Ultra-high-speed multi-channel real-time fluorescent PCR (Polymerase Chain Reaction) circulating system based on photon heating and detection method thereof

Abstract

The invention discloses a superspeed multichannel real-time fluorescent PCR circulation system based on photon heating and a detection method thereof, and belongs to the field of molecular biology detection and photon heating, the system comprises a photon heating module, a photothermal conversion layer, a reaction module, a cooling module, a fluorescent detection module and a control module. The photo-thermal conversion layer is used for absorbing light energy of the photon heating module and converting the light energy into heat energy; the reaction module comprises a plurality of capillary reactors which are fixed through clamps and are horizontally arranged, and capillary tubes are kept in contact with the photothermal conversion layer through the clamps; and the fluorescence detection module is used for carrying out fluorescence collection on partial volume in the heating area limited by the photothermal conversion layer. According to the invention, photothermal conversion and non-contact heating are combined with the horizontal capillary array, so that superspeed operation and multi-channel parallel detection of PCR temperature circulation are realized, and the system is simple in structure and low in cost.

Description

A photon-heated ultra-high-speed multichannel real-time fluorescence PCR cycling system and its detection method Technical Field

[0001] This invention belongs to the field of molecular biology detection and photonic heating technology, and particularly relates to an ultra-high-speed multi-channel real-time fluorescence PCR cyclic system based on photonic heating and its detection method. Background Technology

[0002] Polymerase chain reaction (PCR) and real-time fluorescence PCR are among the core technologies in nucleic acid detection, and have been widely used in clinical diagnosis, infectious disease detection, genotyping, and expression analysis. Currently, most mainstream PCR instruments employ a contact heating method based on thermoelectric cooling pads (TECs), achieving heat conduction through direct contact between the heating element and the reaction tube. This method offers relatively stable temperature control, is compatible with conventional PCR tubes and reagents, and has become standard laboratory equipment. Furthermore, some photonic PCR technologies that have emerged in recent years utilize laser irradiation of photothermal nanomaterials (such as gold nanoparticles and carbon nanotubes) added to the reaction system. These nanomaterials absorb light energy and rapidly generate heat, achieving rapid, localized heating of the reaction solution. The heating rate is significantly higher than traditional contact heating methods, making ultrafast PCR possible.

[0003] However, existing technologies still have significant limitations. Traditional contact heating methods are limited by the thermal inertia of the thermoelectric module and the thermal resistance of the heat transfer medium, with heating and cooling rates typically only 1–2 °C / s. A typical PCR process takes more than one hour to complete, and the system is large, complex, and energy-intensive, making it difficult to meet the demands for rapid, portable, and low-cost on-site testing. On the other hand, while existing photonic PCR technology can achieve millisecond-level heating, it generally relies on specialized photothermal nanomaterials, whose preparation processes are complex and costly, and may affect the compatibility of the reaction system and amplification efficiency. Furthermore, such systems typically employ vertical optical paths or single reaction chamber designs, making it difficult to achieve high-throughput, multi-channel parallel detection, thus limiting their practicality in multi-sample screening and large-scale applications. Therefore, there is an urgent need in this field for a real-time fluorescence PCR system that can achieve ultra-high-speed temperature cycling, possess multi-channel parallel detection capabilities, has a simple structure, low cost, and is compatible with standard PCR reagents. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an ultra-high-speed multi-channel real-time fluorescence PCR cyclic system based on photon heating and its detection method, thereby resolving the issues existing in the prior art.

[0005] To achieve the above objectives, in a first aspect, the present invention provides an ultra-high-speed multi-channel real-time fluorescence PCR cycling system based on photon heating, comprising a photon heating module, a photothermal conversion layer, a reaction module, a cooling module, a fluorescence detection module, and a control module;

[0006] The photothermal conversion layer is disposed on the irradiation path of the photonic heating module and is used to absorb light energy and convert it into heat energy.

[0007] The reaction module includes multiple capillary reactors and clips for fixing the capillary reactors. The capillary reactors are horizontally arranged and are kept in contact with the photothermal conversion layer by the clips.

[0008] The optical path of the fluorescence detection module is configured to be aligned with the heating area defined by the photothermal conversion layer, and to collect fluorescence signals from a portion of the volume within the heating area.

