Fluorescent quantitative PCR instrument based on sample multi-channel detection device

Abstract

The invention relates to the technical field of detection instruments, in particular to a fluorescent quantitative PCR (polymerase chain reaction) instrument based on a sample multichannel detection device, which comprises a thermal cycle module and an optical detection system, the optical detection system consists of a plurality of independent detection units, each unit corresponds to a single reaction hole of the thermal cycle module, and the thermal cycle module is connected with the optical detection system. The device comprises an independent light source, a filtering assembly and a detector. A positioning frame is arranged between the thermal cycle module and the optical detection system, and the optical axis of each detection unit is forcibly aligned with the central axis of the corresponding reaction hole by the frame; a thermal expansion compensation mechanism is integrated at the bottom of the thermal cycle module and counteracts module flatness deviation caused by temperature change through reverse deformation. The method has the detection efficiency effects of high stability, high precision and high repeatability.

Description

Technical Field

[0001] This invention relates to the technical field of detection instruments, and in particular to a fluorescence quantitative PCR instrument based on a multi-channel sample detection device. Background Technology

[0002] Polymerase chain reaction (PCR) technology, especially quantitative real-time PCR (qPCR), has become an indispensable tool in modern molecular biology diagnostics, life science research, and food safety testing. With the increase in throughput and the expansion of application scenarios, the market is placing increasingly stringent demands on the detection accuracy, data repeatability, and long-term operational stability of qPCR instruments.

[0003] In existing technologies, mainstream high-throughput quantitative PCR instruments typically adopt an "integrated" configuration, combining the thermal cycling module responsible for temperature control with the optical detection system responsible for signal acquisition within a single housing. Its basic working principle is as follows: the thermal cycling module carries the multi-well reaction plate, and through Peltier elements or resistance wires, combined with air cooling or semiconductor cooling, it achieves periodic temperature changes in the sample; simultaneously, the optical detection system sequentially excites and acquires signals from each reaction well, thereby monitoring the amplification process in real time.

[0004] In the existing technology, the thermal and mechanical coupling interference between the thermal cycling module and the optical detection system is difficult to overcome, which causes the optical alignment accuracy to drift during continuous operation, affecting the reliability and repeatability of the detection data. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a fluorescence quantitative PCR instrument based on a multi-channel sample detection device that offers high stability, high precision, and high repeatability.

[0006] The above-mentioned objective of this invention is achieved through the following technical solutions: A real-time fluorescence PCR instrument based on a multi-channel sample detection device includes a thermal cycling module and an optical detection system. The optical detection system consists of several independent detection units, each unit corresponding to a single reaction hole of the thermal cycling module, and includes an independent light source, filter components and detector; A positioning frame is provided between the thermal cycling module and the optical detection system, which forces the optical axis of each detection unit to be aligned with the central axis of the corresponding reaction hole. The bottom of the thermal circulation module integrates a thermal expansion compensation mechanism, which offsets the flatness deviation of the module caused by temperature changes through reverse deformation.

[0007] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the thermal expansion compensation mechanism is composed of a double-layer heterogeneous material composite plate, with the upper layer being a high thermal conductivity metal substrate and the lower layer being a ceramic plate with a negative thermal expansion coefficient. The two layers of plates are mechanically interlocked by staggered rivets, with the rivet axis forming an angle of 30-60° with the center line of the reaction hole.

[0008] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the positioning frame comprises a three-layer honeycomb titanium alloy mesh: The top-level grid fixes the optical detection unit; The middle layer mesh has vias to allow light to pass through; The bottom mesh embeds positioning pins for the thermal circulation module. Each mesh layer is connected by tapered guide posts, the surface of which is coated with a self-lubricating ceramic layer.

[0009] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the conical guide column integrates an optical fiber transmission channel, which connects the light source of the top grid and the fluorescence collection lens of the bottom grid. The fluorescence collecting lens is an aspherical lens, and its focal point coincides with the liquid surface in the reaction tube.

[0010] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the independent light source is a vertical cavity surface-emitting laser, and a miniature reflecting prism is set in its output optical path; The reflective prism can be finely adjusted along the optical axis, and the displacement is controlled by a piezoelectric ceramic actuator.

