Integrated optical fiber embedded optofluidic biochip and application method

Abstract

The application relates to the field of on-site rapid detection technology, and discloses an integrated optical fiber embedded optical flow control biochip and an application method, which comprises an optical fiber embedded optical flow control biochip, a micro flow path system, a miniature all-optical fiber optical detection system and a signal and control system. The optical fiber embedded optical flow control biochip is fused with the miniature all-optical fiber optical detection system and the micro flow path system, the volume of the instrument is effectively reduced, accurate optical positioning and complex manufacturing and packaging processes are not needed, the feasibility of mass production of the biochip is greatly improved, the mobility, stability and accuracy of the integrated optical fiber embedded optical flow control biochip system are maximally improved, and a new technical approach is provided for realizing on-site rapid detection of target substances such as nucleic acids and proteins.

Description

Technical Field

[0001] This invention relates to an integrated fiber-optic embedded optofluidic biochip and its application method, and belongs to the field of rapid on-site testing technology. Background Technology

[0002] With the convergence of photonics and microfluidics, optofluidics has emerged as a new, multidisciplinary research field, demonstrating unique advantages, particularly in biochemical sensing. Optofluidic biochips, integrating microfluidic systems and advanced modern optics, are becoming at the forefront of development in precision medicine, biosafety, environmental monitoring, and food testing due to their unique advantages. Optofluidic biochips can flexibly combine and integrate various unit technologies on a controllable micro-platform. Combined with high-performance biomaterials and bioaffinity reaction principles, they can achieve highly sensitive, highly specific, and rapid, cost-effective detection of characteristic targets.

[0003] Optical methods, characterized by high sensitivity and good stability, have become the preferred on-chip detection methods in optofluidics. Fluorescence, spectrophotometry, chemiluminescence, and surface plasmon resonance (SPR) methods have all been applied to the analysis and detection of optofluidic systems. Among these, laser-induced fluorescence technology has become one of the most commonly used detection techniques in optofluidic systems due to its high sensitivity and accuracy, with detection limits even reaching the single-molecule level.

[0004] However, photofluidic biochip detection systems typically employ complex confocal optical systems to detect fluorescence signals. These systems are extremely complex, containing numerous optical separation elements and requiring precise design and precise optical positioning. Furthermore, due to their free-path design, the optical signal is susceptible to scattering or absorption by impurities in the solution. Even a slight misalignment of any optical separation element can disrupt the entire optical system, significantly impacting instrument stability and the accuracy of detection results. Additionally, while photofluidic biochips can be made very small, the extreme complexity of the optical system results in a large overall instrument size and high cost, making it difficult to achieve truly rapid on-site detection of targets. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. Therefore, in response to the above-mentioned problems, the object of the present invention is to provide an integrated fiber-optic embedded optofluidic biochip and its application method that not only simplifies the complex structure of traditional optical biochips but also improves light transmission efficiency.

[0006] In a first aspect, the present invention provides an integrated fiber-optic embedded optofluidic biochip, the chip comprising a functional fiber-optic biosensor and an optofluidic biochip, wherein the functional fiber-optic biosensor is embedded within the optofluidic biochip, and the functional fiber-optic biosensor serves as both a biosensing and identification element and an optical transducer, as well as a transmission device for excitation light and fluorescence.

[0007] Furthermore, the functionalized fiber optic biosensor uses an optical fiber with a special geometry for sensitive fluorescence detection and optical signal transmission of trace target substances based on the principle of homogeneous immunization. When used for sensitive fluorescence detection and optical signal transmission of trace target substances based on the principle of heterogeneous immunization, biorecognition molecules are modified on the surface of the optical fiber. The special geometry refers to the formation of a conical structure at one end of the optical fiber by corrosion with hydrofluoric acid.

[0008] Furthermore, the optofluidic biochip includes a microreaction and detection cell, a mixing pre-reaction cell, and a communicating vessel; the functionalized fiber optic biosensor is embedded in the microreaction and detection cell, and various reagents enter the microreaction and detection cell through the communicating vessel and the mixing pre-reaction cell.

[0009] Furthermore, it also includes a miniature all-fiber optical detection system, which is used to emit excitation light to the functionalized fiber optic biosensor. The excitation light propagates through the functionalized fiber optic biosensor by total internal reflection and forms an evanescent wave on the surface of the functionalized fiber optic biosensor. The evanescent wave excites fluorescent molecules near or bound to the surface of the functionalized fiber optic biosensor to emit fluorescence, and part of the fluorescence is coupled back to the functionalized fiber optic biosensor and collected.

