Multichannel optical fiber biosensor based on fluorescence detection

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a multi-channel optical fiber biosensor based on fluorescence detection, optical fiber sensing units can be cascaded on the same optical fiber, and the sensing units are realized through a packaging structure adopting 3D printing resin. Quantum dot modification, functionalization processing and sensing detection on the surface of the sensing area of the optical fiber sensing unit can be completed in the packaging structure, and integration is achieved. Different antibody materials can be modified on quantum dot materials with different particle sizes so as to obtain fluorescence signals with different emission wavelengths, and qualitative and quantitative detection of various microorganisms and viruses is realized according to the variation of fluorescence intensity of a corresponding emission peak. The sensor is low in cost, short in preparation time and simple in signal acquisition and processing, the packaging structure can ensure the stability of the sensor, the sensor is convenient to carry, and the sensor can be applied to the environmental protection field such as outdoor water quality real-time monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and more specifically relates to a multi-channel optical fiber biosensor based on fluorescence detection. The encapsulation of the sensing unit is realized through a microfluidic encapsulation structure using 3D printing resin. The modification process and sensing detection of the optical fiber sensing unit can be integrated. By fixing corresponding antibodies to quantum dots with different particle sizes modified on the surface of the sensing unit, qualitative and quantitative detection of various microorganisms and viruses can be achieved. It can be used in environmental protection fields such as water quality monitoring. Background Art

[0002] Biological detection technology is one of the key technologies developed in the current detection field. Nowadays, the detection technology for microorganisms has been very mature. The detection of microorganisms and viruses in water quality is an important basis for ensuring water quality hygiene and human healthy life. If the content of microorganisms in water cannot be correctly detected, it may cause great harm to water quality hygiene and human health. Under this background, how to quickly and accurately detect whether the content of harmful microorganisms and viruses in water quality exceeds the standard has attracted extensive research and attention in the industry.

[0003] To quickly detect the existing environmental water quality on-site, it is required that the integrated detection instrument and equipment must be able to qualitatively and quantitatively detect bacteria and viruses in water specifically. Based on PCR and fluorescence-labeled microscopy imaging technology, it can accurately detect whether the bacteria and viruses to be detected exist. By detecting the intensity, lifetime of fluorescence or the number of fluorescence labels, qualitative and quantitative detection can be achieved, which well solves the problem of qualitative and quantitative detection of microorganisms. The antibody-based targeted labeling detection technology can make the labeled antibody bind to the corresponding microorganism or virus to achieve a specific directional change in the optical signal, which fully solves the problem of cross-sensitivity of sensing signals and greatly reduces the risk and hidden danger of detection errors. However, although the existing microorganism detection technologies can achieve rapid, accurate and specific detection of microorganisms, most of them cannot achieve on-site real-time detection. Currently, various methods of microorganism detection require precise instruments and equipment. For on-site detection, these precise instruments and equipment need to be transported. Most of the microorganism detection equipment has a low integration level, so the vibration generated during transportation is likely to damage the internal structure and circuit system of the equipment; many precise detection instruments and equipment cannot work properly in the outdoor on-site environment due to environmental influence, which in turn affects the detection results. Only by solving the problems of detection system integration and anti-interference ability, this biological detection technology encryption technology is expected to be practically applied in the future. Summary of the Invention

[0004] Aiming at the problem that the existing biological sensing system cannot achieve rapid and specific qualitative and quantitative detection of microorganisms outdoors on-site in real time, the present invention proposes a multi-channel optical fiber biosensor based on fluorescence detection.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A multi-channel fiber optic biosensing system based on fluorescence detection, which includes a laser light source module (including a laser light source), a microfluidic power module, a microfluidic channel sensing unit (including a 3D printed resin microfluidic channel encapsulation structure and an internal fiber optic sensing unit), a signal acquisition module (including an optical fiber spectrometer), a computer, and a waste liquid treatment module. The laser light source in the laser light source module can achieve narrow linewidth laser output, and is transmitted to the fiber optic sensing unit through an optical fiber to provide an excitation light signal for the fluorescent material modified on the unit. The fiber optic sensing unit is encapsulated in a 3D printed resin microfluidic channel encapsulation structure to form a microfluidic channel. The 3D printed resin microfluidic channel encapsulation structure has input and output channels for inputting and outputting liquid or air respectively. The optical fiber spectrometer in the signal detection module can detect the fluorescent signals emitted by quantum dots of different particle sizes and present the spectrum of wavelength and fluorescence intensity by the computer connected thereto. The microfluidic power module can control the flow rate of the liquid or gas input into the sensing system, thereby controlling the reaction speed and preventing damage to the sensing unit. The waste liquid treatment module is used to collect the used reaction solution or recycle the sample liquid. The fiber optic sensing unit is inserted into the 3D printed resin microfluidic channel encapsulation structure and is encapsulated at the insertion end and the outlet end with an ultraviolet curable adhesive. Multiple fiber optic sensing units are encapsulated in the corresponding structures and cascaded on the same optical fiber to form an optical signal transmission route, and are connected through a microfluidic pipeline to form a microfluidic route. The microfluidic power module is used for liquid transportation. Different fiber optic sensing units are respectively modified with corresponding quantum dots, functionalized, and combined with different analytes. Different fluorescent emission signals are generated by laser light source excitation, and then the analyte sample liquid is sent into the microfluidic route for calibration and detection. The optical fiber spectrometer receives the optical signal, and finally presents the spectrum at the computer terminal and realizes the specific and rapid qualitative and quantitative detection of the analyte according to the calibration curve.

