Optical fiber system integrating photo-thermal amplification and real-time PCR (Polymerase Chain Reaction) detection

Abstract

The invention discloses a photo-thermal amplification and real-time PCR (polymerase chain reaction) detection integrated optical fiber system, and relates to the field of pathogen testing, the optical fiber system comprises an optical fiber probe, a photo-thermal photosensitizer and a micro reaction chamber; the micro-reaction chamber is used for extracting and discharging a to-be-detected sample by utilizing air pressure, the optical fiber probe is arranged in the micro-reaction chamber, and the optical fiber probe reacts with the to-be-detected sample in the micro-reaction chamber; the photo-thermal photosensitizer is arranged inside or on the outer surface of the optical fiber probe and is used for receiving heat generated by the heating light to heat the optical fiber probe and controlling the reaction temperature of the optical fiber probe and the to-be-detected sample; the optical fiber probe is also used for receiving the exciting light which excites the to-be-detected sample to generate a fluorescence signal, and the optical fiber probe is used for detecting the fluorescence signal. According to the application, sample collection, photo-thermal cycle amplification and real-time fluorescent quantitative detection can be integrated, and rapid and effective detection of a virus sample is realized.

Description

Technical Field

[0001] The present application relates to the field of pathogen testing, and in particular to an optical fiber system integrating photothermal amplification and real-time PCR detection. Background Art

[0002] Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, plays a key role in responding to pandemic viruses due to its speed, sensitivity, and accuracy in detecting and quantifying viral genetic material. Although qPCR technology has many advantages, such as short cycle time, flexible application, and the ability to perform multiplex testing, it still has certain limitations. For example, nucleic acid testing during a viral pandemic requires time-consuming and labor-intensive analysis and well-trained laboratory personnel, posing a challenge for large-scale testing. The cost of qPCR instruments and reagents may be too high for some laboratories. In addition, the technology requires a high level of expertise to design experiments and accurately interpret the results, including selecting appropriate primers and probes, optimizing reaction conditions, and controlling reaction efficiency. Due to the large equipment and complex operating procedures, the use environment of qPCR is also limited, resulting in slow and inconvenient results.

[0003] Therefore, some new technologies have been proposed to replace traditional polymerase chain reaction (PCR) equipment. Thanks to its flexible light guidance, low crosstalk, high sensitivity, and signal detection, fiber optic technology can bring many innovative functions and advantages when integrated with PCR systems. However, current fiber optic PCR technology spatially separates the amplification function and the detection function, resulting in increased contamination risk, high time and resource consumption, reduced sensitivity and specificity, increased variability, and loss of real-time monitoring benefits. In addition, existing fiber optic qPCR devices still have the disadvantages of difficult sampling and high operating requirements, and their amplification function relies on high-risk strong laser heating. Summary of the invention

[0004] The purpose of this application is to provide an optical fiber system that integrates photothermal amplification and real-time PCR detection, which can integrate sample collection, photothermal cycle amplification and real-time fluorescence quantitative detection to achieve rapid and effective detection of virus samples.

[0005] To achieve the above objectives, this application provides the following solutions:

[0006] In a first aspect, the present application provides an optical fiber system integrating photothermal amplification and real-time PCR detection, comprising: an optical fiber probe, a photothermal photosensitizer, and a micro-reaction chamber;

[0007] The micro-reaction chamber uses air pressure to extract and discharge the sample to be tested, the optical fiber probe is arranged in the micro-reaction chamber, and the optical fiber probe reacts with the sample to be tested in the micro-reaction chamber;

[0008] The photothermal photosensitizer is arranged inside or on the outer surface of the optical fiber probe, and the photothermal photosensitizer is used to receive heating light to generate heat to increase the temperature of the optical fiber probe, thereby controlling the reaction temperature between the optical fiber probe and the sample to be tested;

[0009] The optical fiber probe is also used to receive excitation light, the excitation light excites the sample to be tested to generate a fluorescent signal, and the optical fiber probe is used to detect the fluorescent signal.