[0009] Preferably, the photonic heating module includes a laser, and the control module controls the power output and switching timing of the laser through a pulse width modulation signal.

[0010] Preferably, the laser is a visible light laser or a near-infrared laser.

[0011] Preferably, the photothermal conversion layer is composed of a ferrous metal material with high light absorption or a non-metallic material with high thermal conductivity.

[0012] Preferably, the inner diameter of the capillary reactor is on the micrometer scale.

[0013] Preferably, the clip is formed by additive manufacturing technology and has an array of slots, and the capillary reactor is horizontally embedded in the slots.

[0014] Preferably, the cooling module is an air-cooled module, with its air outlet facing the capillary reactor.

[0015] Preferably, the control module is configured to: control the photonic heating module to turn on and adjust its power when heating is required; and control the photonic heating module to turn off and simultaneously start the cooling module when cooling is required, so as to achieve rapid temperature cycling.

[0016] Preferably, the fluorescence detection module includes an imaging sensor, the imaging area of ​​which is defined on the capillary reactor corresponding to the central segment of the heating area.

[0017] Secondly, the present invention provides a method for PCR detection using the system described in the first aspect, comprising the following steps:

[0018] The PCR reaction system was injected into multiple capillary reactors.

[0019] The capillary reactor is horizontally fixed by the clamp and brought into contact with the photothermal conversion layer.

[0020] The control module regulates the photonic heating module to irradiate the photothermal conversion layer to heat the capillary reactor, and regulates the cooling module to cool it, thereby realizing PCR temperature cycling.

[0021] During the temperature cycling process, the fluorescence detection module collects real-time fluorescence signals from a portion of the volume within the heating area.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] This invention provides the technical features of "the photothermal conversion layer being disposed on the irradiation path of the photon heating module" and "the capillary reactor being horizontally disposed and maintaining contact with the photothermal conversion layer through the clamp." The photon heating module provides instantaneous light energy, which the photothermal conversion layer rapidly converts into heat energy. This heat energy is then directly conducted to the trace reaction liquid in the horizontal capillary through the tight contact ensured by the clamp. This significantly reduces the thermal inertia of traditional hot block heating, enabling the system to achieve an average heating and cooling rate of up to 20°C / s. As a result, at least 50 PCR cycles can be completed within 5 minutes, achieving ultra-high-speed operation of PCR temperature cycling.

[0024] This invention significantly reduces the construction and usage costs of the system and facilitates large-scale application through a technical solution that includes "the reaction module comprising multiple capillary reactors and clips for fixing the capillary reactors" and a technical solution that uses common light-absorbing materials for the photothermal conversion layer. The clip structure allows for the use of extremely low-cost standardized capillaries as reactors, and the clips themselves can be manufactured inexpensively using methods such as 3D printing (costing less than $1). Simultaneously, the system utilizes common black light-absorbing material as the photothermal conversion layer, eliminating the need for expensive specialized photothermal nanomaterials and ensuring compatibility with standard PCR reagents, thus reducing costs across the board from hardware consumables to reaction reagents.

[0025] This invention provides the technical features of "the reaction module comprising multiple capillary reactors" and "the capillary reactors being horizontally arranged and kept in contact with the photothermal conversion layer by clamps." The horizontally arranged capillaries are precisely fixed by clamps and ensure uniform contact with the same photothermal conversion layer, resulting in all channels being in almost the same thermal environment. This design allows the system to simultaneously perform parallel temperature cycling and reactions on 24 or more channels, increasing throughput while ensuring the consistency and reliability of amplification across channels.

[0026] This invention improves the signal-to-noise ratio and accuracy of fluorescence detection by configuring the optical path of the fluorescence detection module to be aligned with the heating region defined by the photothermal conversion layer and acquiring fluorescence signals from a portion of the volume within the heating region. Because the heating region is limited, and detection is strictly confined to a portion of the volume within this region, it effectively acquires fluorescence signals emitted by the reaction liquid that has undergone a complete and uniform temperature cycle. This avoids acquiring background signals from temperature gradient regions or non-reaction areas, thereby enhancing the specificity and sensitivity of the detection and laying the foundation for precise quantification. Attached Figure Description

[0027] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0028] Figure 1 is a schematic diagram of the ultra-high-speed multi-channel real-time fluorescence PCR cycling system based on photon heating according to an embodiment of the present invention;

[0029] Figure 2 shows four capillary heating cycle curves according to an embodiment of the present invention.

