A fluorescence detection system and method based on optical fiber transmission

Abstract

A fluorescence detection system and method based on fiber optic transmission is disclosed, relating to the field of fluorescence detection. This invention addresses the problems of complex optical path construction, difficult debugging and calibration of individual optical components, and high installation and maintenance costs associated with existing optical systems. The invention uses a white solid-state LED light source to generate monochromatic excitation light via a condenser lens and excitation light filter wheel. This light is then split into four sets of excitation light transmission fibers via a four-core fiber splitter, and excites a fluorescent dye-containing sample within a light-transmitting element via a fiber focusing lens. The sample's emitted fluorescence is received by the fiber focusing lens at the common end of the fiber, transmitted via the emitted light transmission fiber to a fiber collimator, and focused onto a CCD sensor by the emitted light filter wheel and camera condenser module. Synchronous belt pulleys are used to drive the dual filter wheels for synchronous spectral switching, and a filter wheel isolation bearing provides dynamic shaft isolation. This invention is also applicable to real-time quantitative PCR detection experiments.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, and specifically to a fluorescence detection system and method based on optical fiber transmission. Background Technology

[0002] Fluorescence detection technology utilizes the property that substances emit fluorescence of different wavelengths after absorbing excitation light of a specific wavelength. Qualitative and quantitative analysis of target substances is achieved by detecting fluorescence intensity, spectrum, and other information. In existing technologies, fluorescence detection systems typically employ traditional optical structures, such as lenses and mirrors, to construct the optical path. However, these traditional optical structures have many limitations.

[0003] On the one hand, the optical path setup of traditional optical systems is relatively complex, and the debugging and calibration of various optical components are difficult, requiring professional technicians to spend a lot of time on the operation, which increases the installation and maintenance costs of the system. On the other hand, traditional optical systems have poor flexibility and are difficult to adapt to changes in different detection scenarios and detection requirements. For example, when performing multi-channel detection, the optical path design of traditional optical systems becomes more complex, and different channels are prone to mutual interference, affecting the accuracy of the detection results.

[0004] Furthermore, traditional optical systems suffer from low light transmission efficiency between the light source and the sample, as well as between the sample and the detector. Light is also susceptible to environmental factors such as dust and vibration during transmission, leading to decreased stability and reliability of the detection signal. To overcome these problems, this invention proposes a fluorescence detection system and method based on optical fiber transmission. This system utilizes the excellent light transmission characteristics of optical fibers to simplify the optical path structure and improve the performance and adaptability of the detection system.

[0005] Furthermore, the smooth coordination between the thermal cycling system and optical detection is poor. Traditional water bath or metal block heating and temperature control methods result in slow heating and cooling rates, poor temperature uniformity, and lengthy thermal cycling cycles, ultimately affecting the accuracy and timeliness of the entire qPCR test. Summary of the Invention

[0006] This invention addresses the challenges of complex optical path setups, difficult debugging and calibration of individual optical components, and the high time required for specialized technicians in traditional optical systems, which increases installation and maintenance costs. Furthermore, traditional optical systems suffer from poor flexibility, struggle to adapt to varying detection scenarios and requirements, and exhibit suboptimal coordination between thermal cycling systems and optical detection.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0008] Option 1: This invention proposes a fluorescence detection system based on fiber optic light transmission. The fluorescence detection system includes a white solid-state LED light source, a light source condenser module, an excitation light filter wheel, a four-core fiber optic splitter, an excitation light transmission fiber, a fiber optic focusing lens, a light transmission element, an emission light transmission fiber, a fiber optic collimator, a fiber optic collimator mounting 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 base.

[0009] The white solid-state LED light source emits white light, which is filtered by the excitation light filter wheel and received by the common end of the four-core fiber optic splitter. The branch end is connected to four independent excitation light transmission fibers. The fiber optic focusing lens performs the dual functions of focusing the excitation light and collecting fluorescence. The CCD sensor is divided into four independent photosensitive areas, each corresponding to a four-channel fluorescence signal. The fluorescent dye sample in the light-transmitting element is excited by the fiber optic focusing lens. The fluorescence emitted by the fluorescent dye sample is received by the fiber optic focusing lens and transmitted to the common end of the fiber optic cable. It is then transmitted to the fiber optic collimator through the emission light transmission fiber and focused onto the CCD sensor by the emission light filter wheel and the camera condenser module.

[0010] Furthermore, a preferred embodiment is provided in which the light source focusing lens module is realized by coaxially integrating an achromatic cemented doublet lens group, an aspherical collimating lens, and a calcium fluoride field lens. A plano-convex field lens of a calcium fluoride crystal is configured after the achromatic cemented doublet lens group to focus the light beam into a light spot and match the numerical aperture of the four-core fiber splitter.

[0011] Furthermore, a preferred embodiment is provided, wherein the fiber optic collimator mounting bracket is composed of two cuboid metal bases, and a positioning hole is provided at the center of the cuboid metal base. The four positioning holes are distributed in a square, and the inner wall of each positioning hole is coated with a diamond carbon wear-resistant layer for use with the metal outer sheath of the four sets of fiber optic collimators.