[0010] Optionally, the optical fiber probe includes a core and a cladding wrapping the core, and the core is used to transmit the heating light, the excitation light and the fluorescence signal.

[0011] Optionally, the photothermal photosensitizer is a two-dimensional material, a metal ion, a noble metal or a metal complex.

[0012] Optionally, the micro-reaction chamber is the micro-reaction tube.

[0013] Optionally, the optical fiber system further comprises a temperature monitor disposed inside the optical fiber probe, and the temperature monitor is used to collect the reaction temperature between the optical fiber probe and the sample to be tested and generate a temperature monitoring signal.

[0014] Optionally, the optical fiber system further comprises:

[0015] An optical connection unit, connected to the optical fiber probe, for transmitting excitation light and heating light, and collecting fluorescence signals and temperature monitoring signals;

[0016] An optical path coupling unit is connected to the optical connection unit and is used to combine or decompose the excitation light, the heating light, the fluorescence signal and the temperature monitoring signal.

[0017] Optionally, the optical fiber system further comprises:

[0018] The thermal light source unit transmits heating light to the photothermal photosensitizer through the optical path coupling unit and the optical connection unit.

[0019] Optionally, the optical fiber system further comprises:

[0020] The fluorescence excitation unit emits excitation light to the optical fiber probe through the optical path coupling unit and the optical connection unit.

[0021] Optionally, the optical fiber system further comprises:

[0022] The fluorescence signal monitoring unit receives the fluorescence signal detected by the optical fiber probe through the optical path coupling unit and the optical connection unit, and demodulates the fluorescence signal.

[0023] Optionally, the optical fiber system further comprises:

[0024] The fiber grating temperature measuring unit receives the temperature monitoring signal through the optical path coupling unit and the optical connection unit, and monitors the reaction temperature between the fiber optic probe and the sample to be measured.

[0025] According to the specific embodiments provided in this application, this application has the following technical effects:

[0026] The present application provides an optical fiber system integrating photothermal amplification and real-time PCR detection. The optical fiber probe is heated by generating heat through a photothermal photosensitizer set inside or on the outer surface of the optical fiber probe. Compared with the direct heating method of a high-power laser, the spectral utilization rate of the laser is greatly expanded, and photothermal amplification is realized; the optical fiber probe is used as a fluorescence detection tool to detect the fluorescence signal generated by the sample to be tested, and the detection of real-time PCR is realized; the optical fiber system provided by the present application can realize the integration of photothermal amplification and real-time PCR detection, reducing the risk of contamination. In addition, the present application utilizes the long-distance signal transmission characteristics of optical fiber to realize remote detection, thereby eliminating the need to deploy instruments in crowded biosafety laboratories, facilitating potential distributed pathogen detection, and thus expanding the tool library for responding to a possible new round of epidemics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 A schematic diagram of the structure of an optical fiber system integrating photothermal amplification and real-time PCR detection provided in one embodiment of the present application;

[0029] Figure 2 Another structural schematic diagram of an optical fiber system integrating photothermal amplification and real-time PCR detection provided by an embodiment of the present application;

[0030] Figure 3 A schematic diagram of the structure of a photothermal cycle amplification module, a temperature monitoring module and a fluorescence detection module provided in one embodiment of the present application;

[0031] Figure 4 A schematic diagram of a temperature monitoring spectrum provided in an embodiment of the present application;

[0032] Figure 5 A schematic diagram of remote amplification and detection efficiency of an optical fiber probe provided in one embodiment of the present application. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0035] In an exemplary embodiment, Figure 1-Figure 2 As shown, a fiber optic system integrating photothermal amplification and real-time PCR detection is provided, comprising: a fiber optic probe 1, a photothermal photosensitizer 5 and a micro reaction chamber 4.

[0036] The micro-reaction chamber 4 uses air pressure to extract and discharge the sample to be tested. The optical fiber probe 1 is arranged in the micro-reaction chamber 4 , and the optical fiber probe 1 reacts with the sample to be tested in the micro-reaction chamber 4 .

