Fluorescence detection system and method based on optical fiber light transmission

Abstract

The invention discloses a fluorescence detection system and method based on optical fiber light transmission, and relates to the field of fluorescence detection. The problems that light path construction of an existing optical system is complex, debugging and calibration difficulty of all optical elements is large, and the installation and maintenance cost of a fluorescence detection system is low are solved. A white light solid LED light source is adopted to generate monochromatic exciting light through a collecting lens and an exciting light filter wheel, the monochromatic exciting light is shunted to four groups of exciting light transmission optical fibers through a four-core optical fiber splitter, and a fluorescent dye sample in a light transmission element is excited through an optical fiber focusing lens. Fluorescent light emitted by a sample is received by the optical fiber focus lens to the optical fiber common end and is transmitted to the optical fiber collimator, the emitted light filter wheel and the camera condenser module through the emitted light transmission optical fiber to be focused to the CCD sensor. The synchronous pulley is linked with the double optical filter wheels to realize spectrum synchronous switching, and dynamic isolation of the shaft lever is realized through the optical filter wheel isolation bearing. The method is also suitable for real-time fluorescent quantitative PCR detection experiments.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, and in particular to a fluorescence detection system and method based on optical fiber light transmission. Background Art

[0002] Fluorescence detection technology exploits the property of substances to emit fluorescence of varying wavelengths after absorbing excitation light of a specific wavelength. By measuring fluorescence intensity and spectrum, the system enables qualitative and quantitative analysis of target substances. Existing fluorescence detection systems typically employ traditional optical structures, such as lenses and mirrors, to construct optical paths. However, these traditional optical structures have numerous limitations. On the one hand, the optical path of traditional optical systems is complex to construct, and the debugging and calibration of individual optical components are difficult, requiring specialized technicians to spend a considerable amount of time on the operation, increasing the system's installation and maintenance costs. On the other hand, traditional optical systems lack flexibility and are difficult to adapt to different detection scenarios and changing detection requirements. For example, when performing multi-channel detection, the optical path design of traditional optical systems becomes even more complex, and different channels are prone to mutual interference, affecting the accuracy of detection results. Furthermore, traditional optical systems suffer from low light transmission efficiency between the light source and the sample, and vice versa. Light transmission is easily affected by environmental factors such as dust and vibration, leading to reduced stability and reliability of the detection signal. To overcome these issues, the present invention proposes a fluorescence detection system and method based on optical fiber transmission. These methods utilize the excellent light transmission properties of optical fiber to simplify the optical path structure and improve the performance and adaptability of the detection system.

[0003] Furthermore, the thermal cycling system and optical detection work poorly together. Traditional water bath or metal block heating temperature control methods have slow ramp rates, poor temperature uniformity, and lengthy thermal cycling cycles, ultimately impacting the accuracy and timeliness of the entire qPCR test. Summary of the Invention

[0004] This invention addresses the complex optical path construction of traditional optical systems, the difficulty in debugging and calibrating individual optical components, and the time-consuming operation required by specialized technicians, which increases system installation and maintenance costs. Furthermore, traditional optical systems lack flexibility, making it difficult to adapt to different detection scenarios and changing testing requirements, as well as the inefficient coordination between thermal cycling systems and optical detection.

[0005] To solve the above technical problems, the present invention is achieved through the following technical solutions: Solution 1. The present invention proposes a fluorescence detection system based on optical fiber light transmission, which includes a white light solid LED light source, a light source condenser module, an excitation light filter wheel, a four-core optical fiber splitter, an excitation light transmission optical fiber, an optical fiber focusing mirror, a light-passing element, an emission light transmission optical fiber, an optical fiber collimator, an optical fiber collimator fixing bracket, an emission light filter wheel, a camera condenser module, a CCD sensor, a synchronous pulley, a coupling, a stepper motor, a synchronous belt, a filter wheel isolation bearing, a shaft support bearing, a shaft sleeve, and a support seat; The white light solid LED light source emits white light, which is filtered by the excitation light filter wheel, and the excitation light is received by the common end of the four-core optical fiber splitter, and the branch end is connected to four groups of independent excitation light transmission optical fibers; the optical fiber focusing mirror simultaneously performs the dual functions of focusing the excitation light and collecting fluorescence; the CCD sensor is divided into four independent photosensitive areas, corresponding to four-channel fluorescence signals, and the fluorescent dye sample in the light-transmitting element is excited by the optical fiber focusing mirror. The fluorescence emitted by the fluorescent dye sample is received by the optical fiber focusing mirror to the common end of the optical fiber, transmitted to the optical fiber collimator through the emission light transmission optical fiber, and focused to the CCD sensor through the emission light filter wheel and the camera condenser module.

[0006] Furthermore, a preferred embodiment is provided, in which the light source condenser module is realized by a coaxially integrated achromatic doublet lens group, an aspheric collimating lens and a calcium fluoride field lens, and a calcium fluoride crystal plano-convex field lens is arranged behind the achromatic doublet lens group to converge the light beam into a light spot and match the numerical aperture of the four-core fiber splitter.

[0007] Furthermore, a preferred embodiment is provided, in which the optical fiber collimator fixing frame is composed of two cubic metal bases, a positioning hole is provided at the center of the cubic metal base, and the four positioning holes are distributed in a square. The inner wall of each positioning hole is coated with a diamond carbon wear-resistant layer for adapting to the metal outer sheaths of four groups of optical fiber collimators.