[0030] Figure 3 is a schematic diagram of the heating and cooling rates according to an embodiment of the present invention;

[0031] Figure 4 is a real-time amplification curve of 24 channels according to an embodiment of the present invention;

[0032] Figure 5 is a schematic diagram of the standard curve of an embodiment of the present invention. Detailed Implementation

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] It should be noted that the steps shown in the flowchart in the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although a logical order is shown in the flowchart, in some cases the steps shown or described may be executed in a different order than that shown here.

[0035] Example 1

[0036] As shown in Figure 1, this embodiment provides an ultra-high-speed multi-channel real-time fluorescence PCR cycling system based on photonic heating, including a photonic heating module, a photothermal conversion layer, a reaction module, a cooling module, a fluorescence detection module, and a control module;

[0037] The photonic heating module includes a laser, which is a visible light laser or a near-infrared laser.

[0038] Specifically, the photonic heating module is used to generate laser light as a heating source; it specifically uses a 450 nm semiconductor laser, which is the "laser heating system" in Figure 1. Its power output and switching timing are controlled by the control module through pulse width modulation (PWM) signals.

[0039] The photothermal conversion layer is disposed on the irradiation path of the photonic heating module and is used to absorb light energy and convert it into heat energy.

[0040] Furthermore, the photothermal conversion layer is composed of a ferrous metal material with high light absorption or a non-metallic material with high thermal conductivity.

[0041] Specifically, the photothermal conversion layer is a black light-absorbing and heat-conducting material that is in direct and close contact with the reaction module (capillary) to ensure efficient heat conduction.

[0042] The reaction module includes multiple capillary reactors and clips for fixing the capillary reactors. The capillary reactors are horizontally arranged and are kept in contact with the photothermal conversion layer by the clips.

[0043] Furthermore, the inner diameter of the capillary reactor is on the micrometer scale.

[0044] Furthermore, the clip is formed by additive manufacturing technology and has an array of slots, and the capillary reactor is horizontally embedded in the slots.

[0045] Specifically, the reaction module includes multiple capillary reactors, which are horizontally placed in the array slots of the 3D printed clips and fixed in place by the clips. At the same time, the positioning structure of the clips ensures that the capillaries and the photothermal conversion layer maintain direct and close contact.

[0046] The cooling module is an air-cooled module, and its air outlet is positioned facing the capillary reactor.

[0047] Specifically, the cooling module is used to rapidly cool the reaction module after the laser is turned off; it is a wind-cooling module that is triggered by the control module to deliver directional air from above the capillary / clamp, and the wind speed and duration are adjusted by the control module according to a preset program.

[0048] In this embodiment, both the reaction module and the cooling module are placed in the reaction chamber shown in Figure 1.

[0049] The optical path of the fluorescence detection module is configured to be aligned with the heating area defined by the photothermal conversion layer, and to collect fluorescence signals from a portion of the volume within the heating area.

[0050] Furthermore, the fluorescence detection module includes an imaging sensor, the imaging area of ​​which is defined on the capillary reactor corresponding to the central segment of the heating area.

[0051] Specifically, the fluorescence detection module is used to detect the fluorescence signal generated during PCR amplification in real time. Because the capillary is placed horizontally and the heating area is limited, to ensure that the detected fluorescence signal originates from a solution that has undergone a complete temperature cycle, the detection optical path (imaging area) is precisely aligned and confined to the central segment of the heating area. The fluorescence detection module is the "fluorescence detection system" shown in Figure 1, including a CMOS camera, lens, and optical filters.

[0052] The control module controls the power output and switching timing of the laser through pulse width modulation signals.

[0053] Furthermore, the control module is configured to: control the photonic heating module to turn on and adjust its power when heating is required; and control the photonic heating module to turn off and simultaneously start the cooling module when cooling is required, so as to achieve rapid temperature cycling.