[0012] Furthermore, a preferred embodiment is provided, wherein the achromatic cemented doublet lens group includes a front group and a rear group. The front group is formed by precisely bonding an N-BAF3 optical glass positive lens and an N-SF6 flint glass negative lens together with an ultraviolet curing adhesive. The rear group aspherical field lens is spaced 2.2 mm from the achromatic cemented doublet lens group.

[0013] Furthermore, a preferred embodiment is provided, wherein the main body of the four-core fiber optic splitter is constructed by an integrated metal shell, and the interior of the metal shell is filled with high-refractive-index optical matching adhesive for fixing the excitation light transmission fiber. The four-core fiber optic splitter includes fiber splicing and beam splitting structures. One end of the four-core fiber optic splitter is provided with a main fiber interface, and the main fiber interface is ground and polished. The fiber core in the main fiber interface is connected to the internal beam splitting element. The beam splitting element melts and stretches the main fiber and the four output fibers at high temperature to form a conical transition region.

[0014] Furthermore, a preferred embodiment is provided, wherein the excitation light transmission fiber and the emission light transmission fiber are disposed on the common end constructed at the end of the fiber focusing lens. The excitation light transmission fiber is composed of at least one quartz fiber with a diameter of 300 μm, and the emission light transmission fiber is composed of a single large-core quartz fiber with a diameter of 2 mm. The at least one excitation light transmission fiber is arranged in a circular array around the single emission light transmission fiber.

[0015] Furthermore, a preferred embodiment is provided, wherein the fiber optic focusing lens and the light-transmitting element are connected by a flange. The outer side of the lens barrel of the fiber optic focusing lens and the bottom side of the light-transmitting element are respectively provided with flanges with positioning pin holes. The surface of the flange is machined with a sealing groove and has a built-in fluororubber O-ring. The positioning pin enables the fiber optic focusing lens and the light-transmitting element to be quickly and accurately aligned. The flange is then tightly fixed with evenly distributed hexagonal screws and thread-locking adhesive is applied to complete the fixation.

[0016] Furthermore, a preferred embodiment is provided, wherein both the excitation light filter wheel and the emission light filter wheel are integrally formed from aluminum alloy, and an annular groove is designed inside for fixing the filter. Each filter wheel accommodates 6 filters of different wavelengths, and the wavelength of the excitation light or emission light can be quickly switched by rotation. The excitation light filter wheel and the emission light filter wheel are connected by a synchronous pulley and a synchronous belt.

[0017] Furthermore, in a preferred embodiment, a shaft support bearing is installed between the excitation light filter wheel and the support base, and the shaft support bearing is a deep groove ball bearing;

[0018] A shaft sleeve is provided between the synchronous pulley of the excitation light filter wheel and the shaft support bearing. The shaft sleeve is made of polyetheretherketone material.

[0019] Option 2: A fluorescence detection method based on optical fiber transmission, wherein the fluorescence detection method is implemented based on the fluorescence detection system described in any one of Options 1, and the method specifically includes the following steps:

[0020] Step 1: Turn on the hot air qPCR instrument, set the gradient temperature program, and simultaneously start the white solid-state LED light source and power supply. The white solid-state LED light source has a multi-chip integrated module inside, which is used to emit white light. After being processed by the three-level optical components of the light source condenser module, it forms an incident light field adapted to the four-core fiber optic splitter. Simultaneously, the emission light filter wheel is driven by a stepper motor, and the excitation light filter wheel is linked by a synchronous belt pulley to pre-switch to the excitation light filter corresponding to the target fluorescent dye.

[0021] Step 2: Load the nucleic acid sample labeled with fluorescent dye into 16 independent microfluidic chips using a microsyringe.

[0022] In the reaction chamber, the microfluidic chip is mounted in the positioning slot of the rotating module. The rotating module is started to drive the stepper motor, which rotates the microfluidic chip to the first detection position through the gear transmission mechanism, so that the axes of the four reaction chambers are precisely aligned with the detection holes of the light transmission element. The heating module of the hot air qPCR instrument maintains a constant temperature for the microfluidic chip reaction chamber area to ensure that the PCR reaction is carried out at the set temperature.

[0023] 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 beam splitter structure evenly distributes the light field to the four output fibers. The four sets of excitation light transmission fibers, composed of multiple 300μm diameter fibers, surround the four 2mm diameter transmitting fibers in a circular array, forming four common end structures at the end of the fiber focusing lens, ensuring that the excitation light evenly covers the detection area of ​​the light transmission element.

[0024] Step 4: The fiber optic focusing lens is fixed to the light transmission element via a flange connection. Its aspherical polished surface focuses the excitation light into a 50μm diameter spot, which excites the fluorescent dye in the reaction cell to emit fluorescence. The emitted fluorescence is collected by the fiber optic focusing lens and transmitted to the fiber optic collimator mounting bracket via the central emitting fiber. The fiber optic collimator mounting bracket fixes four fiber optic collimators through the four-quadrant positioning holes of the 7075-T6 aluminum alloy base. The set screw locking mechanism ensures that the positioning accuracy of the fiber optic collimator is ±0.1mm and the angle deviation is <0.5°, thereby realizing the collimated transmission of the fluorescence signal.