[0008] Furthermore, a preferred embodiment is provided, wherein the achromatic doublet lens group includes a front group and a rear group, the front group is formed by precisely gluing an N-BAF3 optical glass positive lens and an N-SF6 flint glass negative lens by UV-curing glue, and the distance between the rear group aspherical field lens and the achromatic doublet lens group is 2.2 mm.

[0009] Furthermore, a preferred embodiment is provided, in which the main body of the four-core fiber optic splitter is packaged in an integrated metal shell, and the interior of the metal shell is filled with optical matching glue with a high refractive index for fixing the excitation light transmission optical fiber. The interior of the four-core fiber optic splitter includes a fiber fusion and splitting structure. A main fiber optic interface is provided at one end of the four-core fiber optic splitter, and the main fiber optic interface is ground and polished. The fiber core in the main fiber optic interface is connected to the internal splitting element; the splitting element melts and stretches the main fiber and the four output fibers at high temperature to form a tapered transition area.

[0010] Furthermore, a preferred embodiment is provided, wherein the excitation light transmitting fiber and the emission light transmitting fiber are arranged on a common end constructed at the end of the fiber focusing lens, the excitation light transmitting fiber is composed of at least one quartz fiber with a diameter of 300 μm, and the emission light transmitting fiber is composed of a single large-core quartz fiber with a diameter of 2 mm, and at least one excitation light transmitting fiber is arranged in a circular array around the single emission light transmitting fiber.

[0011] Furthermore, a preferred embodiment is provided, in which the fiber optic focusing mirror is connected to the light-transmitting element through a flange, and flanges with positioning pin holes are respectively provided on the outer side of the lens barrel of the fiber optic focusing mirror and the bottom side of the light-transmitting element. The surface of the flange is processed with a sealing groove and a built-in fluororubber O-ring. The fiber optic focusing mirror and the light-transmitting element are quickly and accurately aligned through the positioning pins, and the flange is tightly fixed with evenly distributed hexagonal screws, and thread locking glue is applied to complete the fixation.

[0012] Furthermore, a preferred embodiment is provided, in which the excitation light filter wheel and the emission light filter wheel are both made of an integrally formed structure of aluminum alloy, and an annular slot is designed inside to fix the filter. The excitation light filter wheel and the emission light filter wheel are provided with an annular slot inside to fix the filter. Each filter wheel accommodates 6 filters of different wavelengths, and rapid switching of the excitation light or emission light wavelength is achieved by rotation. The excitation light filter wheel and the emission light filter wheel are connected using a synchronous pulley and a synchronous belt.

[0013] Furthermore, a preferred embodiment is provided, wherein a shaft support bearing is installed between the excitation light filter wheel and the support seat, and the shaft support bearing is a deep groove ball bearing; A shaft sleeve is provided between the synchronous pulley of the excitation light filter wheel and the shaft support bearing, and the shaft sleeve is made of polyetheretherketone material.

[0014] Solution 2: A fluorescence detection method based on optical fiber light transmission, the fluorescence detection method is implemented based on the fluorescence detection system described in any one of Solution 1, and the method specifically comprises the following steps: Step 1: Turn on the hot air qPCR instrument, set the gradient temperature program, and simultaneously start the white light solid-state LED light source and power supply. The white light solid-state LED light source is internally provided with a multi-chip integrated module. The multi-chip integrated module is used to emit white light. After being processed by the three-stage optical components of the light source condenser module, it forms an incident light field adapted for the four-core fiber splitter. The emission light filter wheel is synchronously driven by a stepper motor, and the excitation light filter wheel is linked to the synchronous pulley to pre-switch to the excitation light filter corresponding to the target fluorescent dye; Step 2: Load the nucleic acid sample labeled with fluorescent dye into the 16 independent cells of the microfluidic chip through a microinjector. In the reaction pool, the microfluidic chip is mounted in the positioning slot of the rotation module. The rotation module is started to drive the stepper motor, which rotates the microfluidic chip to the first detection position through the gear transmission mechanism. The four reaction pool axes are precisely aligned with the detection holes of the light-transmitting element. The heating module of the hot air qPCR instrument controls the constant temperature of the microfluidic chip reaction pool area to ensure that the PCR reaction is carried out at the set temperature. Step 3: The monochromatic excitation light filtered by the excitation light filter wheel is received by the main fiber interface of the four-core fiber splitter. Its internal fused-tapered optical splitter structure evenly distributes the light field to the four output fibers. Four groups of excitation light transmission fibers, consisting of multiple 300μm diameter optical fibers, surround four 2mm diameter emission fibers in a circular array, forming four sets of common end structures at the end of the fiber focusing mirror to ensure that the excitation light evenly covers the detection area of ​​the light-transmitting element; Step 4: The fiber focusing mirror is fixed to the light-transmitting element via a flange connection. Its aspheric polished surface focuses the excitation light into a 50μm diameter spot, which stimulates the fluorescent dye in the reaction cell to emit fluorescence. The emitted fluorescence is collected by the fiber focusing mirror and transmitted by the central transmitting optical fiber to the fiber collimator fixture. The fiber collimator fixture fixes four fiber collimators through the four-image fixed positioning holes of the aluminum alloy 7075-T6 base. The top screw locking mechanism ensures the positioning accuracy of the fiber collimators to ±0.1mm and the angular deviation to <0.5°, realizing the collimated transmission of the fluorescence signal. Step 5: The collimated fluorescence enters the emission filter wheel and is linked to the excitation filter wheel through a synchronous belt to filter out the fluorescence signal corresponding to the emission wavelength. The double-glued achromatic lens and aspheric field lens of the camera condenser module are combined to focus the fluorescence onto four independent photosensitive areas of the CCD sensor, and four-channel fluorescence signals are synchronously collected, corresponding to the current four reaction cells of the microfluidic chip. The sensor converts the optical signal into an electrical signal, which is then amplified and filtered and transmitted to the data processing unit. Step 6: After completing the testing of the current four reaction pools, the rotation module drives the microfluidic chip to rotate 22.5°, aligning the next set of four reaction pools with the detection wells, and repeating steps 3-5 for testing; this rotation is repeated three times until all 16 reaction pools are tested. During the testing process, the hot air qPCR instrument automatically adjusts the temperature according to the PCR program to complete the denaturation-annealing-extension cycle, and the temperature is kept stable in the extension stage during each fluorescence acquisition; Step 7. After the test is completed, turn off the heating module of the hot air qPCR instrument first, wait for the temperature of the microfluidic chip to drop to room temperature, remove it, and simultaneously turn off the white light solid-state LED light source and the stepper motor drive system. Use data processing software to analyze the four-channel fluorescence signal and calculate the nucleic acid concentration in each reaction pool based on the standard curve.