[0011] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the thermal cycling module includes a split heating unit, each heating unit is independently temperature-controlled and encloses a single reaction well. An aerogel insulation layer is filled between adjacent heating units, and a shape memory alloy spring is embedded in the insulation layer.

[0012] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the bottom of the split heating unit is connected to a heat sink substrate, and the heat sink substrate is embedded with a microfluidic network. The microchannels are arranged in a spiral radial pattern. The inlet of the microchannel is connected to the liquid cooling system, and the outlet of the microchannel is located at the edge of the substrate.

[0013] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the optical detection unit is provided with a rotatable light shield on the top, and the inner wall of the light shield is coated with a light-absorbing material. The light shield is driven by a magnetically coupled torque motor, and its rotation angle covers the range of 0-180°.

[0014] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, the outer edge of the light shield integrates an annular electrostatic adsorption ring, which forms a sealed negative pressure area when energized.

[0015] As a specific embodiment of the fluorescence quantitative PCR instrument based on a multi-channel sample detection device disclosed in this invention, a vibration isolation platform is provided between the positioning frame and the instrument base; The vibration isolation platform includes: Connect to the platform and establish a positioning framework; The lower platform is fixed to the base; The middle layer is a magnetorheological fluid damping layer, the viscosity of which is controlled in real time by an electromagnetic field.

[0016] In summary, the present invention has at least one of the following beneficial technical effects: 1. The core architecture of the fluorescence quantitative PCR instrument disclosed in this invention solves the problem of misalignment caused by thermal stress and mechanical vibration in traditional instruments through the synergistic effect of a rigid positioning frame and a thermal expansion compensation mechanism. The three-layer grid of the positioning frame and the conical guide post form a highly stable reference platform, ensuring that the relative position of the optical system and the reaction well array is constant in three-dimensional space. The thermal compensation mechanism at the bottom actively cancels the material deformation during the thermal cycle, jointly ensuring that the excitation light and fluorescence collection light path can be accurately aligned with each reaction well under any operating condition. This is the basis for achieving high repeatability and high reliability detection data. 2. The thermal cycling module design of the real-time PCR instrument disclosed in this invention achieves precise thermal management at the microscale. The combination of the split heating unit and the aerogel insulation layer minimizes thermal crosstalk between wells, ensuring the temperature uniformity of the 96 reaction wells. Simultaneously, the heat sink substrate with built-in spiral radial microchannels, tightly coupled to the heating unit, constructs an optimized heat dissipation path through efficient heat exchange with the external liquid cooling system. This achieves heating and cooling rates far exceeding those of conventional methods and significantly reduces rate fluctuations, meeting the requirements for rapid and accurate temperature control. 3. The optical detection system of the quantitative PCR instrument disclosed in this invention has been specifically enhanced to address various interference factors in practical applications. First, by replacing the traditional free-space optical path with an optical fiber transmission channel within a tapered guide post, thermal airflow disturbances and ambient stray light are effectively isolated, significantly improving the signal-to-noise ratio. Second, the finely adjustable reflective prism design endows the optical path system with adaptive capabilities, enabling dynamic optimization of the excitation spot according to different reaction vessel specifications, eliminating the influence of edge effects on the detection results. The combination of these two features ensures that all detection channels from the center to the edge obtain a consistent and pure optical signal. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of one embodiment of the present invention; Figure 2 This is an exploded view of one embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of a thermal expansion compensation mechanism according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the positioning frame and laser according to one embodiment of the present invention; Figure 5 This is a schematic diagram of the structure of a tapered guide post according to an embodiment of the present invention; Figure 6 This is a schematic diagram of the structure of a heating unit according to an embodiment of the present invention.

[0018] Figure label: 1. Independent detection unit; 11. Reaction orifice; 12. Laser; 13. Miniature reflective prism; 2. Positioning frame; 21. Conical guide post; 211. Fiber optic transmission channel; 212. Fluorescence collecting lens; 22. Thermal expansion compensation mechanism; 4. Heating unit; 41. Heat sink substrate; 42. Microchannel; 5. Light shield; 51. Adsorption ring; 6. Vibration isolation platform. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings.