[0010] Furthermore, the effective penetration depth of the evanescent wave is 100nm to 200nm.

[0011] Furthermore, the miniature all-fiber optical detection system includes an excitation source, an optical switch, an excitation light-introducing fiber, a fluorescence-collecting fiber, a reference light-introducing fiber, a filter, and a photodetector. The excitation light emitted by the excitation source is split into two paths by the optical switch. One path of excitation light is emitted through the excitation light-introducing fiber to the functionalized fiber optic biosensor. The fluorescence collected by the functionalized fiber optic biosensor is emitted through the fluorescence-collecting fiber to the filter to remove the excitation light and stray light before being detected by the photodetector. The other path of excitation light serves as the reference light and is emitted through the reference light-introducing fiber to the photodetector. The photodetector, based on the time-resolved effect of the optical switch, enables parallel detection of excitation light and fluorescence using the same photodetector.

[0012] Furthermore, it also includes the microfluidic system for inputting the sample to be tested into the photofluidic biochip. The microfluidic system includes microchannels, several microcontrollers, and micropumps. Each of the microcontrollers is respectively disposed on the microchannel to control the injection of buffer solution, sample to be tested, fluorescently labeled biorecognition molecule solution, and regeneration solution. The micropump provides power for reagent injection. By controlling the microcontrollers and micropumps, various reagents are sequentially introduced into the photofluidic biochip for mixing, reaction, and detection.

[0013] Furthermore, it also includes a signal and control system, which includes an electrical signal receiver and amplifier, a signal processing and control unit, and a signal display. The electrical signal receiver and amplifier is used to receive the electrical signal from the photodetector and process and amplify it. The signal processing and control unit is used to receive the signal from the electrical signal receiver and amplifier, process it, and display it through the signal display. In addition, the signal processing and control unit is also used to control the microcontroller valve and micropump of the microfluidic system.

[0014] Secondly, the present invention provides a nucleic acid detection method based on the aforementioned integrated fiber-optic embedded optofluidic biochip, comprising:

[0015] A functionalized fiber optic biosensor is embedded in a microreactor and a detection cell to form a fiber-embedded optofluidic biochip, and the functionalized fiber optic biosensor is connected to an excitation light-introducing fiber and a fluorescence-collecting fiber.

[0016] The buffer solution is introduced into the microreactor and detection cell by controlling the micro-control valve and micro-pump to obtain a baseline signal;

[0017] The micro-control valve and micro-pump control the sample containing the target nucleic acid and the hybridization chain reaction DNA detection reagent to be sequentially introduced into the mixing pre-reaction cell through the communicating vessel for a preset reaction time. At this time, the target nucleic acid initiates the hybridization chain reaction, and the fluorescent molecules labeled on the DNA separate from the quenching molecules.

[0018] The micropump is controlled to pass the sample to be tested into the microreactor and detection cell. The evanescent wave excites the fluorescent molecules labeled on DNA in the homogeneous phase. The excited fluorescence is coupled back to the functionalized fiber optic biosensor, collected and transmitted through the fluorescence collection fiber, and detected by the photodetector.

[0019] The signal detected by the photodetector is processed to obtain the fluorescence signal value of the target nucleic acid;

[0020] The linear relationship between target nucleic acid concentration and fluorescence signal value is used to achieve quantitative detection of target nucleic acid.

[0021] Thirdly, the present invention provides a protein immunoassay method based on the aforementioned integrated fiber-optic embedded optofluidic biochip, comprising:

[0022] A functionalized fiber optic biosensor modified with a primary antibody is embedded in a microreactor and a detection cell to form a fiber-embedded optofluidic biochip. The functionalized fiber optic biosensor is connected to an excitation light-introducing fiber and a fluorescence-collecting fiber.

[0023] The buffer solution is introduced into the microreactor and detection cell by controlling the micro-control valve and micro-pump to obtain a baseline signal;

[0024] The micro-control valve and micro-pump control the sample containing the target protein and the fluorescently labeled primary antibody to pass through the pre-reaction cell sequentially for a preset time, at which point the target protein and the primary antibody bind to form a complex.