[0015] The present invention is beneficial in that: The present invention provides a fluorescence detection system and detection method based on optical fiber light transmission. Through innovative structural design and process optimization, the sensitivity, stability, and detection efficiency of fluorescence detection are significantly improved. A four-core optical fiber splitter and a filter wheel synchronous belt drive structure are used to achieve precise distribution and wavelength matching of multi-channel excitation light. The microfluidic chip rotation module is combined with a four-channel CCD sensor detection to complete the detection of 16 samples. The fluorescence system integrates a hot air qPCR temperature control module, providing an efficient and stable fluorescence detection solution for fields such as genetic testing and biological analysis.

[0016] The present invention is also applicable to fields such as gene detection and biological analysis detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a side view of a fluorescence detection system based on optical fiber light transmission described in embodiment 1.

[0018] Figure 2 This is a front view of a fluorescence detection system based on optical fiber light transmission described in embodiment 1.

[0019] Figure 3 This is a side view of a fiber optic collimator fixing bracket in a fluorescence detection system based on fiber optic light transmission according to embodiment 1.

[0020] Figure 4 This is a diagram showing the internal arrangement of optical fibers in a fluorescence detection system based on optical fiber light transmission as described in embodiment 1.

[0021] Figure 5 This is a flow chart of a fluorescence detection method based on optical fiber light transmission as described in the second embodiment.

[0022] Among them, there are white light solid LED light source 1, light source condenser module 2, excitation light filter wheel 3, four-core optical fiber splitter 4, excitation light transmission optical fiber 5, optical fiber focusing mirror 6, light-passing element 7, emission light transmission optical fiber 8, optical fiber collimator 9, optical fiber collimator fixing bracket 10, emission light filter wheel 11, camera condenser module 12, CCD sensor 13, synchronous pulley 14, coupling 15, stepper motor 16, synchronous belt 17, filter wheel isolation bearing 18, shaft support bearing 19, shaft sleeve 20, and support seat 21. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the implementation methods of this application clearer, the technical solutions in the implementation methods of this application will be clearly and completely described below in combination with the drawings in the implementation methods of this application. Obviously, the described implementation methods are only part of the implementation methods of this application, not all of the implementation methods.

[0024] Implementation method 1: Figure 1 、 Figure 2 As shown, this embodiment is a further explanation of a fluorescence detection system based on optical fiber light transmission, wherein the system specifically includes: a white light solid LED light source 1, a light source condenser module 2, an excitation light filter wheel 3, a four-core optical fiber splitter 4, an excitation light transmission optical fiber 5, an optical fiber focusing lens 6, a light-passing element 7, an emission light transmission optical fiber 8, an optical fiber collimator 9, an optical fiber collimator fixing bracket 10, an emission light filter wheel 11, a camera condenser module 12, a CCD sensor 13, a synchronous pulley 14, and a coupling 15. , stepper motor 16, synchronous belt 17, filter wheel isolation bearing 18, shaft support bearing 19, shaft sleeve 20, support seat 21; the white light solid LED light source 1 emits white light, which is filtered by the excitation light filter wheel 3, and the excitation light is received by the common end of the four-core optical fiber splitter 4, and the branch end is connected to four groups of independent excitation light transmission optical fibers 5; the optical fiber focusing mirror 6 simultaneously undertakes the dual functions of excitation light focusing and fluorescence collection; the CCD sensor 13 is divided into four independent photosensitive areas, corresponding to four-channel fluorescence signals respectively.

[0025] The light source condenser module 2 consists of three coaxially integrated optical components: a fused silica aspheric collimating lens directly facing the light-emitting surface of the white light LED, compressing the 120° divergent beam to ±5° to improve light flux collection efficiency. Following this is an achromatic doublet lens assembly, with the positive lens made of N-BK7 optical glass and the negative lens made of N-SF11 flint glass. Optimizing the curvature of the bonded surfaces eliminates axial chromatic aberration in the 400-700nm band. A calcium fluoride crystal plano-convex field lens is placed behind the doublet lens assembly to converge the light beam into a spot and match the numerical aperture of the four-core fiber splitter 4. The three-stage lens assembly is nested within an aluminum alloy barrel. Precision-machined stepped grooves on the barrel's inner wall sequentially position the front aspheric lens, the middle doublet lens assembly, and the rear field lens. Aluminum alloy spacers provide physical isolation and stress buffering between the lenses. The barrel's front end is secured with an aluminum alloy pressure ring and a fluororubber seal.