[0025] Step 5: The collimated fluorescence enters the emission light filter wheel and is linked with the excitation light filter wheel through a synchronous belt to filter out the fluorescence signal corresponding to the emission wavelength. The double cemented achromatic lens and aspherical field lens of the camera condenser module focus the fluorescence onto the four independent photosensitive areas of the CCD sensor, and simultaneously collect four-channel fluorescence signals, corresponding to the four reaction cells of the microfluidic chip. The sensor converts the light signal into an electrical signal, which is then amplified, filtered, and transmitted to the data processing unit.

[0026] Step 6: After completing the detection of the current 4 reaction chambers, rotate the microfluidic chip 22.5° using the rotating module to align the next set of 4 reaction chambers with the detection wells. Repeat steps 3-5 for detection. Repeat this process 3 times until all 16 reaction chambers have been 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, keeping the temperature stable in the extension stage during each fluorescence acquisition.

[0027] Step 7: After the test is completed, first turn off the heating module of the hot air qPCR instrument. After the microfluidic chip temperature drops to room temperature, take it out and simultaneously turn off the white light solid LED light source and the stepper motor drive system. Analyze the four-channel fluorescence signal using data processing software and calculate the nucleic acid concentration in each reaction cell based on the standard curve.

[0028] The advantages of this invention are:

[0029] This invention provides a fluorescence detection system and method based on fiber optic light transmission. Through innovative structural design and process optimization, it significantly improves the sensitivity, stability, and detection efficiency of fluorescence detection. A four-core fiber optic splitter and filter wheel synchronous belt drive structure are employed to achieve precise distribution and wavelength matching of multi-channel excitation light. A microfluidic chip rotation module, combined with a four-channel CCD sensor, enables 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 gene detection, bioanalysis, and other fields.

[0030] This invention is also applicable to fields such as gene detection and bioanalysis. Attached Figure Description

[0031] Figure 1 This is a side view of a fluorescence detection system based on optical fiber transmission as described in Embodiment 1.

[0032] Figure 2 This is a front view of a fluorescence detection system based on optical fiber transmission as described in Embodiment 1.

[0033] Figure 3 This is a side view of the fiber optic collimator mounting bracket in a fluorescence detection system based on fiber optic light transmission, as described in Embodiment 1.

[0034] Figure 4 This is a diagram showing the internal arrangement of optical fibers in a fluorescence detection system based on optical fiber transmission, as described in Embodiment 1.

[0035] Figure 5 This is a flowchart of a fluorescence detection method based on optical fiber transmission as described in Embodiment 2.

[0036] Among them, there are: 1. White solid-state LED light source; 2. Light source condenser lens module; 3. Excitation light filter wheel; 4. Four-core fiber optic splitter; 5. Excitation light transmission fiber; 6. Fiber optic focusing lens; 7. Light transmission element; 8. Emitting light transmission fiber; 9. Fiber optic collimator; 10. Fiber optic collimator fixing frame; 11. Emitting light filter wheel; 12. Camera condenser lens module; 13. CCD sensor; 14. Synchronous belt pulley; 15. Coupling; 16. Stepper motor; 17. Synchronous belt; 18. Filter wheel isolation bearing; 19. Shaft support bearing; 20. Shaft sleeve; and 21. Support base. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them.

[0038] Implementation Method 1, such as Figure 1 , Figure 2 As shown, this embodiment is a further explanation of a fluorescence detection system based on fiber optic light transmission. The system specifically includes: a white solid-state LED light source 1, a light source condenser module 2, an excitation light filter wheel 3, a four-core fiber optic splitter 4, an excitation light transmission fiber 5, a fiber optic focusing lens 6, a light transmission element 7, an emission light transmission fiber 8, a fiber optic collimator 9, a fiber optic collimator mounting 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. The system includes a stepper motor 16, a synchronous belt 17, a filter wheel isolation bearing 18, a shaft support bearing 19, a shaft sleeve 20, and a support base 21. The white solid-state LED light source 1 emits white light, which is filtered by the excitation light filter wheel 3 and received by the common end of the four-core fiber optic splitter 4. The branch ends are connected to four independent excitation light transmission fibers 5. The fiber optic focusing lens 6 simultaneously performs the dual functions of excitation light focusing and fluorescence collection. The CCD sensor 13 is divided into four independent photosensitive areas, each corresponding to a different channel of fluorescence signal.

[0039] The light source focusing lens module 2 consists of three coaxially integrated optical components: a fused silica aspherical collimating lens directly faces the light-emitting surface of the white LED light source, compressing the 120° diverging beam to ±5° to improve light flux collection efficiency; subsequently, an achromatic cemented doublet lens group is set, with the positive lens being N-BK7 optical glass and the negative lens being N-SF11 flint glass, eliminating axial chromatic aberration in the 400-700nm band through cemented surface curvature optimization; after the cemented doublet lens group, a calcium fluoride crystal plano-convex field lens is configured to converge the beam into a spot and match a four-core fiber optic splitter with a numerical aperture of 4. The three lenses are nested inside an aluminum alloy lens barrel, with a stepped groove precisely machined on the inner wall of the barrel to sequentially position the front aspherical lens, the middle cemented doublet lens group, and the rear field lens, with each lens physically isolated and stress-buffered by aluminum alloy spacers; the front end of the lens barrel is locked with an aluminum alloy pressure ring and a fluororubber sealing ring.