[0025] A micropump is controlled to pass the protein-primary antibody complex into the microreaction and detection cell. The complex is captured by the primary antibody on the functionalized fiber optic biosensor. The fluorescently labeled primary antibody bound to the functionalized fiber optic biosensor is excited by evanescent wave. The excited fluorescence is coupled back to the functionalized fiber optic biosensor, collected and transmitted by the fluorescence collection fiber, and detected by the photodetector.

[0026] The detected signal is processed to obtain the fluorescence signal value of the target protein;

[0027] The linear relationship between the concentration of the target protein and the fluorescence signal value is used to achieve its quantitative detection.

[0028] Because the present invention adopts the above technical solution, it has the following characteristics:

[0029] 1. This invention organically integrates advanced fiber optics, optofluidic technology and biosensor chip technology to propose an integrated fiber-embedded optofluidic biochip. This integrated fiber-embedded optofluidic biochip achieves a tight combination of functional fiber biosensors and optofluidic chip system. The functional fiber biosensors serve as both biosensing and identification elements and optical transducers, as well as transmission devices for excitation light and fluorescence, greatly improving the transmission efficiency of light energy and reducing light loss, thereby efficiently improving the detection sensitivity of the instrument.

[0030] 2. The integrated fiber-embedded optical fluidic biochip of the present invention also includes a microfluidic system, a miniature all-fiber optical detection system, and a signal and control system. The fiber-embedded optical fluidic biochip system integrates sample introduction, mixing pre-reaction, micro-reaction, and detection, and combines the miniature all-fiber optical detection system and the microfluidic system. While effectively reducing the size of the instrument, it eliminates the need for precise optical positioning and complex manufacturing and packaging processes, greatly improving the feasibility of mass production of biochips. It maximizes the overall portability, stability, and accuracy of the integrated fiber-embedded optical fluidic biochip system, providing a new technical approach for the rapid on-site detection of target substances such as nucleic acids and proteins.

[0031] In summary, this invention can be widely applied to the detection of trace target substances. Attached Figure Description

[0032] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. In the drawings:

[0033] Figure 1 This is a schematic diagram of the integrated fiber-optic embedded optofluidic biochip according to an embodiment of the present invention, wherein the reference numerals are:

[0034] 1-Functionalized fiber optic biosensors;

[0035] 2-Optical fluidic biochip: 21-Fiber optic connector, 22-Microreaction and detection cell, 23-Mixed pre-reaction cell, 24-Communicating device;

[0036] 3-Microflow system: 31-Micro control valve one, 32-Micro control valve two, 33-Micro control valve three, 34-Micro control valve three, 35-Micro pump;

[0037] 4-Miniature all-fiber optical detection system: 41-Excitation source, 42-Optical switch, 43-Excitation light introduction fiber, 44-Fluorescence collection fiber, 45-Reference light introduction fiber, 46-Filter, 47-Photodetector;

[0038] 5-Signal and Control Systems: 51-Electrical signal receiver and amplifier, 52-Signal processing and control unit, 53-Signal display. Detailed Implementation

[0039] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0040] Although terms such as first, second, third, etc., may be used in this document to describe multiple elements, components, regions, layers, and / or segments, these elements, components, regions, layers, and / or segments should not be limited by these terms. These terms may be used only to distinguish one element, component, region, layer, or segment from another. Unless the context clearly indicates otherwise, terms such as "first," "second," and other numerical terms used herein do not imply order or sequence. Therefore, the first element, component, region, layer, or segment discussed below may be referred to as the second element, component, region, layer, or segment without departing from the teachings of the exemplary embodiments.

[0041] For ease of description, spatial relative terms may be used in the text to describe the relationship of one element or feature relative to another element or feature as shown in the figure. These relative terms include, for example, "inside," "outside," "middle," "outer," "below," "above," etc. Such spatial relative terms are intended to include different orientations of the device in use or operation, other than those depicted in the figure.

[0042] Existing integrated optical systems are extremely complex, with numerous separate optical components, requiring precise design and high precision in optical positioning. Furthermore, the overall instrument size is large and the cost is high, making it difficult to achieve truly rapid on-site detection of targets. This invention provides an integrated fiber-optic embedded optofluidic biochip and its application method. The chip incorporates a functionalized fiber-optic biosensor, which serves as both a biosensing and identification element and an optical transducer, as well as a transmission device for excitation light and fluorescence. Therefore, the integrated fiber-optic embedded optofluidic biochip provided by this invention not only simplifies the complex system structure of traditional optical biochips, significantly reduces instrument size, improves light transmission efficiency, and reduces light loss, but also enhances the sensitivity and accuracy of target substance detection, and improves the stability and portability of the instrument.