[0026] like Figure 3 As shown, the optical fiber collimator fixing frame 10 adopts two precisely machined square metal bases, and the material is aluminum alloy 7075-T6. In terms of base processing technology, a five-axis linkage CNC machining center is used to process four quadrant precision positioning holes at the center position. The four positioning holes are strictly distributed in a square to ensure the consistency of the optical path of the four groups of optical fiber collimators 9 after installation. The inner wall of each positioning hole is coated with a diamond-like carbon wear-resistant layer, which can tightly fit the metal outer sheath of the four groups of optical fiber collimators 9. While ensuring a stable connection, it effectively reduces the wear on the optical fiber collimator 9 during the plugging and unplugging process and extends its service life. The optical fiber collimator 9 is fixed with two metal base top screw locking mechanisms. Four high-precision top screws are distributed on each base. The top screw head is inlaid with a polytetrafluoroethylene PTFE gasket with moderate hardness, which can provide sufficient locking force and avoid damaging the outer sheath of the optical fiber collimator. By tightening the top screw, the fiber collimator 9 can be precisely positioned in the X, Y, and Z directions, ensuring that the angular deviation between the collimators is less than 0.5°. This carefully designed fiber collimator fixture 10 provides a stable and reliable optical path reference for high-throughput simultaneous fluorescence detection. During simultaneous multi-channel detection, it effectively suppresses optical path deviation caused by external factors such as vibration and temperature changes, ensuring that the four sets of fluorescence signals are transmitted along a highly consistent path, avoiding crosstalk between channels and significantly improving the accuracy and repeatability of fluorescence detection data.

[0027] The camera condenser module 12 utilizes a composite configuration of a doublet achromatic lens and an aspheric field lens. The front doublet lens group is composed of an N-BAF3 optical glass positive lens and an N-SF6 flint glass negative lens precisely bonded together using UV-curable adhesive, eliminating residual chromatic dispersion of less than 0.8% in the 500-700nm band. The rear aspheric field lens group is separated from the doublet lens by 2.2mm to correct for off-axis coma. Both lens groups are enclosed in a ceramic lens barrel. The doublet lens is radially restrained by an Invar alloy retaining ring, while the aspheric field lens is axially preloaded by a titanium-nickel shape memory alloy spring. The front end of the barrel is dynamically connected to the output flange of the emission filter wheel using a three-jaw flexible coupling, while the rear end is coupled to the quartz protective window of the CCD sensor 13 via a metal bellows hermetic seal. Its primary function is to converge the fluorescence filtered by the emission filter wheel, concentrating it onto the effective photosensitive area of ​​the CCD sensor 13, thereby improving fluorescence signal collection efficiency and detection sensitivity. Due to the use of high-precision optical materials and precise mechanical structure, optical aberration and chromatic aberration are greatly reduced, and the clarity and resolution of imaging are improved, so that fluorescence signals can be detected and analyzed more accurately, providing more reliable data support for fluorescence detection. At the same time, its modular design is also easy to install, disassemble and maintain, reducing the maintenance cost and difficulty of the system, and further improving the practicality and stability of the entire fluorescence detection system.

[0028] The white light solid-state LED light source 1 adopts multi-chip integrated packaging technology, internally integrating LED chips of different wavelengths. Through precise ratio and optical design, its luminescence spectrum can fully cover the excitation requirements of six commonly used fluorescent dyes. Specifically, for common fluorescent dyes such as FITC, fluorescein isothiocyanate, excitation peak 490nm, TRITC, tetramethylrhodamine isothiocyanate, excitation peak 550nm, Alexa Fluor series dyes, Cy series dyes, etc., the white light solid-state LED light source 1 can provide high-intensity and high-stability excitation light at the corresponding excitation wavelength. In addition, the white light solid-state LED light source 1 also has a flexible light intensity adjustment function. Through digital signal control, it can achieve 0-100% linear adjustment of light intensity to adapt to fluorescence detection scenarios with different concentrations and different sensitivity requirements. Whether it is the detection of trace biomarkers or the analysis of high-concentration environmental pollutants, the white light solid-state LED light source 1 can provide excitation light with appropriate intensity and spectral characteristics, giving full play to the performance advantages of fluorescence detection systems based on optical fiber light transmission, and providing a solid light source foundation for fluorescence detection in multiple fields and scenarios.

[0029] The main body of the four-core fiber splitter 4 is encapsulated in an integrated metal shell, and the interior of the shell is filled with a high-refractive-index optical matching glue. This glue can not only effectively fix the optical fiber, but also reduce the reflection loss of light at the interface of different media, thereby improving the overall light transmission efficiency. From the internal structure, the four-core fiber splitter 4 contains a precise fiber fusion and splitting structure. At the end that receives the excitation light, there is a main fiber interface. The fiber end face at the interface is ultra-precision ground and polished to ensure that the incident excitation light can be fully and efficiently coupled into the splitter. The core of the main fiber is connected to the internal splitting element. The splitting element is a splitter manufactured based on the fused cone technology. Its core part is to melt and stretch the main fiber and the four output fibers at high temperature to form a tapered transition area. In this area, the light field is redistributed. According to the geometric size and refractive index distribution of the optical fiber, the incident excitation light is evenly distributed to the four output fibers in a specific proportion, achieving a one-to-four splitting effect. The four-core fiber splitter 4 realizes the splitting process through the fused taper technology, which distributes light directly inside the optical fiber, avoiding the light reflection and scattering loss caused by the use of traditional optical components such as splitter prisms and beam splitters. The integrated structural design and the use of internal optical matching glue enhance the mechanical stability and environmental adaptability of the splitter, and effectively reduce the changes in the optical path caused by external factors; its compact structure greatly simplifies the optical path layout of the entire fluorescence detection system, so that the excitation light is efficiently converted from a single channel to a four-channel output, laying the foundation for subsequent multi-channel simultaneous detection. In conjunction with other components in the system, it can realize parallel excitation and detection of multiple detection sites, and the detection efficiency is improved by 4 times compared with single-channel detection.