[0054] Specifically, the control module controls the photonic heating module, cooling module, and fluorescence detection module to achieve a preset PCR temperature cycling program. Its control logic is as follows: based on the preset temperature-time program, it sends power adjustment and on / off commands to the laser (achieving rapid and precise heating through PWM modulation), simultaneously triggering the start / stop of the cooling module and fan speed adjustment; and at the end of the annealing / extension phase of each temperature cycle, it controls the fluorescence detection module to acquire signals from a local volume in the center of the heated area. PCR amplification is completed within the heated area corresponding to the photothermal conversion layer.

[0055] Example 2

[0056] Based on the inventive concept of Embodiment 1, this embodiment provides an ultra-high-speed multi-channel real-time fluorescence PCR cycling system based on photon heating, specifically including:

[0057] (1) Photonic heating module: A 20W visible light laser is used as the photonic heating source;

[0058] (2) Photothermal conversion layer: A black metal coating is prepared on the substrate surface as a photothermal conversion layer;

[0059] (3) Reaction module: Open the door and horizontally embed multiple fused silica capillaries into the array slots of the 3D printed clips. The clips fix the capillaries and ensure that each capillary is in close contact with the photothermal conversion layer. Each capillary independently constitutes a PCR reactor (single reaction volume < 1 μL).

[0060] (4) Cooling module: Equipped with an air-cooling module, installed above the clip, with the air outlet oriented towards the capillary tube area;

[0061] (5) Detection module: A CMOS camera is used as a fluorescence detection device, and its optical path is aligned with the center section of the heating area of ​​the capillary to realize real-time monitoring of the local volume of 40.2 nL;

[0062] (6) Control module: Run the operation software through the touch screen, set the operation parameters, send commands to the Arduino controller to connect the laser, air-cooling module and CMOS camera, and preset the temperature-time program;

[0063] (7) System Operation: Close the hatch, engage the safety button, start the laser power supply, and control the laser (PWM modulation power) and the start / stop of the air-cooling module through the control module to achieve rapid temperature change; complete no less than 50 PCR cycles within 5 minutes. A schematic diagram of the four capillary heating cycle curves is shown in Figure 2. The heating and cooling rates are shown in Figure 3.

[0064] (8) Detection and analysis: The control software uses a CMOS camera to collect fluorescence signals at the end of each annealing / extension stage to complete real-time monitoring.

[0065] In this embodiment, the thermal cycling program includes: an initial run at 95 °C for 20 s, followed by 50 cycles (0.5 s at 95 °C, 2 s at 60 °C). The heating / cooling rate is 20 °C / s, with a total time of 239 seconds. Thermocouple verification shows temperature consistency across the 24 channels, with a variation of <5%.

[0066] In this embodiment, quantitative performance testing included: serial dilutions of lambda DNA (5 pg / µL to 5 ng / µL), a standard curve R² = 0.9952, and an amplification efficiency of 95.3%. Reliable detection was achieved at the lowest concentration of 5 pg / µL (heating volume ~3.66 × 10³ copies, detection volume ~366 copies). All 24-channel Ct variants were <5%. Temperature cycling was performed, and real-time fluorescence detection of the 24 channels was achieved using an imaging sensor. The 24-channel real-time amplification curve is shown in Figure 4. The standard curve is shown in Figure 5.

[0067] In this embodiment, the card holder is 3D printed, with a cost of less than $1.

[0068] The beneficial effects of this embodiment:

[0069] This embodiment enables ultra-high-speed operation of PCR reaction, significantly shortening the detection time; it achieves 50 cycles within 5 minutes, which is significantly faster than existing systems.

[0070] The system has a simple structure and low manufacturing cost. It uses ordinary black light-absorbing material and 3D printed card holders, resulting in extremely low cost (card holder < $1); it is suitable for large-scale promotion.

[0071] This embodiment supports multi-channel parallel detection, with 24 channels for parallel detection, significantly increasing throughput. It is compatible with standard PCR reagents, requires no specialized nanomaterials, and reduces detection costs. This embodiment is suitable for on-site point-of-care testing and resource-constrained environments, facilitating on-site deployment.