[0043] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0044] like Figure 1 As shown, the integrated fiber-optic embedded optofluidic biochip provided in this embodiment includes a functional fiber-optic biosensor 1, an optofluidic biochip 2, a microfluidic system 3, a miniature all-fiber optical detection system 4, and a signal and control system 5.

[0045] A functionalized fiber optic biosensor 1 is embedded in an optofluidic biochip 2 to form a fiber-embedded optofluidic biochip. The functionalized fiber optic biosensor 1 serves as both a biosensing and recognition element and an optical transducer, as well as a transmission device for excitation light and fluorescence.

[0046] The microfluidic system 3 is used to acquire the sample to be tested and input it into the photofluidic biochip 2.

[0047] The miniature all-fiber optical detection system 4 is used to emit excitation light to the functional fiber biosensor 1. The excitation light forms an evanescent wave in the functional fiber biosensor 1. The evanescent wave excites fluorescent molecules near or bound to the surface of the functional fiber biosensor 1 to emit fluorescence, and collects the fluorescence excited by the sample to be tested.

[0048] The signal and control system 5 is used to automatically control the microflow system 3 and process the electrical signals collected by the miniature all-fiber optical detection system 4 to generate visualized fluorescence signal curves, etc.

[0049] In a preferred embodiment of the present invention, the miniature all-fiber optical detection system 4 includes an excitation light source 41, a 1×2 optical switch 42, an excitation light introduction fiber 43, a fluorescence collection fiber 44, a reference light introduction fiber 45, a filter 46, and a photodetector 47, wherein:

[0050] The excitation source 41 is connected to a 1×2 optical switch 42 via an optical fiber. The excitation light emitted by the excitation source 41 is split into two beams after passing through the 1×2 optical switch 42. One beam of excitation light is transmitted to the functionalized fiber optic biosensor 1 via the excitation light guide fiber 43. The fluorescence collected by the functionalized fiber optic biosensor 1 is transmitted to the filter 46 via the fluorescence collection fiber 44 to filter out the excitation light and stray light. The filter 46 then detects and converts the excitation light and stray light into a measurable electrical signal by the photodetector 47. The other beam of excitation light serves as a reference light and is transmitted to the photodetector 47 via the reference source guide fiber 45 for detection. The reference light is used to correct the fluctuation of the excitation source 41 and improve the detection stability of the system.

[0051] Furthermore, the wavelength and power of the excitation source 41 can be set according to the actual sample to be tested, and are not limited here.

[0052] Furthermore, filter 46 can be a bandpass filter.

[0053] Furthermore, based on the time-resolved effect of the 1×2 optical switch 42, the photodetector 47 can realize the parallel detection of excitation light and fluorescence. That is, based on the time-resolved effect, the fluorescence excited by the reference light and the excitation light alternately enters the photodetector 47, thereby realizing the parallel detection of the two types of light.

[0054] In a preferred embodiment of the present invention, the microfluidic system 3 is used for the introduction and mixing of various reagents such as buffer solutions, test samples, and regeneration solutions. It includes microchannels, microcontroller valve 31, microcontroller valve 32, microcontroller valve 33, microcontroller valve 34, and micropump 35. Microcontroller valves 31, 32, 33, and 34 are respectively positioned on the microchannels to control the introduction of reagents such as buffer solutions, test samples, fluorescently labeled biorecognition molecule solutions, and regeneration solutions. Micropump 35 provides power for reagent introduction. Through the control of each microcontroller valve and micropump, various reagents such as buffer solutions, test samples, and regeneration solutions are sequentially introduced into the photofluidic biochip 2 for mixing, reaction, and detection, thereby achieving the detection of target substances. It should be noted that the number of microcontroller valves in this example is set to four; however, this is not a limitation and can be adjusted as needed.

[0055] In a preferred embodiment of the present invention, the optofluidic biochip 2 integrates sample preparation, reaction, and detection functions for the mixing, reaction, and detection of target substances. It includes an optical fiber connector 21, a micro-reaction and detection cell 22, a mixing pre-reaction cell 23, and a 1×4 communicating vessel 24. A functionalized optical fiber biosensor 1 is embedded in the micro-reaction and detection cell 22 and connected to an excitation light-introducing optical fiber 43 and a fluorescence-collecting optical fiber 44 via the optical fiber connector 21. Various reagents, under the control of the micro-control valves and micro-pumps 35 of the microfluidic system 3, sequentially enter the micro-reaction and detection cell 22 through the mixing pre-reaction cell 23 via the communicating vessel 24.