[0007] For further optimization of this technical solution, the fiber optic sensing unit can adopt structures such as tapered optical fiber and D-shaped optical fiber to improve the sensitivity of sensing. An interferometric fiber optic sensing unit can also be used for encapsulation to prepare the sensing unit.

[0008] For further optimization of this technical solution, the 3D printed resin microfluidic channel structure is a structure formed by photocuring 3D printing, which has a micron-level precision and structure controllability, and has good transparency to observe whether there is any abnormality during the detection of the fiber optic sensing unit, and has more photocuring materials available for manufacturing, providing the encapsulation structure with more adaptability to liquids.

[0009] The technical solution is further optimized, and the 3D printing structure can adopt a more special microfluidic structure design to achieve the control of flow rate and microfluidic route, which can meet the detection needs of more occasions or more special substances to be tested.

[0010] The technical solution is further optimized. The optical fiber sensing unit can use materials with good binding properties and can achieve different fluorescence emissions using the same excitation light, such as quantum dots, carbon dots, etc., which are fixed on the surface of the optical fiber by doping, physical deposition, chemical modification, etc., and generate fluorescence through the action of evanescent waves and return to the core to be transmitted to the spectrometer. By modifying specific reactants on fluorescent substances with different emission wavelengths, it is used to specifically detect viruses or microorganisms to be tested. By inputting samples into multiple sensing sites of the microfluidic circuit through a power system, specific qualitative and quantitative detection of multiple microorganisms and viruses can be achieved for the same sample.

[0011] This technical solution is further optimized. The UV curing glue used for bonding can be replaced by an adhesive with hydrophobic and waterproof properties, so that the sensor can still maintain structural stability in different external environments. The low refractive index curing glue will also have a smaller stress effect on the sensor unit. The type of packaging curing glue and packaging conditions can be adjusted according to the detection environment.

[0012] With further optimization of this technical solution, the fiber optic sensing unit modified with indirect reactants can be used to monitor the various rates of some substances in the manufacturing process, and can then be used in the manufacture of chemical products and some industrial production, not limited to biological detection, thus broadening the application field.

[0013] Different from the prior art, the above technical solution has the following advantages:

[0014] 1. The present invention utilizes the flexibility of 3D printing technology and the performance of resin materials to realize the manufacture of customized microfluidic packaging structures, and the material of the structure can be replaced according to the needs of the occasion to adapt to the working environment. The manufacturing method is not limited to melt curing printing, and light curing and other methods can be used. The manufacturing can be completed without opening a mold, which greatly shortens the time and cost of traditional microfluidic chip manufacturing and solves the problem of high cost and long time consumption of traditional microfluidic structure manufacturing.

[0015] 2. The present invention adopts an integrated packaging structure to manufacture sensors, and can connect multiple packaging structures through microfluidic pipes to form a microfluidic route that runs through multiple sensing units. By inputting the same sample liquid, multi-channel simultaneous detection of specific biological detection of different analytes can be achieved at multiple sites. The packaging structure can also effectively protect the optical fiber sensing unit from the influence of factors such as vibration during transportation, which is convenient for long-distance transportation and real-time measurement outdoors, solving the problem that traditional optical fiber biochemical sensors have poor stability and are difficult to perform real-time measurements.