[0037] The photothermal photosensitizer 5 is disposed inside or on the outer surface of the optical fiber probe 1. Figure 1 The mesophotothermal photosensitizer 5 is arranged inside the optical fiber probe 1. Figure 2 The photothermal photosensitizer 5 is arranged on the outer surface of the optical fiber probe 1. The photothermal photosensitizer 5 is used to receive the heating light 9 to generate heat to heat up the optical fiber probe 1, control the reaction temperature between the optical fiber probe 1 and the sample to be tested, and realize photothermal amplification.

[0038] The optical fiber probe 1 is also used to receive the excitation light 7, the excitation light 7 excites the sample to be tested to generate a fluorescent signal 8, and the optical fiber probe 1 is used to detect the fluorescent signal 8. As a fluorescence detection tool, the optical fiber probe 1 transmits the excitation light 7 and detects the fluorescent signal 8 to achieve real-time PCR detection. Figure 5 As shown, the optical fiber probe 1 can also be extended to achieve remote thermal cycle amplification and real-time PCR detection.

[0039] In a specific embodiment, the optical fiber probe 1 includes a core 2 and a cladding 3 that wraps the core 2. The core 2 is used to transmit heating light 9, excitation light 7, and fluorescent signal 8. The cladding 3 limits the total reflection propagation of light in the core 2, reduces loss, and protects the core 2.

[0040] The diameter of the fiber core 2 is 105-400 microns, the radial thickness of the cladding 3 is 20-40 microns, and the length of the optical fiber probe 1 is 0.5-3 centimeters.

[0041] In a specific embodiment, the photothermal photosensitizer 5 is a two-dimensional material, metal ions, noble metals or metal composites. The two-dimensional material is graphene, carbon nanotubes or black phosphorus, etc.; the metal ions are erbium ions, ytterbium ions, bismuth ions or cobalt ions, etc.; the noble metal is gold or silver, etc.; the metal composite material is copper oxide or iron oxide, etc.

[0042] Through the photothermal effect of the photothermal photosensitizer 5, the photothermal photosensitizer 5 can be excited by low-power near-infrared light (heating light 9) to generate heat, control the reaction temperature, and achieve efficient and safe thermal cycling.

[0043] In a specific embodiment, the micro-reaction chamber 4 is a micro-reaction tube of various tubular shapes, preferably a silica capillary tube. The micro-reaction chamber 4 can not only directly extract and discharge the sample to be tested by air pressure, but also serve as a reaction space for photothermal amplification and real-time PCR detection, and can be quickly disassembled and replaced to achieve a fast and convenient sample collection function.

[0044] In a specific implementation, the optical fiber system further includes: a temperature monitor 6, which is used to collect the reaction temperature between the optical fiber probe 1 and the sample to be tested, and generate a temperature monitoring signal to achieve real-time monitoring and feedback of the temperature. The temperature monitor 6 is arranged inside the optical fiber probe 1, and the temperature monitor 6 is a fiber grating or interferometer sensing structure. Specifically, Figure 4 As shown, the temperature monitor 6 is a fiber Bragg grating, which monitors the reaction temperature by detecting the change of the refractive index of the liquid.

[0045] In a specific implementation, Figure 3 As shown, it also includes: an optical connection unit 10, an optical path coupling unit 11, a heat source light unit 12, a fluorescence excitation unit 13, a fiber grating temperature measurement unit 14 and a fluorescence signal monitoring unit 15.

[0046] The optical connection unit 10, the heat source light unit 12 and the optical path coupling unit 11 constitute a photothermal cycle amplification module, wherein the heat source light unit 12 is a 980nm pump laser.

[0047] The optical connection unit 10 , the fiber Bragg grating temperature measurement unit 14 and the optical path coupling unit 11 constitute a temperature monitoring module, wherein the fiber Bragg grating temperature measurement unit 14 is a fiber optical spectrum analyzer or an optical wavelength meter.