[0056] In use, in the sample introduction area, various reagents and test samples are sequentially introduced into the mixing pre-reaction cell 23 under the control of microvalve and micropump of the microfluidic system 3. After mixing and pre-reaction, they enter the micro-reaction and detection cell 22, which is embedded with a functionalized fiber optic biosensor 1. Unlike traditional biochips, due to the limited penetration depth of evanescent waves, the reaction and detection of the fused fiber optic optofluidic chip are carried out simultaneously, eliminating the need to separate the reaction zone and the detection zone. This effectively saves detection time and improves detection efficiency. After the reaction is completed, the waste liquid is discharged by the microfluidic system.

[0057] In a preferred embodiment of the present invention, the functionalized fiber optic biosensor 1 is embedded in the optofluidic chip 2 and directly connected to the excitation light-introducing fiber and the fluorescence-collecting fiber via the fiber optic connector 21. The excitation light enters the functionalized fiber optic biosensor 1 through the excitation light-introducing fiber, propagates therein by total internal reflection, and forms an evanescent wave on the surface of the functionalized fiber optic biosensor 1. Its effective penetration depth is approximately 100 nm to 200 nm. The evanescent wave excites fluorescent molecules of the sample to be tested near or bound to the surface of the functionalized fiber optic biosensor 1 to emit fluorescence. Part of the fluorescence is coupled back to the functionalized fiber optic biosensor 1, collected and transmitted by it, and enters the fluorescence-collecting fiber. After being detected by the photodetector 47, it is converted into a measurable electrical signal.

[0058] Furthermore, the functionalized fiber optic biosensor 1 consists of an optical fiber with a special geometry, and biorecognition molecules can be modified on its surface for sensitive fluorescence detection and optical signal transmission of trace target substances based on homogeneous or heterogeneous immune principles. The special geometry refers to the optical fiber being etched into a tapered structure by hydrofluoric acid. For example, the diameter of the optical fiber can be etched from 600 micrometers to 220 micrometers to form a tapered structure. This is just one example, and it is not limited to this. It can be set according to actual needs.

[0059] In a preferred embodiment of the present invention, the signal and control system 5 is used to convert the photoelectric converter into electrical signals for collection and processing, generating and displaying a visualized fluorescence signal curve. The signal and control system 5 includes an electrical signal receiver and amplifier 51, a signal processing and control unit 52, and a signal display 53. The electrical signal receiver and amplifier 51 receives, processes, and amplifies the electrical signal from the photodetector 47. The signal processing and control unit 52 receives and processes the signal from the electrical signal receiver and amplifier 51, and displays the processing result on the signal display 53. Simultaneously, the signal processing and control unit 52 is also used for controlling micro-control valves and micro-pumps 34 in the microfluidic system 3.

[0060] In summary, the integrated fiber-optic embedded optofluidic chip of this invention integrates sample preparation, reaction, and detection functions into one unit. It is formed by embedding a functionalized fiber-optic biosensor into the optofluidic chip. This integrated fiber-optic embedded optofluidic biochip achieves a tight integration of the functionalized fiber-optic biosensor and the optofluidic chip system. The functionalized fiber-optic biosensor serves as both a biosensing and identification element and an optical transducer, as well as a transmission device for excitation light and fluorescence, greatly improving the light energy transmission efficiency and reducing light loss, thereby significantly improving the detection sensitivity of the instrument.

[0061] The application method of the integrated fiber-optic embedded optical fluidic chip of the present invention will be described in detail below through specific embodiments. It should be noted that although Embodiment 1 and Embodiment 2 are illustrated using nucleic acid and organic matter detection as examples, those skilled in the art should understand that the biochip in these embodiments can also be used for other biochemical experiments that require optical detection, and this is just one example, not a limitation.

[0062] Example 1: This example provides a nucleic acid detection method based on an integrated fiber-optic embedded optofluidic biochip, which detects target nucleic acids based on the DNA hybridization chain reaction principle, including:

[0063] S1. Embed the functionalized fiber optic biosensor 1 into the microreactor and detection cell 22 to form a fiber-embedded optofluidic biochip 2, and connect the functionalized fiber optic biosensor 1 to the excitation light introduction fiber 43 and the fluorescence collection fiber 44 through the fiber optic connector 21.