[0040] like Figure 3 As shown, the fiber optic collimator mounting bracket 10 uses two precision-machined cuboid metal bases made of 7075-T6 aluminum alloy. In the base machining process, a five-axis CNC machining center is used to machine four-quadrant precision positioning holes at the center. These four positioning holes are arranged in a strict square pattern to ensure the optical path consistency of the four sets of fiber optic 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 sets of fiber optic collimators 9. This ensures a stable connection while effectively reducing wear on the fiber optic collimators 9 during insertion and removal, extending their service life. The fiber optic collimators 9 are fixed using a two-base set screw locking mechanism. Each base has four high-precision set screws, and the heads of the set screws are inlaid with PTFE gaskets of moderate hardness, providing sufficient locking force while avoiding damage to the fiber optic collimator sheath. By tightening the set screw, the fiber optic collimator 9 can be precisely positioned in the X, Y, and Z directions, ensuring that the angular deviation between collimators is less than 0.5°. This meticulously designed fiber optic collimator holder 10 provides a stable and reliable optical path reference for high-throughput fluorescence synchronous detection. During multi-channel simultaneous detection, it can effectively suppress optical path offset 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.

[0041] The camera focusing lens module 12 adopts a composite configuration of a cemented doublet achromatic lens and an aspherical field lens. The front group of the cemented doublet lens is precisely bonded together with an N-BAF3 optical glass positive lens and an N-SF6 flint glass negative lens using UV-curable adhesive, eliminating residual chromatic aberration (<0.8%) in the 500-700nm wavelength range. The rear aspherical field lens is 2.2mm apart from the cemented doublet lens, correcting off-axis coma. Both lens groups are encapsulated within a ceramic lens barrel. The cemented doublet lens is radially limited by an Invar alloy positioning ring, while the aspherical field lens is axially pre-tightened by a titanium-nickel shape memory alloy spring. The front end of the lens barrel is dynamically connected to the output flange of the emission filter wheel using a three-jaw flexible coupling, and the rear end is coupled to the quartz protective window of the CCD sensor 13 via a metal bellows hermetically sealed structure. Its main function is to converge the fluorescence filtered by the emission filter wheel, allowing the fluorescence to concentrate on the effective photosensitive area of ​​the CCD sensor 13, improving the collection efficiency and detection sensitivity of the fluorescence signal. Thanks to the use of high-precision optical materials and a sophisticated mechanical structure, optical aberrations and chromatic aberrations are greatly reduced, improving the clarity and resolution of the image. This enables more accurate detection and analysis of fluorescence signals, providing more reliable data support for fluorescence detection. At the same time, its modular design facilitates installation, disassembly, and maintenance, reducing system maintenance costs and difficulty, and further enhancing the practicality and stability of the entire fluorescence detection system.

[0042] The aforementioned white solid-state LED light source 1 employs multi-chip integrated packaging technology, internally integrating LED chips of different wavelengths. Through precise proportioning and optical design, its emission spectrum comprehensively covers the excitation requirements of six commonly used fluorescent dyes. Specifically, for common fluorescent dyes such as FITC (fluorescein isothiocyanate), with an excitation peak of 490nm; TRITC (rhodamine tetramethylisothiocyanate), with an excitation peak of 550nm; Alexa Fluor series dyes; and Cy series dyes, the white solid-state LED light source 1 can provide high-intensity and high-stability excitation light at the corresponding excitation wavelengths. Furthermore, the white solid-state LED light source 1 also features flexible intensity adjustment capabilities. Through digital signal control, it can achieve linear intensity adjustment from 0-100% to adapt to fluorescence detection scenarios with different concentrations and sensitivity requirements. Whether for the detection of trace biomarkers or the analysis of high-concentration environmental pollutants, the white solid-state LED light source 1 can provide excitation light with suitable intensity and spectral characteristics, fully leveraging the performance advantages of fiber-optic-based fluorescence detection systems and providing a solid light source foundation for fluorescence detection in multiple fields and scenarios.