[0048] The optical connection unit 10, the fluorescence excitation unit 13, the fluorescence signal monitoring unit 15 and the optical path coupling unit 11 constitute a fluorescence detection module, wherein the fluorescence excitation unit 13 is a 465nm blue light source, and the fluorescence signal monitoring unit 15 is a spectrum analyzer.

[0049] Specifically, the optical connection unit 10 is connected to the optical fiber probe 1, and is used to transmit the excitation light 7 and the heating light 9, and collect the fluorescence signal 8 and the temperature monitoring signal.

[0050] Specifically, the optical path coupling unit 11 is respectively connected to the optical connection unit 10, the heat source light unit 12, the fluorescence excitation unit 13, the fiber grating temperature measurement unit 14 and the fluorescence signal monitoring unit 15, and is used to merge or decompose the excitation light 7, the heating light 9, the fluorescence signal 8 and the temperature monitoring signal.

[0051] Specifically, the heat source unit 12 transmits the heating light 9 to the photothermal photosensitizer 5 through the optical path coupling unit 11 and the optical connection unit 10. The heat source light unit 12 transmits the heating light 9 to the optical fiber probe 1 to excite the photothermal photosensitizer 5 to generate heat.

[0052] Specifically, the fluorescence excitation unit 13 emits the excitation light 7 to the optical fiber probe 1 through the optical path coupling unit 11 and the optical connection unit 10 .

[0053] Specifically, the fluorescence signal monitoring unit 15 receives the fluorescence signal 8 detected by the optical fiber probe 1 through the optical path coupling unit 11 and the optical connection unit 10, and demodulates the fluorescence signal 8 to achieve real-time monitoring of the qPCR process.

[0054] Specifically, the fiber Bragg grating temperature measuring unit 14 receives the temperature monitoring signal through the optical path coupling unit 11 and the optical connection unit 10 to monitor the reaction temperature between the fiber optic probe 1 and the sample to be measured.

[0055] The optical fiber system integrating photothermal amplification and real-time PCR detection provided in this application has the following advantages:

[0056] (1) The photothermal function of the optical fiber system provided in the present application relies on a photothermal photosensitizer. Compared with the direct heating method using high-power lasers, the heating method of the optical fiber system greatly expands the spectral utilization of the laser and reduces product costs. Due to the high photothermal conversion of the photothermal photosensitizer, the pump energy utilization efficiency is significantly improved, and a safe optical fiber amplification function can be achieved using only low-power near-infrared light. The heating originates from the optical fiber in the test liquid rather than the liquid directly absorbing the laser, and the thermal cycle is smooth and controllable.

[0057] (2) The fiber optic system provided in this application has integrated functions including: photothermal driven cyclic amplification, signal acquisition, temperature feedback, sample collection, microreactor and result reading, and the entire qPCR procedure can be implemented in one fiber optic system.

[0058] (3) The microreactor uses air pressure to extract and discharge samples, integrating the sample collection function into the system. This strategy is not only convenient, it can achieve rapid sample transfer, saving program time and resources, but also has the feature of "disposable after use", avoiding the possibility of environmental pollution.

[0059] (4) This application utilizes the long-distance signal transmission characteristics of optical fiber, and the proposed strategy realizes remote testing capabilities. This method can eliminate the need to deploy instruments in crowded biosafety laboratories and help realize distributed pathogen testing to respond to a possible new round of large-scale viral epidemics.

[0060] (5) Compared with conventional qPCR technology, the fiber optic system provided in the present application has comparable functions and utilities. It not only integrates amplification and detection functions, but also can perform the photothermal cycle amplification process in a shorter time, thereby speeding up the qPCR procedure.

[0061] (6) Different from conventional qPCR technology, the fiber optic system provided in this application uses a fiber optic probe that is not only a light transmission tool, but is also a partial tool for amplification and detection, which can effectively improve detection sensitivity and accuracy and improve efficiency.