[0030] like Figure 4As shown, the common end constructed by the excitation light transmission fiber 5 and the emission light transmission fiber 8 at the end of the fiber focusing mirror 6 is the core structure for achieving efficient optical signal transmission and conversion. The excitation light transmission fiber 5 is composed of multiple quartz optical fibers with a diameter of 300μm. This type of optical fiber has the characteristics of low attenuation and high numerical aperture, which can ensure that the excitation light is transmitted to the detection area with minimal energy loss. The emission light transmission fiber 8 adopts a single large-core quartz optical fiber with a diameter of 2mm. The design of its core diameter size fully considers the fluorescence collection efficiency. The larger core diameter can effectively capture the divergent fluorescence emitted by the fluorescent dye. In the structural design of the common end of the optical fiber, multiple relatively thin excitation light transmission fibers 5 are surrounded by a single relatively thick emission light transmission fiber in a precise circular array. At the same time, in order to further optimize the light field distribution, the array arrangement of the excitation light transmission fiber 5 follows specific geometric rules, and the spacing error between its axis and the axis of the emission light transmission fiber 8 is controlled within ±5μm, so that the excitation light can evenly cover the end face area of ​​the emission light transmission fiber 8, avoiding the difference in fluorescence excitation efficiency caused by uneven distribution of excitation light energy. This unique fiber common end structural design realizes the spatial separation and efficient transmission of excitation light and emission fluorescence, significantly improving the detection performance of the system. In the actual detection process, the excitation light can be accurately transmitted to the fiber focusing mirror 6, and after focusing, it efficiently excites the fluorescent dye, and the generated fluorescence can be quickly captured and transmitted by the emission light transmission fiber 8 in the center, reducing the scattering and loss of fluorescence during the transmission process. Compared with the traditional structure, this common end design effectively enhances the detection accuracy and reliability of the entire fluorescence detection system.

[0031] The fiber optic focusing mirror 6 and the light-transmitting element 7 are connected by a flange. The main body of the fiber optic focusing mirror 6 is made of high-precision optical glass and processed through an aspheric grinding process. This can effectively reduce aberrations and enhance the focusing effect. A precise optical coating layer is designed inside to achieve efficient transmission of excitation light and directional convergence of emitted light. The flange connection structure adopts a split design. The outer side of the fiber optic focusing mirror 6 barrel and the bottom side of the light-transmitting element 7 are respectively provided with flanges with locating pin holes. The surface of the flange is anodized to form a wear-resistant layer. At the same time, a high-precision sealing groove is machined and a fluororubber O-ring is built in to ensure airtightness and optical stability after connection. During connection, the locating pins are used to quickly and accurately align the fiber optic focusing mirror 6 and the light-transmitting element 7. The flange is then tightly fixed with evenly distributed hexagonal screws and thread locking glue is applied to prevent loosening due to vibration. This connection method not only achieves a firm connection between the two, but also ensures that the coaxiality error between the optical elements is less than 0.2°, providing a guarantee for the stable transmission of optical signals. The flange connection method is convenient for disassembly, assembly and maintenance. When it is necessary to replace the light-transmitting element 7 or to clean or calibrate the fiber focusing mirror 6, the disassembly and installation can be completed quickly, which greatly shortens the system maintenance time. The fiber focusing mirror 6 simultaneously plays the dual role of focusing the excitation light and converging the emitted light, which effectively simplifies the optical path structure of the system. Compared with the traditional separate focusing and converging structure, this integrated design reduces the energy loss and optical path deviation when light is transmitted between multiple optical elements, and improves the overall transmission efficiency of the optical signal by more than 30%. In addition, through the optimization of optical design and precise connection technology, the cooperation between the fiber focusing mirror 6 and the light-transmitting element 7 can accurately control the focused spot size of the excitation light and the convergence angle of the emitted light, so that the excitation light forms a high-energy density spot in the light-transmitting element, significantly improving the fluorescence excitation efficiency and detection sensitivity.