[0064] S2. Control the micro-control valve 31 and micro-pump 35 to pass the buffer solution into the micro-reaction and detection cell 22 to obtain the baseline signal.

[0065] S3, along with microcontrollers 32 and 33 and micropump 35, sequentially introduce the test sample containing the target nucleic acid and the hybridization chain reaction DNA detection reagent into the mixing pre-reaction cell 23 via the communicating vessel 24. The reaction lasts for 5 to 3 minutes. At this time, the target nucleic acid initiates the hybridization chain reaction, and the fluorescent molecules labeled on the DNA separate from the quenching molecules.

[0066] S4. The sample to be tested is introduced into the micro-reaction and detection cell 22 using the micropump 35. The fluorescent molecules labeled on the DNA in the homogeneous phase are excited by evanescent wave. The excited fluorescence is coupled back to the functionalized fiber optic biosensor 1, collected and transmitted by the fluorescence collection fiber 44, detected by the photodetector 47, and displayed on the signal display 53 after signal processing. This is the fluorescence signal value of the target nucleic acid.

[0067] S5. Quantitative detection of target nucleic acid is achieved by utilizing the linear relationship between target nucleic acid concentration and fluorescence signal value.

[0068] Example 2: This example provides a protein immunoassay method based on an integrated fiber-optic embedded optofluidic biochip, implemented using a sandwich immunoassay mechanism, including:

[0069] S1. Embed the primary antibody-modified functional fiber optic biosensor 1 into the microreaction and detection cell 22 to form a fiber-embedded optofluidic biochip 2, and connect it to the excitation light introduction fiber 43 and the fluorescence collection fiber 44 through the fiber optic connector 21.

[0070] S2. The buffer solution is introduced into the micro-reaction and detection cell 22 through the micro-control valve 31 and the micro-pump 35 to obtain the baseline signal.

[0071] S3. Using microcontrol valve 2 32, microcontrol valve 33 and micropump 35, the sample containing the target protein and the fluorescently labeled primary antibody are sequentially passed through the pre-reaction cell and reacted for 5 to 3 minutes. At this time, the target protein and the primary antibody bind to form a complex.

[0072] S4. The protein-primary antibody complex is introduced into the microreaction and detection cell 22 using the micropump 35. The complex is captured by the primary antibody on the functionalized fiber optic biosensor 1. The fluorescently labeled primary antibody bound to the functionalized fiber optic biosensor 1 is excited by evanescent wave excitation. The excited fluorescence is coupled back to the functionalized fiber optic biosensor 1, collected and transmitted by the fluorescence collection fiber 44, detected by the photodetector 47, and displayed on the signal display 53 after signal processing, which is the fluorescence signal value of the target protein.

[0073] S4. Quantitative detection is achieved by utilizing the linear relationship between the concentration of the target protein and the fluorescence signal value.

[0074] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In the description of this specification, the terms "a preferred embodiment," "furthermore," "specifically," "in this embodiment," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the embodiments in this specification. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An integrated fiber-optic embedded optofluidic biochip, characterized in that, The chip includes a functionalized fiber optic biosensor and an optofluidic biochip. The functionalized fiber optic biosensor is embedded in the optofluidic biochip and serves as both a biosensing and recognition element and an optical transducer, as well as a transmission device for excitation light and fluorescence. It also includes a miniature all-fiber optical detection system, which is used to emit excitation light to the functionalized fiber optic biosensor. The excitation light propagates through the functionalized fiber optic biosensor by total internal reflection and forms an evanescent wave on the surface of the functionalized fiber optic biosensor. The evanescent wave excites fluorescent molecules near or bound to the surface of the functionalized fiber optic biosensor to emit fluorescence, and part of the fluorescence is coupled back to the functionalized fiber optic biosensor and collected. The miniature all-fiber optical detection system includes an excitation source, an optical switch, an excitation light-introducing fiber, a fluorescence-collecting fiber, a reference light-introducing fiber, a filter, and a photodetector. The excitation light emitted by the excitation source is split into two paths by the optical switch. One path of excitation light is transmitted through the excitation light-introducing fiber to the functionalized fiber optic biosensor. The fluorescence collected by the functionalized fiber optic biosensor is transmitted through the fluorescence-collecting fiber to the filter to remove the excitation light and stray light before being detected by the photodetector. The other path of excitation light serves as the reference light and is transmitted through the reference light-introducing fiber to the photodetector. The photodetector, based on the time-resolved effect of the optical switch, enables parallel detection of excitation light and fluorescence using the same photodetector. It also includes a microfluidic system for inputting the sample to be tested into the photofluidic biochip. The microfluidic system includes a microchannel, several microcontrollers and a micropump. Each of the microcontrollers is respectively set on the microchannel to control the injection of buffer solution, sample to be tested, fluorescently labeled biorecognition molecule solution and regeneration solution. The micropump provides power for reagent injection. By controlling the microcontrollers and micropump, various reagents are sequentially introduced into the photofluidic biochip for mixing, reaction and detection. It also includes a signal and control system, which includes an electrical signal receiver and amplifier, a signal processing and control unit, and a signal display. The electrical signal receiver and amplifier is used to receive the electrical signal from the photodetector and process and amplify it. The signal processing and control unit is used to receive the signal from the electrical signal receiver and amplifier, process it, and display it through the signal display. In addition, the signal processing and control unit is also used to control the microcontroller valve and micropump of the microfluidic system.