[0020] To better understand the technical solutions demonstrated in the embodiments of this invention, the working principle of a conventional quantitative PCR instrument is first introduced. When the thermal cycling module of a conventional quantitative PCR instrument performs repeated heating and cooling cycles, for example, frequently switching between 94℃, 60℃, and 72℃, its material inevitably undergoes significant thermal expansion and contraction. This deformation is particularly pronounced in large-area modules with dozens or even hundreds of reaction wells, leading to macroscopic warping or twisting of the module. Since the optical detection system and the thermal module are structurally rigidly connected, the deformation of the module directly disrupts the pre-calibrated optical path alignment—that is, the excitation light path no longer precisely passes perpendicularly through the center of the reaction well, and the fluorescence collection light path cannot efficiently capture signals from the sample. Furthermore, internal vibrations of the instrument exacerbate this misalignment. The result is a significant attenuation of the detection signal at the edge wells, an increase in the CV value of the inter-well signal, and increasingly severe data drift during long-term continuous operation.

[0021] Please see Figures 1-6 This invention discloses a fluorescence quantitative PCR instrument based on a multi-channel sample detection device, comprising a thermal cycling module and an optical detection system. The optical detection system includes multiple independent detection units 1, each corresponding to a reaction well 11 in the thermal cycling module, forming parallel detection channels. Each independent detection unit 1 is equipped with an independent vertical-cavity surface-emitting laser 12 as a light source, a filter assembly, and a high-sensitivity detector, ensuring independent acquisition and processing of signals from each channel and avoiding cross-interference.

[0022] The thermal cycling module and the optical detection system are rigidly connected via a positioning frame 2. The positioning frame 2 adopts a three-layer honeycomb titanium alloy mesh structure. The top mesh fixes each optical detection unit through slots to ensure that the unit spacing matches the array of reaction holes 11. The middle mesh has uniformly opened through holes with a diameter slightly larger than the optical path diameter to provide an unobstructed channel for excitation light and fluorescence transmission. The bottom mesh is embedded with the positioning pins of the thermal cycling module through an interference fit to achieve initial alignment between the modules.

[0023] To further enhance optical transmission stability, a quartz optical fiber transmission channel 211 is pre-embedded inside the tapered guide post 21. The two ends of the optical fiber transmission channel 211 are coupled to the laser output end of the top-layer grid and the fluorescence collecting lens 212 of the bottom-layer grid, respectively. In some feasible methods, the fluorescence collecting lens 212 adopts an aspherical design, and through aberration correction, the focal point is precisely placed in the center region of the liquid surface in the reaction tube, thereby improving fluorescence collection efficiency. Compared with traditional free-space optical paths, this optical fiber transmission scheme can effectively isolate air turbulence generated during thermal cycling, and tests have shown that it can improve the detection signal-to-noise ratio by more than 20%.

[0024] An active thermal expansion compensation mechanism 22 is integrated at the bottom of the thermal cycling module. This mechanism is composed of two layers of functional materials: an upper layer of high thermal conductivity aluminum alloy substrate that directly supports the array of reaction holes 11; and a lower layer of ceramic plate with a negative thermal expansion coefficient. The two layers are mechanically interlocked by obliquely arranged titanium alloy rivets, with the rivet axes forming an angle of 45°±15° with the centerline of the reaction holes 11. When the temperature rises, the thermal expansion of the metal substrate and the contraction of the ceramic plate create a torque balance. This torque balance, through the bending deformation of the composite material, actively compensates for flatness errors, ensuring that the flatness deviation of the module remains within ±10μm throughout the entire thermal cycling process.

[0025] Each independent detection unit 1 has an optical path adjustment system comprising a miniature reflective prism 13. The prism is mounted on a piezoelectric ceramic-driven translation stage and can be precisely displaced within ±0.5 mm along the optical axis via program control. This mechanism dynamically adjusts the excitation spot size according to the specifications of the reaction tube used, ensuring consistent illumination intensity between the edge and center apertures.

[0026] The thermal circulation module adopts a split heating scheme, with each reaction hole 11 heated by an independent heating unit 4. A silica aerogel insulation layer is filled between the heating units 4. In some feasible embodiments, shape memory alloy springs can be arranged between the heating units 4. When the temperature exceeds 60°C, the restoring force generated by the phase change of the springs can increase the contact pressure between the heating units 4, enhancing lateral heat conduction and ensuring that the temperature difference between the reaction holes 11 in steady state does not exceed ±0.3°C.