[0032] The excitation light filter wheel 3 and the emission light filter wheel 11 are both integrally formed from aluminum alloy, with an annular slot designed inside for fixing the filters. Each filter wheel can accommodate six filters of different wavelengths, and rapid switching of the excitation light or emission light wavelength can be achieved through rotation. The two are connected to the synchronous belt 17 via a synchronous pulley 14 of the same specifications, and the synchronous pulley 14 is made of high-strength polyurethane. A Kevlar fiber reinforcement layer is embedded inside the synchronous belt 17 to improve durability while ensuring transmission accuracy. During installation, the synchronous belt 17 is pre-tightened with a tensioning pulley, and the tensioning force is controlled at 8-10N to ensure that there is no slippage between the synchronous pulley 14 and the synchronous belt 17 during system operation, so that the excitation light filter wheel 3 and the emission light filter wheel 11 can rotate at the same angle, ensuring accurate matching of the excitation light and emission light wavelengths. The excitation filter wheel axle is connected to the emission filter wheel disc via a filter wheel isolation bearing 18. This bearing utilizes a double-row angular contact ball bearing structure and is filled with high-temperature-resistant lithium-based grease, capable of withstanding both radial and axial loads. This bearing not only isolates the rotation of the two filter wheels, preventing mutual interference, but also provides stable support for the filter wheels, ensuring coaxiality during rotation. This effectively reduces the impact of system operating noise and vibration on the optical path. The stepper motor 16 is connected to the emission filter wheel 11 via a coupling. When the stepper motor drives the emission filter wheel 11 to rotate, the coupling 15 ensures smooth and accurate power transmission. The coupling 15 features a quick-release design, facilitating equipment maintenance and component replacement. A high-precision shaft support bearing 19, constructed from deep-groove ball bearings, is installed between the excitation filter wheel 3 and the device support seat 21. The outer ring of the bearing utilizes a transition fit with the mounting hole of the device support seat 21, while the inner ring utilizes an interference fit with the excitation filter wheel axle. Dustproof seals are installed at both ends of the shaft support bearing 19 to prevent dust from entering the bearing and extend its service life. A customized shaft sleeve 20 is provided between the synchronous pulley of the excitation light filter wheel and the shaft support bearing 19. The sleeve is made of polyetheretherketone material, which has excellent wear resistance and self-lubricating properties. It can effectively isolate the synchronous pulley and the bearing to avoid direct contact between the two to cause friction and wear. At the same time, it plays an axial positioning role to ensure the stability of the synchronous pulley during rotation. This structural design enables the filter wheel system to ensure the accuracy and stability of the switching between the excitation light and emission light wavelengths, meeting the requirements of fluorescence detection for spectral consistency; the modular component design facilitates maintenance and upgrades, and the filter wheel, bearings, couplings and other components can be independently disassembled and replaced, reducing equipment maintenance costs.

[0033] Implementation method 2: Figure 5 As shown, a fluorescence detection method based on optical fiber light transmission includes the following steps: Step 1: Turn on the hot air qPCR instrument and set the cycle parameters for a gradient temperature program, such as 95°C pre-denaturation, 55-65°C annealing, and 72°C extension. Simultaneously, turn on the white light solid-state LED light source 1 and the detection system power supply. The white light solid-state LED light source 1 houses a multi-chip integrated module for emitting white light. This light is then processed by the tertiary optical components of the light source condenser module 2, where a fused silica aspheric lens compresses the beam, an achromatic doublet lens eliminates chromatic aberration, and a calcium fluoride field lens focuses the light spot. This forms an incident light field adapted for the four-core fiber splitter 4. A stepper motor drives the emission filter wheel, which is then linked to the excitation filter wheel 3 via a synchronous pulley 14 to pre-select the excitation filter corresponding to the target fluorescent dye, such as HEX.

[0034] Step 2: Fluorescent dye-labeled nucleic acid samples are loaded into the 16 independent reaction wells of the microfluidic chip using a microinjector. The microfluidic chip is mounted in the positioning slots of the rotation module. The rotation module's drive motor is activated, and a gear transmission mechanism rotates the microfluidic chip to the first detection position, precisely aligning the axes of the four reaction wells with the detection holes of the optical element. The heating module of the hot air qPCR instrument maintains constant temperature control over the chip's reaction wells, ensuring that the PCR reaction proceeds at the set temperature.

[0035] Step 3: Monochromatic excitation light, such as 490 nm, filtered by the excitation light filter is received by the main fiber interface of the four-core fiber splitter 4. Its internal fused-tapered beam splitter structure evenly distributes the light field to the four output fibers. Four sets of excitation light transmission fibers, consisting of multiple 300 μm diameter fibers, are arranged in a circular array around four sets of 2 mm diameter emission fibers. Four sets of common terminals are formed at the end of the fiber focusing lens, ensuring that the excitation light evenly covers the detection area of ​​the optical element.

[0036] Step 4: The fiber optic focusing lens 6 is connected to the light-transmitting element 7 via a flange connection. Its aspheric polished surface focuses the excitation light into a 50μm diameter spot, stimulating the fluorescent dye, such as HEX, in the reaction cell to emit fluorescence. The emitted fluorescence is collected by the fiber optic focusing lens 6 and transmitted by the central transmitting optical fiber to the fiber optic collimator fixture 10. The fiber optic collimator fixture 10 fixes four fiber optic collimators 9 through four fixed positioning holes in the aluminum alloy 7075-T6 base. The top screw locking mechanism ensures the collimator positioning accuracy of ±0.1mm and the angular deviation of <0.5°, realizing the collimated transmission of the fluorescence signal.

[0037] Step 5: The collimated fluorescence enters the emission filter wheel 11, which is linked to the excitation filter wheel 5 via a timing belt 17 to select the fluorescence signal corresponding to the emission wavelength, such as HEX-550. The camera condenser module 12's double-cemented achromatic lens, consisting of an N-BAF3 positive lens and an N-SF6 negative lens, combined with an aspheric field lens, focuses the fluorescence onto four independent photosensitive areas of the CCD sensor 13, synchronously collecting four channels of fluorescence signals corresponding to the four reaction cells currently in the microfluidic chip. The CCD sensor 13 converts the optical signal into an electrical signal, which is then amplified and filtered before being transmitted to the data processing unit.