[0072] Example 3

[0073] This embodiment provides a method for PCR detection using the system described in Embodiment 1, including the following steps:

[0074] S1. Inject the PCR reaction system into multiple capillary reactors;

[0075] S2. The capillary reactor is horizontally fixed by the clamp and brought into contact with the photothermal conversion layer;

[0076] Specifically, the capillary reactor is placed horizontally in the array slots of the 3D printed clip, and the capillary is fixed by the positioning structure of the clip, ensuring that it maintains close and uniform contact with the photothermal conversion layer; the photothermal conversion layer is irradiated by a laser, and the output power of the laser is adjusted by the control module according to the target temperature (PWM modulation), so that it absorbs light energy and heats the capillary reactor through heat conduction.

[0077] S3. The control module regulates the photonic heating module to irradiate the photothermal conversion layer to heat the capillary reactor, and regulates the cooling module to cool it, thereby realizing PCR temperature cycling.

[0078] Specifically, in this embodiment, the laser and cooling modules are linked and controlled by the control module to achieve rapid heating and cooling cycles in PCR: when heating is required, the laser is turned on and adjusted to the corresponding power; when cooling is required, the laser is turned off and the air-cooling module is started simultaneously; through closed-loop temperature sensing feedback, the laser power and cooling intensity are dynamically adjusted to achieve the preset temperature-time curve.

[0079] S4. During the temperature cycling process, the fluorescence detection module collects real-time fluorescence signals from a portion of the volume within the heating area.

[0080] Specifically, at the end of the annealing / extension phase of each temperature cycle, the fluorescence detection module is controlled to collect fluorescence signals in real time from the local volume of the central section of the heating region in the capillary reactor; real-time amplification curves are generated based on the changes in fluorescence signals to achieve qualitative or quantitative analysis.

[0081] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A high-speed, multi-channel real-time fluorescence PCR cycling system based on photon heating, characterized in that, The system includes a photonic heating module, a photothermal conversion layer, a reaction module, a cooling module, a fluorescence detection module, and a control module. The photothermal conversion layer is disposed on the irradiation path of the photonic heating module and is used to absorb light energy and convert it into heat energy. The reaction module includes multiple capillary reactors and clamps for fixing the capillary reactors. The capillary reactors are horizontally arranged and maintain contact with the photothermal conversion layer through the clamps. The optical path of the fluorescence detection module is configured to be aligned with the heating area defined by the photothermal conversion layer and to collect fluorescence signals from a portion of the volume within the heating area.

2. The system according to claim 1, characterized in that, The photonic heating module includes a laser, and the control module controls the power output and switching timing of the laser through a pulse width modulation signal.

3. The system according to claim 2, characterized in that, The laser is a visible light laser or a near-infrared laser.

4. The system according to claim 1, characterized in that, The photothermal conversion layer is composed of a ferrous metal material with high light absorption or a non-metallic material with high thermal conductivity.

5. The system according to claim 1, characterized in that, The inner diameter of the capillary reactor is on the micrometer scale.

6. The system according to claim 1, characterized in that, The clip is formed by additive manufacturing technology and has an array of slots, and the capillary reactor is horizontally embedded in the slots.

7. The system according to claim 1, characterized in that, The cooling module is an air-cooled module, and its air outlet is positioned facing the capillary reactor.

8. The system according to claim 1, characterized in that, The control module is configured to: control the photonic heating module to turn on and adjust its power when heating is required; and control the photonic heating module to turn off and simultaneously start the cooling module when cooling is required, so as to achieve rapid temperature cycling.

9. The system according to claim 1, characterized in that, The fluorescence detection module includes an imaging sensor, the imaging area of ​​which is defined on the capillary reactor corresponding to the central segment of the heating area.

10. A method for PCR detection using the system according to any one of claims 1 to 9, characterized in that, The method includes the following steps: injecting the PCR reaction system into multiple capillary reactors; horizontally fixing the capillary reactors with clamps and bringing them into contact with the photothermal conversion layer; controlling the photon heating module to irradiate the photothermal conversion layer to heat the capillary reactors and controlling the cooling module to cool them, thereby achieving PCR temperature cycling; during the temperature cycling process, real-time fluorescence signal acquisition of a portion of the heated area using the fluorescence detection module.

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