[0043] The four-core fiber optic splitter 4 is encapsulated in an integrated metal shell, with the interior filled with a high-refractive-index optical matching adhesive. This adhesive not only effectively fixes the optical fibers but also reduces light reflection loss at interfaces between different media, improving overall optical transmission efficiency. Internally, the four-core fiber optic splitter 4 incorporates a precise fiber fusion splicing and splitting structure. At the end receiving the excitation light, there is a main fiber interface. The fiber end face at this interface undergoes ultra-precision grinding and polishing to ensure that the incident excitation light can be coupled completely and efficiently into the splitter. The core of the main fiber is connected to the internal splitting element, which is a splitter manufactured using fused taper technology. Its core component involves melting and stretching the main fiber and four output fibers at high temperature to form a tapered transition region. Within this region, the light field is redistributed. Based on the fiber geometry and refractive index distribution, the incident excitation light is evenly distributed into the four output fibers in a specific ratio, achieving a four-way splitting effect. This four-core fiber optic splitter utilizes fused taper technology to achieve beam splitting, directly distributing light within the fiber. This avoids light reflection and scattering losses associated with traditional optical components such as beam splitters and beam splitters. The integrated structural design and the use of internal optical matching adhesive enhance the splitter's mechanical stability and environmental adaptability, effectively reducing optical path changes caused by external factors. Its compact structure greatly simplifies the optical path layout of the entire fluorescence detection system, enabling efficient conversion of excitation light from a single channel to four-channel output. This lays the foundation for subsequent multi-channel simultaneous detection. In conjunction with other components in the system, it can achieve parallel excitation and detection of multiple detection sites, improving detection efficiency by four times compared to single-channel detection.

[0044] like Figure 4As shown, the common end constructed at the end of the fiber focusing lens 6 for the excitation light transmission fiber 5 and the emission light transmission fiber 8 is the core structure for achieving efficient optical signal transmission and conversion. The excitation light transmission fiber 5 is composed of multiple quartz fibers with a diameter of 300μm. This type of 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 uses a single large-core quartz fiber with a diameter of 2mm. Its core diameter design fully considers the fluorescence collection efficiency. The larger core diameter can effectively capture the divergent fluorescence emitted from the fluorescent dye. In the structural design of the fiber common end, multiple thinner excitation light transmission fibers 5 are arranged in a precise circular array around a single thicker emission light transmission fiber. Meanwhile, to further optimize the light field distribution, the array arrangement of the excitation light transmission fiber 5 follows specific geometric rules, with the spacing error between its axis and the axis of the emission light transmission fiber 8 controlled within ±5μm. This ensures that the excitation light can uniformly cover the end face region of the emission light transmission fiber 8, avoiding differences in fluorescence excitation efficiency caused by uneven excitation light energy distribution. This unique fiber common end structure design achieves spatial separation and efficient transmission of excitation light and emitted fluorescence, significantly improving the system's detection performance. In actual detection, the excitation light can be accurately transmitted to the fiber focusing lens 6, where it is focused and efficiently excites the fluorescent dye. The generated fluorescence is then quickly captured and transmitted by the central emission light transmission fiber 8, reducing fluorescence scattering and loss during transmission. Compared with traditional structures, this common end design effectively enhances the detection accuracy and reliability of the entire fluorescence detection system.

[0045] The fiber optic focusing lens 6 and the optical transmission element 7 are connected via flanges. The main body of the fiber optic focusing lens 6 is made of high-precision optical glass and processed using an aspherical grinding process, which effectively reduces aberrations and improves the focusing effect. It features a precision optical coating layer inside, enabling efficient transmission of excitation light and directional focusing of emitted light. The flange connection structure adopts a split design, with flanges featuring locating pin holes on the outer side of the fiber optic focusing lens 6 and the bottom side of the optical transmission element 7. The flange surface is anodized to form a wear-resistant layer and is machined with a high-precision sealing groove, incorporating a built-in fluororubber O-ring to ensure airtightness and optical stability after connection. During connection, the locating pins enable rapid and precise alignment of the fiber optic focusing lens 6 and the optical transmission element 7. Then, evenly distributed hexagonal screws are used to tightly secure the flanges, and thread-locking adhesive is applied to prevent loosening due to vibration. This connection method not only ensures a firm bond between the two components but also guarantees that the coaxiality error between the optical elements is less than 0.2°, providing a guarantee for stable optical signal transmission. The flange connection facilitates disassembly and maintenance. When replacing the optical transmission element 7 or cleaning and calibrating the fiber optic focusing lens 6, disassembly and installation can be completed quickly, significantly reducing system maintenance time. The fiber optic focusing lens 6 simultaneously focuses the excitation light and converges the emission light, effectively simplifying the system's optical path structure. Compared to traditional separate focusing and converging structures, this integrated design reduces energy loss and optical path deviation during light transmission between multiple optical elements, improving the overall optical signal transmission efficiency by more than 30%. Furthermore, through optimized optical design and precise connection technology, the cooperation between the fiber optic focusing lens 6 and the optical transmission element 7 can accurately control the focused spot size of the excitation light and the convergence angle of the emission light, enabling the excitation light to form a high-energy-density spot within the optical transmission element, significantly improving fluorescence excitation efficiency and detection sensitivity.