[0038] Step 6: After testing the current four reaction wells, the rotation module drives the microfluidic chip 22.5° to align the next set of four reaction wells with the test wells, and repeat steps 3-5. This rotation is repeated three times until all 16 reaction wells are tested. During the test, the hot air qPCR instrument automatically adjusts the temperature according to the PCR program to complete the denaturation-annealing-extension cycle, maintaining a stable temperature during the extension phase during each fluorescence acquisition.

[0039] Step 7. After the test is complete, turn off the heating module of the hot air qPCR instrument and remove the microfluidic chip after the temperature cools to room temperature. Simultaneously turn off the white light solid-state LED light source 1 and the stepper motor drive system. Use data processing software to analyze the four-channel fluorescence signals and calculate the nucleic acid concentration in each reaction cell based on the standard curve, such as the Ct value analysis in real-time fluorescence quantitative PCR.

[0040] In summary, the present invention provides a fluorescence detection system and detection method based on optical fiber light transmission, which significantly improves the sensitivity, stability and detection efficiency of fluorescence detection through innovative structural design and process optimization. A four-core optical fiber splitter and a filter wheel synchronous belt drive structure are used to achieve precise distribution and wavelength matching of multi-channel excitation light. The microfluidic chip rotation module is combined with four-channel CCD detection to complete the detection of 16 samples. The system integrates a hot air qPCR temperature control module, providing an efficient and stable fluorescence detection solution for fields such as genetic testing and biological analysis.

[0041] Those skilled in the art will understand that the above description is only a preferred embodiment of the present invention, and the features described in the various embodiments and / or technical solutions of the present disclosure can be combined or combined in various ways, even if such a combination or combination is not explicitly described in the present disclosure. It is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0042] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the accompanying technical solutions are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A fluorescence detection system based on optical fiber light transmission, characterized in that: The fluorescence detection system comprises a white light solid LED light source (1), a light source condenser module (2), an excitation light filter wheel (3), a four-core optical fiber splitter (4), an excitation light transmission optical fiber (5), an optical fiber focusing mirror (6), a light-transmitting element (7), an emission light transmission optical fiber (8), an optical fiber collimator (9), an optical fiber collimator fixing frame (10), an emission light filter wheel (11), a camera condenser module (12), a CCD sensor (13), a synchronous pulley (14), a coupling (15), a stepping motor (16), a synchronous belt (17), a filter wheel isolation bearing (18), a shaft support bearing (19), a shaft sleeve (20), and a support seat (21); The white light solid LED light source (1) emits white light, which is filtered by the excitation light filter wheel (3), and the excitation light is received by the common end of the four-core optical fiber splitter (4), and the branch end is connected to four groups of independent excitation light transmission optical fibers (5); the optical fiber focusing mirror (6) simultaneously assumes the dual functions of excitation light focusing and fluorescence collection; the CCD sensor (13) is divided into four independent photosensitive areas, corresponding to four-channel fluorescence signals, and the optical fiber focusing mirror (6) excites the fluorescent dye sample in the light-transmitting element (7); the fluorescence emitted by the fluorescent dye sample is received by the optical fiber focusing mirror (6) to the common end of the optical fiber, transmitted to the optical fiber collimator (9) through the emission light transmission optical fiber (8), and focused to the CCD sensor (13) through the emission light filter wheel (11) and the camera condenser module (12).

2. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The light source condenser module (2) is realized by coaxially integrating an achromatic doublet lens group, an aspheric collimating lens and a calcium fluoride field lens, and a calcium fluoride crystal plano-convex field lens is arranged behind the achromatic doublet lens group to converge the light beam into a light spot and match the numerical aperture of the four-core optical fiber splitter (4).

3. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The optical fiber collimator fixing frame (10) is composed of two square metal bases, and a positioning hole is provided at the center of the square metal base. The four positioning holes are distributed in a square shape, and the inner wall of each positioning hole is plated with a diamond carbon wear-resistant layer for adapting to the metal outer sheaths of the four sets of optical fiber collimators (9).

4. The fluorescence detection system based on optical fiber light transmission according to claim 2, characterized in that: The achromatic doublet lens group includes a front group and a rear group. The front group is made of an N-BAF3 optical glass positive lens and an N-SF6 flint glass negative lens precisely bonded by ultraviolet curing glue. The distance between the rear aspherical field lens and the achromatic doublet lens group is 2.2 mm.

5. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The main body of the four-core optical fiber splitter (4) is formed by an integrated metal shell packaging. The interior of the metal shell is filled with optical matching glue with a high refractive index for fixing the excitation light transmission optical fiber (5). The interior of the four-core optical fiber splitter (4) includes an optical fiber fusion and splitting structure. One end of the four-core optical fiber splitter (4) is provided with a main optical fiber interface, and the main optical fiber interface is ground and polished. The fiber core in the main optical fiber interface is connected to the internal splitting element; the splitting element melts and stretches the main optical fiber and the four output optical fibers at high temperature to form a tapered transition area.

6. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The excitation light transmission optical fiber (5) and the emission light transmission optical fiber (8) are arranged on a common end constructed at the end of the optical fiber focusing mirror (6), the excitation light transmission optical fiber (5) is composed of at least one quartz optical fiber with a diameter of 300 μm, and the emission light transmission optical fiber (8) is composed of a single large-core quartz optical fiber with a diameter of 2 mm, and at least one excitation light transmission optical fiber (5) is surrounded by the single emission light transmission optical fiber (8) in a circular array.

7. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The optical fiber focusing mirror (6) is connected to the light-transmitting element (7) through a flange. The outer side of the lens barrel of the optical fiber focusing mirror (6) and the bottom side of the light-transmitting element (7) are respectively provided with flanges with positioning pin holes. The surface of the flange is processed with a sealing groove and a built-in fluororubber O-ring. The optical fiber focusing mirror (6) and the light-transmitting element (7) are quickly and accurately aligned through the positioning pins. The flange is tightly fixed by evenly distributed hexagonal screws and is applied with thread locking glue to complete the fixation.

8. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: The excitation light filter wheel (3) and the emission light filter wheel (11) are both made of an aluminum alloy integrally formed structure, and an annular slot is designed inside for fixing the filter. The excitation light filter wheel (3) and the emission light filter wheel (11) are provided with an annular slot inside for fixing the filter. Each filter wheel accommodates 6 filters of different wavelengths, and the wavelength of the excitation light or emission light is quickly switched by rotation. The excitation light filter wheel (3) and the emission light filter wheel (11) are connected using a synchronous pulley (14) and a synchronous belt (17).

9. The fluorescence detection system based on optical fiber light transmission according to claim 1, characterized in that: A shaft support bearing (19) is installed between the excitation light filter wheel (3) and the support seat (21), and the shaft support bearing (19) is a deep groove ball bearing; A shaft sleeve (20) is provided between the synchronous pulley (14) of the excitation light filter wheel (3) and the shaft support bearing (19), and the shaft sleeve (20) is made of polyetheretherketone material.

10. A fluorescence detection method based on optical fiber light transmission, characterized in that: The fluorescence detection method is implemented based on the fluorescence detection system according to any one of claims 1 to 9, and the method specifically comprises the following steps: Step 1: Turn on the hot air qPCR instrument, set the gradient temperature program, and start the white light solid LED light source (1) and the power supply at the same time. The white light solid LED light source (1) is provided with a multi-chip integrated module inside. The multi-chip integrated module is used to emit white light. After being processed by the three-stage optical components of the light source condenser module (2), an incident light field adapted to the four-core optical fiber splitter (4) is formed. The emission light filter wheel (11) is driven synchronously by the stepper motor (16), and the excitation light filter wheel (3) is linked via the synchronous pulley (14) to pre-switch to the excitation light filter corresponding to the target fluorescent dye; Step 2: Load the nucleic acid sample labeled with fluorescent dye into 16 independent reaction pools of the microfluidic chip through a microinjector. The microfluidic chip is mounted in the positioning slot of the rotation module. The rotation module is started to drive the stepper motor (16). The microfluidic chip is rotated to the first detection position through the gear transmission mechanism so that the axes of the four reaction pools are precisely aligned with the detection holes of the light-transmitting element. The heating module of the hot air qPCR instrument controls the temperature of the reaction pool area of ​​the microfluidic chip to ensure that the PCR reaction is carried out at the set temperature. Step 3: The monochromatic excitation light filtered by the excitation light filter wheel (3) is received by the main fiber interface of the four-core fiber splitter (4). The internal fused cone splitting structure evenly distributes the light field to the four output optical fibers. Four groups of excitation light transmission optical fibers (5) composed of multiple optical fibers with a diameter of 300 μm are arranged in a circular array around four 2 mm diameter emission optical fibers, forming four groups of common end structures at the end of the optical fiber focusing mirror (6), ensuring that the excitation light evenly covers the detection area of ​​the light-transmitting element. Step 4, the optical fiber focusing mirror (6) is connected to the fixed light-transmitting element (7) through a flange, and its aspheric polished surface focuses the excitation light into a 50 μm diameter spot, which excites the fluorescent dye in the reaction pool to emit fluorescence. The emitted fluorescence is collected by the optical fiber focusing mirror (6) and transmitted by the central emission optical fiber to the optical fiber collimator fixing frame (10). The optical fiber collimator fixing frame (10) fixes four optical fiber collimators (9) through the four-image limiting positioning holes of the aluminum alloy 7075-T6 base. The top screw locking mechanism ensures that the positioning accuracy of the optical fiber collimator (9) is ±0.1 mm and the angle deviation is <0.5°, thereby realizing the collimated transmission of the fluorescence signal; Step 5: The collimated fluorescence enters the emission light filter wheel (11), and is linked to the excitation light filter wheel (3) through a synchronous belt to screen out the fluorescence signal corresponding to the emission wavelength. The double-glued achromatic lens and the aspheric field mirror of the camera condenser module (12) are compositely configured to focus the fluorescence onto the four independent photosensitive areas of the CCD sensor (13), and synchronously collect four-channel fluorescence signals corresponding to the current four reaction pools of the microfluidic chip. The CCD sensor (13) converts the optical signal into an electrical signal, which is then amplified and filtered and transmitted to the data processing unit; Step 6. After completing the detection of the current four reaction pools, the rotation module drives the microfluidic chip to rotate 22.5° to align the next group of four reaction pools with the detection wells, and repeat steps 3 to 5 for detection; rotate three times in sequence until all 16 reaction pools are detected. During the detection process, the hot air qPCR instrument automatically adjusts the temperature according to the PCR program to complete the denaturation-annealing-extension cycle, and maintains the temperature stable in the extension stage during each fluorescence acquisition; Step 7: After the test is completed, first turn off the heating module of the hot air qPCR instrument, take out the microfluidic chip after the temperature drops to room temperature, and simultaneously turn off the white light solid LED light source (1) and the drive system of the stepper motor (16). The four-channel fluorescence signal is analyzed by data processing software, and the nucleic acid concentration in each reaction pool is calculated based on the standard curve.

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