2. The integrated fiber-optic embedded optofluidic biochip according to claim 1, characterized in that, The optofluidic biochip includes a microreaction and detection cell, a mixing pre-reaction cell, and a communicating vessel; the functionalized fiber optic biosensor is embedded in the microreaction and detection cell, and various reagents enter the microreaction and detection cell through the communicating vessel and the mixing pre-reaction cell.

3. The integrated fiber-optic embedded optofluidic biochip according to claim 2, characterized in that, The effective penetration depth of evanescent waves is 100 nm to 200 nm.

4. A nucleic acid detection method based on the integrated fiber-optic embedded optofluidic biochip according to any one of claims 1 to 3, characterized in that, include: A functionalized fiber optic biosensor is embedded in a microreactor and a detection cell to form a fiber-embedded optofluidic biochip, and the functionalized fiber optic biosensor is connected to an excitation light-introducing fiber and a fluorescence-collecting fiber. The buffer solution is introduced into the microreactor and detection cell by controlling the micro-control valve and micro-pump to obtain a baseline signal; The micro-control valve and micro-pump control the sample containing the target nucleic acid and the hybridization chain reaction DNA detection reagent to be sequentially introduced into the mixing pre-reaction cell through the communicating vessel for a preset reaction time. At this time, the target nucleic acid initiates the hybridization chain reaction, and the fluorescent molecules labeled on the DNA separate from the quenching molecules. The micropump is controlled to pass the sample to be tested into the microreactor and detection cell. The evanescent wave excites the fluorescent molecules labeled on DNA in the homogeneous phase. The excited fluorescence is coupled back to the functionalized fiber optic biosensor, collected and transmitted through the fluorescence collection fiber, and detected by the photodetector. The signal detected by the photodetector is processed to obtain the fluorescence signal value of the target nucleic acid; The linear relationship between target nucleic acid concentration and fluorescence signal value is used to achieve quantitative detection of target nucleic acid.

5. A protein immunoassay method based on an integrated fiber-optic embedded optofluidic biochip as described in any one of claims 1 to 3, characterized in that, include: A functionalized fiber optic biosensor modified with a primary antibody is embedded in a microreactor and a detection cell to form a fiber-embedded optofluidic biochip. The functionalized fiber optic biosensor is connected to an excitation light-introducing fiber and a fluorescence-collecting fiber. The buffer solution is introduced into the microreactor and detection cell by controlling the micro-control valve and micro-pump to obtain a baseline signal; The micro-control valve and micro-pump control the sample containing the target protein and the fluorescently labeled primary antibody to pass through the pre-reaction cell sequentially for a preset time, at which point the target protein and the primary antibody bind to form a complex. A micropump is controlled to pass the protein-primary antibody complex into the microreaction and detection cell. The complex is captured by the primary antibody on the functionalized fiber optic biosensor. The fluorescently labeled primary antibody bound to the functionalized fiber optic biosensor is excited by evanescent wave. The excited fluorescence is coupled back to the functionalized fiber optic biosensor, collected and transmitted by the fluorescence collection fiber, and detected by the photodetector. The detected signal is processed to obtain the fluorescence signal value of the target protein; The linear relationship between the concentration of the target protein and the fluorescence signal value is used to achieve its quantitative detection.

Images