[0027] The split heating unit 4 has a copper alloy heat sink substrate 41 welded to its bottom, and a microchannel cooling network 42 is etched inside the substrate. The microchannels 42 adopt a spiral radial layout, with the inlets centrally connected to the external liquid cooling unit and the outlets distributed at the four corners of the substrate. This channel design allows the coolant to flow along the thermal gradient direction, achieving an average heating rate of 4.5℃ / s and a cooling rate of 3.2℃ / s, with a rate fluctuation of less than 5% throughout the process.

[0028] An active light-shielding system is installed at the top of the optical detection unit. The light-shielding cover 5 is made of carbon fiber composite material, with a carbon black light-absorbing coating sprayed on the inner surface. It can achieve a fixed angle rotation of 0-180° through a magnetically coupled torque motor. The edge of the light-shielding cover 5 is inlaid with a silver electrode ring electrostatic adsorption ring 51. When a 500V DC voltage is applied, a negative pressure zone of -5kPa can be formed above the reaction tube cap, which can both prevent the tube cap from shifting and block water vapor condensation.

[0029] The entire positioning frame 2 is decoupled from the instrument base via a vibration isolation platform 6, which employs a sandwich structure: the upper and lower platforms are made of alumina ceramic plates, with carbonyl iron powder magnetorheological fluid poured in between. Vibration frequency is monitored by sensors, and the intensity of the electromagnetic coil current is controlled accordingly, adjusting the viscosity of the damping fluid in real time. This system can reduce the transmissibility of mechanical vibrations below 50Hz to below 0.1, and control the resonant amplitude of the optical system within a range of 5μm.

[0030] The working process of this real-time PCR instrument is as follows: First, initialization and sample loading are performed. The user places the transparent reaction tube containing the reaction mixture into each reaction port 11 of the thermal cycling module. After starting the instrument, the rigid positioning frame 2 ensures that each reaction port 11 is precisely positioned directly below its corresponding independent optical detection unit.

[0031] Then, the split heating unit 4 is independently powered on under the command of the control system, directly transferring heat energy to the corresponding reaction hole 11, causing the solution inside the tube to heat up rapidly. At this time, the double-layer composite plate of the thermal expansion compensation mechanism 22 starts to work. The upper high thermal conductivity metal plate expands, but the lower negative thermal expansion ceramic plate contracts. The two are restrained by the oblique rivets, producing opposite deformation, thereby maintaining the overall flatness of the module and preventing warping.

[0032] When the target temperature is reached, heating unit 4 enters a precise PID temperature control mode. Simultaneously, the aerogel insulation layer and shape memory alloy spring work together to equalize the temperature of adjacent units. When cooling is required, the control system activates the external liquid cooling unit, pumping coolant into the spiral radial microchannels 42 of the heat sink substrate 41 to rapidly remove heat, achieving rapid and uniform cooling.

[0033] During the annealing / stretching phase of each thermal cycle, the vertical-cavity surface-emitting laser 12 of a specific detection channel emits excitation light of a specific wavelength. The light enters the fiber optic transmission channel 211, is deflected by a miniature reflecting prism 13, and then precisely illuminates the bottom of the corresponding reaction tube vertically downwards. The position of the prism can be finely adjusted by a piezoelectric ceramic actuator to adapt to different tube shapes.

[0034] Inside the reaction tube, the fluorescent dye is excited to emit fluorescence at a longer wavelength. This fluorescence is efficiently captured by the aspherical fluorescence collecting lens 212 at the bottom, focused, and then guided upwards through the optical fiber transmission channel 211. The fluorescence eventually penetrates the filter assembly and is received by the high-sensitivity detector at the top, where it is converted into an electrical signal.

[0035] Throughout the process, the magnetorheological fluid vibration isolation platform 6 operates continuously, actively absorbing vibrations from the equipment motor or the desktop. At the moment of optical detection, the light shield 5 rotates to the closed position, and its electrostatic adsorption ring 51 is energized, adsorbing the tube cap to form a temporary seal, creating a dark, dust-free, and vibration-free local detection environment. After completion, it is opened for the next detection.

[0036] The real-time fluorescence signal obtained by the detector is transmitted to the data processing system. After processing by the algorithm, the fluorescence amplification curve of each well is plotted, and finally the accurate quantification of the starting template DNA is achieved.