[0016] 3. The present invention cascades multiple fiber optic sensing units on a single sensing optical fiber. The same excitation light energy is used to excite quantum dots modified with different reactants on different sensing units to generate different fluorescence emission peaks corresponding to substances for measurement, and the signals between the sensing units will not interfere with each other. The quantum dots have a high quantum yield and a fast response speed of the fluorescence signal, and can simultaneously and specifically and rapidly monitor the qualitative and quantitative analysis of multiple substances to be measured in real time. Moreover, only the fluorescence signal of one optical fiber needs to be monitored, which greatly reduces the difficulty of signal demodulation and processing, optimizes the problem that traditional fiber optic biosensors require multiple sensors for detection, and simplifies the process of signal demodulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of a multi-channel fiber optic biosensing system;

[0018] Figure 2 It is a schematic diagram of the principle of fluorescence biological detection;

[0019] Figure 3 It is a cross-sectional view of the microfluidic packaging component in the transverse direction;

[0020] Figure 4 It is a cross-sectional view of the microfluidic packaging component in the longitudinal direction;

[0021] Figure 5 It is a structure and detection diagram of the fiber optic sensing unit.

[0022] Description of the reference numerals:

[0023] 1: Microfluidic power module 2: Microfluidic pipeline 3: 3D printing resin microfluidic channel structure 4: Laser light source module 5: Computer 6: Signal acquisition module 7: Waste liquid treatment module 8: Quantum dots of different particle sizes modified with reactants 9: Different substances to be measured 10: Quantum dots combined with substances to be measured 11: Transmission optical fiber 12: Extension arm

[0024] 13: Optical fiber fixing groove 14: Base 15: Optical fiber packaging channel 16: Microfluidic channel 17: Curing glue

[0025] 18: Fiber optic sensing area 19: Core 20: Quantum dots modified with reactants or antibodies 21: Microorganisms or viruses

[0026] 22: Water molecules DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To describe in detail the technical content, structural features, achieved objectives and effects of the technical solution, the following will be described in detail with specific embodiments in conjunction with the accompanying drawings.

[0028] Refer to Figure 1As shown in the figure, it is a schematic diagram of a multi-channel fiber optic biosensing system. The fiber optic biosensing system includes a microfluidic power module 1, microfluidic pipelines 2, a 3D printed resin microfluidic channel structure 3, a laser light source module 4, a computer 5, a signal acquisition module 6, and a waste liquid treatment module 7. The microfluidic power module 1 injects the reaction solution or sample solution from the microfluidic pipeline 2 into the entire microfluidic route through a peristaltic pump, flowing through each 3D printed resin microfluidic channel structure 3. Gas can also be injected into the microfluidic pipeline 2 through the power system to achieve drying and removal of excess waste liquid. The injected solution can combine with the end face of the processed fiber optic sensing unit sensing area (the specific structure is as shown in Figure 5 ) in the 3D printed resin microfluidic channel structure 3 and be modified with fluorescent quantum dots, functionalized, and used to detect biochemical substances. The laser light source module 4, the fiber optic sensing unit in the 3D printed resin microfluidic channel structure 3, and the signal processing module 6 are connected by a transmission fiber 11 to form an optical path for transmitting and receiving optical signals. The fluorescent substance on the sensing area of the fiber optic sensing unit generates fluorescence signals through the excitation light provided by the laser light source module 4. The reaction substances modified on the quantum dots combine with the object to be measured, resulting in the quenching of the fluorescence signal, which returns to the fiber core in the form of an evanescent wave and is transmitted.

[0029] The fluorescence signal returns to the fiber core and is transmitted to the fiber optic spectrometer in the signal processing module 6 to complete the acquisition and processing of the signal. The connected computer 5 presents the fluorescence signal in the form of a spectrum, fits the calibration curve, and calculates the concentration of the current object to be measured based on the calibration curve and the intensity of the corresponding fluorescence emission peak to complete the specific detection of qualitative and quantitative analysis. The reaction solution or the sample solution after detection is discharged by the microfluidic power system 1 to the waste liquid treatment module 7 for collection and unified treatment. This system can not only integrate the processes of modification, functionalization, and detection, but also has good portability, making it suitable for on-site real-time monitoring.