[0046] Both the excitation light filter wheel 3 and the emission light filter wheel 11 are integrally formed from aluminum alloy and have an internal annular groove for fixing the filters. Each filter wheel can accommodate 6 filters of different wavelengths, and rotation enables rapid switching between excitation and emission light wavelengths. They are connected to a synchronous belt 17 via a synchronous pulley 14 of the same specification, made of high-strength polyurethane. The synchronous belt 17 has an embedded Kevlar fiber reinforcement layer, improving durability while ensuring transmission accuracy. During installation, the synchronous belt 17 is pre-tensioned using a tensioning pulley, with the tension controlled at 8-10N to ensure no slippage between the synchronous pulley 14 and the synchronous belt 17 during system operation. This ensures that the excitation light filter wheel 3 and the emission light filter wheel 11 rotate at the same angle, guaranteeing precise matching of the excitation and emission light wavelengths. The excitation light filter wheel shaft and the emission light filter wheel disk are connected by a filter wheel isolation bearing 18. This bearing adopts a double-row angular contact ball bearing structure, filled with high-temperature resistant lithium-based grease, and can withstand radial and axial bidirectional loads. This achieves rotational isolation between the two filter wheels, preventing mutual interference, and 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 light filter wheel 11 via a coupling 15. When the stepper motor drives the emission light filter wheel 11 to rotate, the coupling 15 ensures the smoothness and accuracy of power transmission. The coupling 15 adopts a quick-release design for easy equipment maintenance and component replacement. A high-precision shaft support bearing 19 is installed between the excitation light filter wheel 3 and the device support base 21. This bearing is a deep groove ball bearing. The outer ring of the bearing has an transition fit with the mounting hole of the device support base 21, and the inner ring has an interference fit with the excitation light filter wheel shaft. Dustproof seals are provided at both ends of the shaft support bearing 19 to prevent dust from entering the bearing and extend its service life. A custom-made shaft sleeve 20 is installed between the synchronous pulley of the excitation filter wheel and the shaft support bearing 19. The sleeve is made of polyetheretherketone (PEEK), which has excellent wear resistance and self-lubricating properties. It effectively isolates the synchronous pulley and bearing, preventing direct contact and friction, and also provides axial positioning, ensuring the stability of the synchronous pulley during rotation. This structural design ensures the accuracy and stability of the wavelength switching between excitation and emission light in the filter wheel system, meeting the spectral consistency requirements of fluorescence detection. The modular component design facilitates maintenance and upgrades; the filter wheel, bearings, couplings, and other components can be independently disassembled and replaced, reducing equipment maintenance costs.

[0047] Implementation Method 2, such as Figure 5 As shown, a fluorescence detection method based on optical fiber transmission includes the following steps:

[0048] Step 1: Turn on the hot air qPCR instrument and set the gradient temperature program, such as 95℃ pre-denaturation, 55-65℃ annealing, and 72℃ extension cycle parameters. Simultaneously, start the white solid-state LED light source 1 and the detection system power supply. The white solid-state LED light source 1 has a multi-chip integrated module for emitting white light. After passing through the three-stage optical components of the light source condenser module 2—a fused silica aspherical lens compressing the beam, an achromatic doublet lens group eliminating chromatic aberration, and a calcium fluoride field lens converging the light spot—it forms an incident light field adapted to the four-core fiber optic splitter 4. Simultaneously, a stepper motor drives the emission light filter wheel, which in turn drives the excitation light filter wheel 3 via a synchronous belt pulley 14, pre-switching to the target fluorescent dye, such as HEX, and the corresponding excitation light filter.

[0049] Step 2: Nucleic acid samples labeled with fluorescent dye are loaded into the 16 independent reaction chambers of the microfluidic chip using a microsyringe. The microfluidic chip is mounted in the positioning slots of the rotating module. The drive motor of the rotating module is activated, and the microfluidic chip is rotated to the first detection position via a gear transmission mechanism, ensuring precise alignment of the axes of the four reaction chambers with the detection wells of the light-transmitting element. The heating module of the hot-air qPCR instrument maintains a constant temperature in the chip reaction chamber area, ensuring the PCR reaction proceeds at the set temperature.

[0050] Step 3: The monochromatic excitation light, such as 490nm, filtered by the excitation light filter wheel, 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, composed of multiple 300μm diameter fibers, surround four sets of 2mm diameter transmitting fibers in a circular array, forming four common end structures at the end of the fiber focusing lens, ensuring that the excitation light uniformly covers the detection area of ​​the optical transmission element.

[0051] Step 4: The fiber optic focusing lens 6 is connected to and fixed to the light-transmitting element 7 via a flange. Its aspherical polished surface focuses the excitation light into a 50μm diameter spot, exciting 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 to the fiber optic collimator mounting bracket 10 through the central emitting fiber. The fiber optic collimator mounting bracket 10 fixes four fiber optic collimators 9 through the four-quadrant positioning holes of the aluminum alloy 7075-T6 base. The set screw locking mechanism ensures that the collimator positioning accuracy is ±0.1mm and the angle deviation is <0.5°, realizing the collimated transmission of the fluorescence signal.