[0037] Through the precise coordination of the above structure and process, the device achieves a comprehensive performance improvement from thermal control and optical detection to mechanical stability, ensuring the accuracy, speed and reliability of experimental results.

[0038] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A real-time fluorescence PCR instrument based on a multi-channel sample detection device, characterized in that, Includes a thermal cycling module and an optical inspection system. The optical detection system consists of several independent detection units (1), each unit corresponding to a single reaction hole (11) of the thermal cycling module, including an independent light source, filter components and detector; A positioning frame (2) is provided between the thermal cycling module and the optical detection system. This frame forces the optical axis of each detection unit to be aligned with the central axis of the corresponding reaction hole (11). The bottom of the thermal cycle module integrates a thermal expansion compensation mechanism (22), which offsets the module flatness deviation caused by temperature changes through reverse deformation.

2. The real-time PCR instrument based on a multi-channel sample detection device according to claim 1, characterized in that: The thermal expansion compensation mechanism (22) is composed of a double-layer heterogeneous material composite plate, with the upper layer being a high thermal conductivity metal substrate and the lower layer being a ceramic plate with a negative thermal expansion coefficient. The two layers of plates are mechanically interlocked by staggered rivets, with the rivet axis forming an angle of 30-60° with the center line of the reaction hole (11).

3. A real-time PCR instrument based on a multi-channel sample detection device according to claim 2, characterized in that: The positioning frame (2) comprises a three-layer honeycomb titanium alloy mesh: The top-level grid fixes the optical detection unit; The middle layer mesh has vias to allow light to pass through; The bottom mesh embeds positioning pins for the thermal circulation module. Each layer of mesh is connected by tapered guide posts (21), and the surface of the guide posts is coated with a self-lubricating ceramic layer.

4. A real-time PCR instrument based on a multi-channel sample detection device according to claim 3, characterized in that: The tapered guide post (21) integrates an optical fiber transmission channel (211), which connects the light source of the top layer grid to the fluorescence collection lens (212) of the bottom layer grid. The fluorescence collecting lens (212) is an aspherical lens, and its focal point coincides with the liquid surface in the reaction tube.

5. A real-time PCR instrument based on a multi-channel sample detection device according to claim 4, characterized in that: The independent light source is a vertical cavity surface-emitting laser (12), and a miniature reflecting prism (13) is provided in its output optical path. The reflective prism can be finely adjusted along the optical axis, and the displacement is controlled by a piezoelectric ceramic actuator.

6. A real-time PCR instrument based on a multi-channel sample detection device according to claim 1, characterized in that: The thermal circulation module includes a split heating unit (4), each heating unit (4) is independently temperature controlled and encloses a single reaction hole (11). An aerogel insulation layer is filled between adjacent heating units (4), and a shape memory alloy spring is embedded in the insulation layer.

7. A real-time PCR instrument based on a multi-channel sample detection device according to claim 6, characterized in that: The split heating unit (4) is connected to the heat sink substrate (41) at the bottom, and the heat sink substrate (41) is embedded with a microchannel (42) network; The microchannels (42) are distributed in a spiral radial pattern. The inlet of the microchannels (42) is connected to the liquid cooling system, and the outlet of the microchannels (42) is located at the edge of the substrate.

8. A real-time PCR instrument based on a multi-channel sample detection device according to claim 1, characterized in that: The optical detection unit is provided with a rotatable light shield (5) on top, and the inner wall of the light shield (5) is coated with light-absorbing material; The light shield (5) is driven by a magnetically coupled torque motor, and its rotation angle covers the range of 0-180°.

9. A real-time PCR instrument based on a multi-channel sample detection device according to claim 8, characterized in that: The outer edge of the light shield (5) integrates an annular electrostatic adsorption ring (51), which forms a sealed negative pressure area when energized.

10. A real-time PCR instrument based on a multi-channel sample detection device according to claim 1, characterized in that: A vibration isolation platform (6) is provided between the positioning frame (2) and the base of the instrument. The vibration isolation platform (6) includes: Connect the positioning framework to the platform (2); The lower platform is fixed to the base; The intermediate layer is a magnetorheological fluid damping layer, the viscosity of which is controlled in real time by an electromagnetic field.

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