which is used to select excitation light of a specific wavelength and filter other stray light, thereby improving the signal-to-noise ratio.

[0016] Flow cell: usually made of quartz, with a small volume (μL level) and a short optical path. The chromatographic effluent is illuminated by the excitation light here to produce fluorescence.

[0017] Emission monochromator: This includes a grating or filter that selects the specific fluorescence wavelength emitted by the sample and excludes solvent scattered light and other interferences.

[0018] Detector: A photomultiplier tube (PMT) detector or a photodiode (PD) detector is used to convert fluorescence signals into electrical signals and amplify weak signals to improve sensitivity.

[0019] Signal processing system: includes amplifiers, analog-to-digital converters (ADCs), and data acquisition software, which are used to process electrical signals, output chromatographic peaks, and calculate peak areas or heights.

[0020] Optical components: including lenses, mirrors and apertures, used to focus and guide the light path and reduce light loss.

[0021] In an embodiment of the present invention, a light path design method for improving the sensitivity of a fluorescence detector specifically includes the following steps: S1, the flow cell is optically bonded with quartz glass; Among them, the circulation pool is square, including a first side and a second side for receiving excitation light and emitting excitation light, and a third side and a fourth side for detecting fluorescence; a photodiode detector (PD detector) is located on one side of the second side; a photomultiplier tube detector (PMT detector) is located on one side of the third side; and an excitation light source is located on one side of the first side. In this embodiment, the excitation light source is a xenon lamp.

[0022] S2, evaporating an alloy film on the first side and the third side, and evaporating a reflective film on the fourth side to reflect the scattered fluorescence back to the PMT detector; like Figure 1 As shown, Figure 1 The black continuous and intermittent thick lines in the middle are the evaporated metal film, the square hole in the middle is the flow path, and the direction of the blue arrow is the optical path length.

[0023] S3. The excitation light illuminates the flow cell from the first side at a specific angle, where the angle is determined by the longitudinal size of the flow cell; a smaller size can reduce the illumination size.

[0024] S4. A beam splitter is added on the fourth side. The excitation light transmitted through the flow cell is reflected back to the flow cell by the beam splitter to form secondary excitation.

[0025] Add a beam splitter behind the flow cell (for example, transmission: reflection = 1:9), and most of the excitation light that passes through the flow cell will be reflected back to the flow cell, forming secondary excitation, which increases the excitation light path, increases the fluorescence excitation amount, and improves the sensitivity. Figure 2 As shown, Figure 2 The middle broken line is a beam splitter, which can be metal-coated or other forms. The transmission and reflection ratio of the beam splitter is 1:9.

[0026] Another embodiment further includes utilizing a PD detector to detect the intensity of the excitation light source for use in fluorescence calculations. While typical fluorescence light sources are very strong, this can easily cause the PD detector to exceed its limits. In this embodiment, only a small portion of the excitation light passes through the spectrometer, thus avoiding the possibility of the PD detector exceeding its limits. The PD detector detects the energy of the xenon lamp and is used in fluorescence calculations. For example, the fluorescence value FU = PMT value / PD value, where FU represents the fluorescence value, PMT represents the detection value of the photomultiplier tube detector, and PD represents the detection value of the photodiode detector, can significantly improve instrument stability and reduce drift, thereby indirectly improving detection accuracy and sensitivity.

[0027] In another embodiment, when analyzing a high-concentration sample, the beam splitter is moved out of the light path via a transmission mechanism. Furthermore, a diffuser is inserted simultaneously with the beam splitter's removal from the light path, and the difference between the transmittance of the diffuser and the transmittance of the beam splitter does not exceed a preset threshold. The preset threshold is a pre-set, low value to ensure that the transmittances of the diffuser and the beam splitter are similar.

[0028] When analyzing high-concentration samples, the fluorescence signal may be too strong, causing saturation even when the PMT magnification is adjusted to the lowest level. In this embodiment, a transmission mechanism, such as motor rotation or the use of two electromagnets, can be designed to adjust the beam splitter to a position outside the optical path. This reduces the optical path and the amount of fluorescence, making it possible to detect samples with slightly higher concentrations. In addition, while the beam splitter is removed from the optical path, a diffuser is inserted into the optical path. The diffuser's transmittance is controlled to be close to that of the beam splitter, ensuring that the PD detector does not exceed the limit.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for designing an optical path to improve the sensitivity of a fluorescence detector, characterized in that: The following steps are involved: The flow cell is optically bonded with quartz glass; The flow cell is square in shape and includes a first side and a second side for receiving and emitting excitation light, and a third side and a fourth side for detecting fluorescence; a photodiode detector is located on one side of the second side; and a photomultiplier tube detector is located on one side of the third side; Alloy films are evaporated on the first side and the third side, and a reflective film is evaporated on the fourth side to reflect scattered fluorescence back to a photomultiplier tube detector; Excitation light illuminates the flow cell from the first side at a specific angle, wherein the angle is determined by the longitudinal dimension of the flow cell; A beam splitter is added on the fourth side, and the excitation light transmitted through the flow cell is reflected back to the flow cell by the beam splitter to form secondary excitation.

2. The method according to claim 1, characterized in that The transmission and reflection ratio of the beam splitter is a fixed value.

3. The method according to claim 1, characterized in that Also includes: The intensity of the excitation light source is detected using a photodiode detector, and the fluorescence value is calculated according to the formula FU=PMT value / PD value, where FU represents the fluorescence value, PMT value represents the detection value of the photomultiplier tube detector, and PD value represents the detection value of the photodiode detector.

4. The method according to claim 3, characterized in that The excitation light source is a xenon lamp.

5. The method according to claim 1, wherein Also includes: When analyzing high-concentration samples, the beam splitter is moved out of the light path through the transmission mechanism.

6. The method according to claim 5, characterized in that Also includes: When the beam splitter is moved out of the light path, the diffuser is inserted synchronously, and the difference between the transmittance of the diffuser and the transmittance of the beam splitter does not exceed a preset threshold.

7. The method according to claim 5, characterized in that The transmission mechanism uses motor rotation or electromagnet cooperation to achieve position adjustment of the beam splitter.

8. The method according to claim 1, characterized in that The beam splitter is a vapor-deposited metal film.

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