[0052] Step 5: The collimated fluorescence enters the emission light filter wheel 11, which is synchronized with the excitation light filter wheel 5 via the synchronous belt 17 to filter out the fluorescence signal corresponding to the emission wavelength, such as HEX-550. The double-cemented achromatic lens of the camera condenser module 12, with an N-BAF3 positive lens and an N-SF6 negative lens cemented together, combined with an aspherical field lens, focuses the fluorescence onto the four independent photosensitive areas of the CCD sensor 13, simultaneously acquiring four channels of fluorescence signals, corresponding to the four reaction cells of the microfluidic chip. The CCD sensor 13 converts the optical signal into an electrical signal, which is then amplified, filtered, and transmitted to the data processing unit.

[0053] Step 6: After completing the detection of the current 4 reaction chambers, rotate the microfluidic chip 22.5° using the rotating module to align the next group of 4 reaction chambers with the detection wells. Repeat steps 3-5 for detection. Repeat this process 3 times until all 16 reaction chambers have been detected. During the detection process, the hot-air qPCR instrument automatically adjusts the temperature according to the PCR program, completing the denaturation-annealing-extension cycle, maintaining a stable temperature during the extension phase for each fluorescence acquisition.

[0054] Step 7: After the test is completed, first turn off the heating module of the hot air qPCR instrument, and remove the microfluidic chip after it has cooled to room temperature. Simultaneously turn off the white solid-state LED light source 1 and the stepper motor drive system. Analyze the four-channel fluorescence signals using data processing software, and calculate the nucleic acid concentration in each reaction chamber based on the standard curve, such as the Ct value analysis for real-time quantitative PCR.

[0055] In summary, this invention provides a fluorescence detection system and method based on fiber optic transmission. Through innovative structural design and process optimization, it significantly improves the sensitivity, stability, and detection efficiency of fluorescence detection. A four-core fiber optic splitter and filter wheel synchronous belt drive structure are employed to achieve precise distribution and wavelength matching of multi-channel excitation light. A microfluidic chip rotation module, combined with a four-channel CCD detector, can 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 gene detection, bioanalysis, and other fields.

[0056] Those skilled in the art will understand that the above description is merely a preferred embodiment of the present invention, and the features described in the various embodiments and / or technical solutions of this disclosure can be combined or combined in various ways, even if such combinations or combinations are not explicitly described in this disclosure. This is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended technical solutions are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims of the invention and their equivalents, the invention also intends to include these modifications and modifications.

Claims

1. A fluorescence detection system based on optical fiber transmission, characterized in that, The fluorescence detection system includes a white solid-state LED light source (1), a light source condenser module (2), an excitation light filter wheel (3), a four-core fiber optic splitter (4), an excitation light transmission fiber (5), a fiber optic focusing lens (6), a light transmission element (7), an emission light transmission fiber (8), a fiber optic collimator (9), a fiber optic collimator mounting bracket (10), an emission light filter wheel (11), a camera condenser module (12), a CCD sensor (13), a synchronous belt pulley (14), a coupling (15), a stepper motor (16), a synchronous belt (17), a filter wheel isolation bearing (18), a shaft support bearing (19), a shaft sleeve (20), and a support base (21). The white solid-state LED light source (1) emits white light, which is filtered by the excitation light filter wheel (3) and received by the common end of the four-core fiber optic splitter (4). The branch end is connected to four independent excitation light transmission fibers (5). The fiber optic focusing lens (6) simultaneously performs the dual functions of excitation light focusing and fluorescence collection. The CCD sensor (13) is divided into four independent photosensitive areas, which correspond to four channels of fluorescence signals. The fluorescent dye sample in the light transmission element (7) is excited by the fiber optic focusing lens (6). The fluorescence emitted by the fluorescent dye sample is received by the fiber optic focusing lens (6) and transmitted to the fiber optic collimator (9) through the emission light transmission fiber (8). It is then focused onto the CCD sensor (13) through the emission light filter wheel (11) and the camera condenser module (12). The main body of the four-core fiber optic splitter (4) is constructed by an integrated metal shell. The metal shell is filled with high-refractive-index optical matching adhesive to fix the excitation light transmission fiber (5). The four-core fiber optic splitter (4) includes fiber optic splicing and beam splitting structures. One end of the four-core fiber optic splitter (4) is provided with a main fiber interface, and the main fiber interface is ground and polished. The fiber core in the main fiber interface is connected to the internal beam splitting element. The beam splitting element melts and stretches the main fiber and the four output fibers at high temperature to form a conical transition region. The excitation light transmission fiber (5) and the emission light transmission fiber (8) are disposed on the common end constructed at the end of the fiber focusing lens (6). The excitation light transmission fiber (5) is composed of at least one quartz fiber with a diameter of 300 μm, and the emission light transmission fiber (8) is composed of a single large-core quartz fiber with a diameter of 2 mm. At least one excitation light transmission fiber (5) is arranged in a circular array around the single emission light transmission fiber (8). The excitation light filter wheel (3) and the emission light filter wheel (11) are both made of aluminum alloy and are integrally formed. They are designed with annular grooves inside for fixing the filters. The excitation light filter wheel (3) and the emission light filter wheel (11) are provided with annular grooves inside for fixing the filters. Each filter wheel can accommodate 6 filters of different wavelengths. The wavelength of the excitation light or emission light can be quickly switched by rotation. The excitation light filter wheel (3) and the emission light filter wheel (11) are connected by a synchronous pulley (14) and a synchronous belt (17).