[0062] (7) Unlike conventional qPCR technology, the fiber optic system provided in this application is portable and low-cost, does not rely on large laboratory instruments, and can be used for pathogen detection even in remote areas.

[0063] (8) The optical fiber system provided in the present application uses optical fiber as a tool for signal transmission. Ultra-long-distance detection can be achieved by customizing the length of the optical fiber. This remote diagnosis method greatly reduces the risk of working in dangerous environments.

[0064] (9) The optical fiber system provided in this application has a simple structure and is easy to operate. Even non-professionals can operate it, and it can realize data visualization and automated analysis through simple spectroscopy, which reduces the need for professional knowledge and skills and improves the efficiency and accuracy of epidemiological surveys.

[0065] (10) The optical fiber system provided in this application has the potential for distributed detection. qPCR procedures can be performed in multiple environments or spaces, and can be linked to a centralized computer through coupled optical fibers to further achieve multiple data analysis.

[0066] (11) The optical fiber system provided in this application has high scalability in terms of wavelength, space and time selectivity, and can be combined with a variety of sensing detection technologies to achieve integrated and accurate diagnosis of different pathogens.

[0067] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. At the same time, for those skilled in the art, according to the ideas of this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting this application.

Claims

1. An optical fiber system integrating photothermal amplification and real-time PCR detection, characterized in that: include: Fiber optic probes, photothermal photosensitizers, and micro-reaction chambers; The micro-reaction chamber uses air pressure to extract and discharge the sample to be tested, the optical fiber probe is arranged in the micro-reaction chamber, and the optical fiber probe reacts with the sample to be tested in the micro-reaction chamber; The photothermal photosensitizer is arranged inside or on the outer surface of the optical fiber probe, and the photothermal photosensitizer is used to receive heating light to generate heat to increase the temperature of the optical fiber probe, thereby controlling the reaction temperature between the optical fiber probe and the sample to be tested; The optical fiber probe is also used to receive excitation light, the excitation light excites the sample to be tested to generate a fluorescent signal, and the optical fiber probe is used to detect the fluorescent signal.

2. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 1, characterized in that: The optical fiber probe comprises a core and a cladding wrapping the core, and the core is used to transmit the heating light, the excitation light and the fluorescence signal.

3. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 1, characterized in that: The photothermal photosensitizer is a two-dimensional material, a metal ion, a noble metal or a metal complex.

4. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 1, characterized in that: The micro reaction chamber is the micro reaction tube.

5. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 1, characterized in that: The optical fiber system further comprises a temperature monitor arranged inside the optical fiber probe, and the temperature monitor is used to collect the reaction temperature between the optical fiber probe and the sample to be tested and generate a temperature monitoring signal.

6. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 5, characterized in that: The optical fiber system further comprises: An optical connection unit, connected to the optical fiber probe, for transmitting excitation light and heating light, and collecting fluorescence signals and temperature monitoring signals; An optical path coupling unit is connected to the optical connection unit and is used to combine or decompose the excitation light, the heating light, the fluorescence signal and the temperature monitoring signal.

7. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 6, characterized in that: The optical fiber system further comprises: The thermal light source unit transmits heating light to the photothermal photosensitizer through the optical path coupling unit and the optical connection unit.

8. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 6, characterized in that: The optical fiber system further comprises: The fluorescence excitation unit emits excitation light to the optical fiber probe through the optical path coupling unit and the optical connection unit.

9. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 6, characterized in that: The optical fiber system further comprises: The fluorescence signal monitoring unit receives the fluorescence signal detected by the optical fiber probe through the optical path coupling unit and the optical connection unit, and demodulates the fluorescence signal.

10. The optical fiber system integrating photothermal amplification and real-time PCR detection according to claim 6, characterized in that: The optical fiber system further comprises: The fiber grating temperature measuring unit receives the temperature monitoring signal through the optical path coupling unit and the optical connection unit, and monitors the reaction temperature between the fiber optic probe and the sample to be measured.

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