[0030] Refer to Figure 2 As shown in the figure, it is a schematic diagram of fluorescence biological detection principle. Fluorescent materials can be modified on the surface of the optical fiber through physical deposition, chemical modification, sputtering thin film, doping, etc. Quantum dots not only have good fluorescence quantum yields, but also can change their fluorescence emission wavelengths without changing their excitation wavelengths just by changing their particle sizes. Therefore, different reactants can be modified on the surface of quantum dots to specifically bind to different objects to be measured. Quantum dots 8 with different particle sizes modified with reactants generate fluorescence in different emission bands after being excited by excitation light. Since quantum dots with different particle sizes are respectively modified on independent sensing units, there will be no problem of cross-sensitivity during the detection process, and the finally collected fluorescence signals will not be crosstalked.

[0031] Quantum dots with different particle sizes modifying different reactants will bind to different analytes 9 during the detection process. The binding of the analyte will cause quenching of the fluorescence emission of the corresponding quantum dots. The fluorescence intensity of the quantum dots 10 bound to the analyte will decrease to varying degrees, and the degree of decrease depends on the binding amount of the analyte. Therefore, calibration can be carried out according to the fluorescence intensity and the binding amount of the analyte, and qualitative and quantitative detection of the analyte can be achieved based on the change in fluorescence intensity.

[0032] Please refer to Figure 3 and Figure 4 which are the transverse sectional view and the longitudinal sectional view of the microfluidic encapsulation component. Its main function is to place the transmission optical fiber at the center position of the structure and fix it with glue, and fix the optical fiber sensing unit connected to the transmission optical fiber at the center position inside the microfluidic channel for the detection of the liquid to be measured. A preferred embodiment of the optical fiber biosensor microfluidic encapsulation structure of the present invention is adopted. The design and physical realization of the microfluidic encapsulation structure are achieved by using 3D printing technology. The base 14 of this embodiment with dimensions of 30×16×1.5 mm serves as a support, with a total height of 6 mm. The optical fiber groove 13 is a semi-cylindrical groove with a diameter of 0.3 mm and a length of 0.5 mm. When placing a single-mode transmission optical fiber with a diameter of 125 μm with a cladding on the optical fiber groove 13, it can limit its horizontal movement and prevent the transmission optical fiber 11 from shifting due to vibrations or other reasons during the encapsulation process of the structure, resulting in a change in the position of the sensing unit inside, playing a role in restricting the degrees of freedom. The size of the groove can be adjusted according to the type and size of the transmission optical fiber 11. The encapsulation is sealed with a curing glue 17. In order to facilitate rapid encapsulation, an ultraviolet curing glue is selected, which can be cured within 3 minutes under the action of an ultraviolet lamp and has good waterproof performance to prevent liquid from seeping out from the inside. The optical fiber encapsulation channel 15 is a square cavity structure with dimensions of 1.2×1.2 mm and a length of 4 mm, which can introduce the transmission optical fiber 11 from both sides and can effectively encapsulate the sensing area of the optical fiber sensing unit completely inside the component, and there is enough space to prevent the optical fiber structure from fitting against the inner wall of the structure and affecting the performance. The size can be adjusted according to the size of the optical fiber sensing unit.

[0033] The microfluidic channel 16 is a square cavity structure with dimensions of 1.2×1.2 mm and a length of 4 mm, which is the same as the size of the optical fiber encapsulation channel 15, facilitating the 3D printer to ensure the printing accuracy during the printing process. The size can be adjusted accordingly according to the flow rate required by the microfluidics. The extension arm 12 is an extension structure with a length of 16 mm, located at both ends of the encapsulation structure respectively. Its main function is to reinforce the transmission optical fibers on both sides of the optical fiber sensing unit after the encapsulation is completed. The curing glue at the end points of the extension arm can effectively ensure that the optical fiber structure will not break due to external vibrations, jitters or external forces generated during the structure movement, ensuring the stability of the optical fiber sensing unit.

[0034] Refer to Figure 5As shown, it is the structure and detection diagram of the fiber optic sensing unit. The fiber optic sensing area 18 uses the fiber with the etched cladding as the substrate. In the etched area, by means of physical deposition, chemical modification, etc., the quantum dots 20 modified with reactants or antibodies are fixed on the surface of the fiber optic sensing area. The excitation light is input from the light source through the fiber core 19 and excites the quantum dots modified on the surface of the fiber optic sensing area to generate fluorescence signals in the form of evanescent waves. The sample solution with microorganisms or viruses 21 is input into the microfluidic channel through the microfluidic power system. When passing through the sensing area of the fiber optic sensing unit, it is specifically captured by the quantum dots with reactants or antibodies and undergoes a fluorescence quenching reaction. Since the reactants or antibodies do not have specificity for water molecules 22 and other microorganisms or viruses, water molecules and non-specific substances will not be captured, and then flow to the next fiber optic sensing unit for detection on the other side of the microfluidic channel. The quantum dots modified on each fiber optic sensing unit can achieve specific detection for different analytes and there will be no crosstalk between them, thus realizing multi-channel and multi-site sensing.