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

3. The fluorescence detection system based on optical fiber transmission according to claim 1, characterized in that, The fiber collimator mounting bracket (10) is composed of two cubic metal bases. The center of the cubic metal base is provided with a positioning hole. 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, which is used to fit the metal outer sheath of the four sets of fiber collimators (9).

4. The fluorescence detection system based on optical fiber transmission according to claim 2, characterized in that, The achromatic cemented doublet lens group includes a front group and a rear group. The front group is made of N-BAF3 optical glass positive lens and N-SF6 flint glass negative lens precisely bonded together with UV curing adhesive. The rear group aspherical field lens is 2.2mm away from the achromatic cemented doublet lens group.

5. The fluorescence detection system based on optical fiber transmission according to claim 1, characterized in that, The fiber optic focusing lens (6) and the light transmission element (7) are connected by a flange. The outer side of the lens barrel of the fiber optic focusing lens (6) and the bottom side of the light transmission element (7) are respectively provided with flanges with positioning pin holes. The flanges are machined with sealing grooves and have built-in fluororubber O-rings. The fiber optic focusing lens (6) and the light transmission element (7) are quickly and accurately aligned by positioning pins. The flanges are then tightly fixed by evenly distributed hexagonal screws and thread locking adhesive is applied to complete the fixing.

6. The fluorescence detection system based on optical fiber 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 base (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). The shaft sleeve (20) is made of polyether ether ketone material.

7. A fluorescence detection method based on optical fiber transmission, characterized in that, The fluorescence detection method is implemented based on the fluorescence detection system according to any one of claims 1-6, and the method specifically includes the following steps: Step 1: Turn on the hot air qPCR instrument, set the gradient temperature program, and simultaneously start the white solid LED light source (1) and power supply. The white solid LED light source (1) is equipped with a multi-chip integrated module. The multi-chip integrated module is used to emit white light. After being processed by the three-level optical components of the light source condenser module (2), it forms an incident light field that is adapted to the four-core fiber optic splitter (4). Simultaneously, the emission light filter wheel (11) is driven by the stepper motor (16), and the excitation light filter wheel (3) is linked by the synchronous belt pulley (14) to pre-switch to the excitation light filter corresponding to the target fluorescent dye. Step 2: The nucleic acid sample labeled with fluorescent dye is loaded into the 16 independent reaction chambers of the microfluidic chip through a microsyringe. The microfluidic chip is mounted in the positioning slot of the rotating module. The rotating module is started to drive the stepper motor (16) and rotate the microfluidic chip to the first detection position through the gear transmission mechanism, so that the axes of the four reaction chambers are precisely aligned with the detection holes of the light transmission element. The heating module of the hot air qPCR instrument maintains a constant temperature for the reaction chamber 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). Its internal fused tapered beam splitting structure evenly distributes the light field to the four output fibers. The four groups of excitation light transmission fibers (5) composed of multiple fibers with a diameter of 300μm surround the four 2mm diameter transmitting fibers in a circular array. Four common end structures are formed at the end of the fiber focusing lens (6) to ensure that the excitation light evenly covers the detection area of ​​the light transmission element. Step 4: The fiber focusing lens (6) is fixed to the light transmission element (7) through the flange connection. Its aspherical polished surface focuses the excitation light into a 50μm diameter spot, which excites the fluorescent dye in the reaction cell to emit fluorescence. After the emitted fluorescence is collected by the fiber focusing lens (6), it is transmitted to the fiber collimator fixing frame (10) through the central emitting fiber. The fiber collimator fixing frame (10) fixes four fiber collimators (9) through the four-quadrant positioning holes of the aluminum alloy 7075-T6 base. The top screw locking mechanism ensures that the positioning accuracy of the fiber collimator (9) is ±0.1mm and the angle deviation is <0.5°, so as to realize the collimated transmission of the fluorescence signal. Step 5: The collimated fluorescence enters the emission light filter wheel (11) and is linked with the excitation light filter wheel (3) through a synchronous belt to filter out the fluorescence signal corresponding to the emission wavelength. The double cemented achromatic lens and aspherical field lens of the camera condenser module (12) are combined to focus the fluorescence onto the four independent photosensitive areas of the CCD sensor (13) and simultaneously collect four-channel fluorescence signals, corresponding to the four reaction cells of the microfluidic chip. The CCD sensor (13) converts the light signal into an electrical signal, which is then amplified, filtered, and transmitted to the data processing unit. Step 6: After completing the detection of the current 4 reaction cells, rotate the microfluidic chip 22.5° using the rotating module to align the next set of 4 reaction cells with the detection wells. Repeat steps 3 to 5 for detection. Repeat this process 3 times until all 16 reaction cells have been 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, keeping 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. After the microfluidic chip temperature drops to room temperature, take it out and simultaneously turn off the white solid LED light source (1) and the driving system of the stepper motor (16). Analyze the four-channel fluorescence signal through data processing software and calculate the nucleic acid concentration in each reaction cell based on the standard curve.

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