[0035] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or terminal device. Without further limitation, the elements defined by the statement "including..." or "comprising..." do not exclude the existence of additional elements in the process, method, article or terminal device including the said elements. In addition, in this article, "greater than", "less than", "exceeding", etc. are understood not to include the present number; "above", "below", "within", etc. are understood to include the present number.

[0036] Although the above embodiments have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the above description is only the embodiments of the present invention, and does not limit the patent protection scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A multi-channel optical fiber biosensor based on fluorescence detection, characterized in that: It includes a laser light source module, a microfluidic power module, a microfluidic channel, a microfluidic channel sensor unit, a signal acquisition module, a computer and a waste liquid treatment module. The wavelength of the laser light source module is determined according to the absorption wavelength of the quantum dots of the fluorescent material used. The absorption wavelength of the quantum dots is 350-400nm, which can maximize the fluorescence of the quantum dots. The microfluidic power module delivers the reaction liquid or the sample to be tested into the sensing range through the microfluidic pipeline for detection; The microfluidic channel sensing unit is a fiber optic sensing unit cascaded at multiple sites on an optical fiber, which can be modified at the same time after the reaction solution is injected and can also perform quantitative detection of multiple analytes at the same time after the sample solution is injected; The signal acquisition module includes a portable spectrometer capable of detecting broadband fluorescence spectra for detecting fluorescence signals generated by quantum dots with different emission wavelengths; The computer is connected to the portable spectrometer for displaying and storing the spectrum, calculating the calibration curve according to the signal and detecting the concentration or content of the object to be tested; The waste liquid treatment module is used to collect the discarded reaction solution or sample solution after detection.

2. The multi-channel optical fiber biosensor based on fluorescence detection according to claim 1, characterized in that: The optical fiber sensing unit is prepared by a cladding corrosion method. The fluorescence signal is collected and transmitted by modifying quantum dots on the surface of the optical fiber sensing area after corrosion to excite the evanescent field. The quantum dot material modified with the reactant is fixed on the surface of the optical fiber sensing area by silanization or vapor deposition to excite fluorescence and collect it on the same optical fiber. When the reactant reacts with the object to be measured, fluorescence quenching is caused to achieve sensing.

3. The multi-channel optical fiber biosensor based on fluorescence detection as claimed in claim 1, characterized in that: The packaging structure of the microfluidic channel sensor unit is manufactured by 3D printing, and the manufacturing method is a traditional heating and welding method or a light curing method.

4. The multi-channel optical fiber biosensor based on fluorescence detection as claimed in claim 1, characterized in that: The optical fiber sensor and the microfluidic channel in the microfluidic channel sensing unit are in a cross shape. After the optical fiber is inserted and packaged, only the microfluidic channel can realize the circulation of liquid and gas. The connection angle between the optical fiber packaging structure and the microfluidic channel is adjusted according to needs.

5. The multi-channel optical fiber biosensor based on fluorescence detection as claimed in claim 1, characterized in that: All of the optical fiber sensing units are located at different positions on the same optical fiber. By changing the particle size of the quantum dots, the fluorescence emission peak can be changed, and the fluorescence of different emission peaks can be stimulated by using a light source of the same wavelength. The reaction degree of the reactants modified on the corresponding quantum dots can be calculated according to the spectral signal intensity of different wavelengths, and then the concentration of the analyte can be detected.

6. The multi-channel optical fiber biosensor based on fluorescence detection according to claim 1, characterized in that: After the microfluidic channel structure is packaged, it can be connected to the pipeline combination from the side to form a microfluidic channel array to optimize the microfluidic route. The size of the microfluidic channel structure can be changed according to the different requirements for the flow rate of the detected sample and the speed of the flow to form a microfluidic chip. After the connection, it is only necessary to input the reaction solution or sample solution at the entrance of the microfluidic pipeline by the microfluidic power system to realize the simultaneous modification and functionalization of multiple optical fiber sensing units, and to achieve simultaneous response to different analytes in the same sample. To remove the waste liquid, it is only necessary to input nitrogen or other gases into the microfluidic power system to push the internal waste liquid into the waste liquid treatment system for treatment.

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