Enhanced Fabry-Perot microcavity biosensor for low concentration detection

Abstract

The invention belongs to the technical field of optical biosensors, and particularly relates to an ultralow-concentration Fabry-Perot microcavity biosensor. The sensing microcavity comprises a square capillary quartz tube, and the upper surface and the lower surface of the square capillary quartz tube are plated with high-reflectivity films; a liquid core area of the square tube is a micro-flow channel, and a polyethylene glycol polymerization promoting biomolecule solution is introduced into the micro-flow channel, so that the Fabry-Perot microcavity biosensor is obtained. According to the invention, the square capillary quartz tube provides high-precision parallelism between the two reflectors, so that diffraction and walk-off loss can be reduced, the mode volume in the cavity can be reduced, the light energy density can be increased, strong interaction between light and substances can be realized, and the quality factor can be improved; polyethylene glycol can promote the polymerization of biomolecular protein, enhance the reaction of an antigen-antibody compound, and further promote the aggregation of an immune compound. The biosensor is simple in structure, convenient to prepare, good in repeatability and stability, high in response speed and free of surface modification and chemical labeling.

Description

Technical Field

[0001] The invention belongs to the technical field of optical sensors, and in particular relates to a Fabry-Perot microcavity sensor. Background Art

[0002] With the increasing demand for biomedicine, ultra-low concentration bioassay technology has become a key technology in early diagnosis, precision medicine and drug development. In clinical samples, the concentration of biomarkers is usually in the picomolar to attomole level, which poses a huge challenge to traditional detection methods. Optical microcavities have become a powerful tool for highly sensitive detection through strong light-matter interactions. These interactions can amplify the signal response, and even small changes in the mode field can cause significant responses in the resonant spectrum, such as wavelength shift, linewidth broadening and light intensity changes, thereby achieving ultrasensitive biomarker detection. Among them, the Fabry-Perot microcavity with a high quality factor has a high overlap with the target analyte through the light field, has a high quality factor and a small mode volume, and can achieve strong light-matter interactions, thus providing a simple and efficient method for ultrasensitive biological detection.

[0003] Fabry-Perot microcavity biosensors usually amplify the optical signal through a gain medium or fluorescent marker to suppress the walk-off loss caused by the tilt of the two mirrors. However, label-based biosensors require additional chemical modification steps, which may be toxic to biomolecules and cause photobleaching of fluorescent materials during long-term testing, thereby generating signal artifacts and increasing the risk of interference. In contrast, label-free biomonitoring assays directly measure biomolecular interactions by monitoring changes in optical signals, with the advantages of non-invasiveness, simplicity, cost-effectiveness, high repeatability, and fast response time.

[0004] Immunoturbidimetry is a label-free biosensing technique that enhances antigen-antibody reactions by using biomolecule polymerization promoters. The increase in the number and size of nanoparticles generated during the reaction causes the incident light to scatter and absorb, which in turn causes changes in the turbidity of the solution. By measuring changes in the intensity of transmitted or scattered light, the concentration of the target biomolecule can be quantitatively analyzed. However, immunoturbidimetry faces the problem of insufficient resolution in the detection of low-concentration biological samples, which becomes an important challenge. Summary of the invention

[0005] The object of the present invention is to provide a polyethylene glycol enhanced Fabry-Perot microcavity sensor capable of realizing ultra-low concentration biomolecule detection.

[0006] The Fabry-Perot microcavity sensor provided by the present invention enhances the interaction between light and matter through a high-quality factor Fabry-Perot microcavity, thereby realizing the detection of small refractive index changes, and further realizing the detection of low-concentration biological molecules; and enhances the antigen-antibody complex reaction through the polymerization of polyethylene glycol on biological molecules, promotes the aggregation of immune complexes, thereby increasing the size and concentration of antigen-antibody nanoparticles, and realizing the amplification of optical signals.

[0007] The enhanced Fabry-Perot microcavity sensor for ultra-low concentration biological detection provided by the present invention has a structure as follows: Figure 1 As shown; specifically comprising: a square capillary quartz tube with a hollow structure, the central area of ​​which serves as a microfluidic channel; the upper and lower surfaces of the square quartz tube are coated with a high reflectivity film, which serves as a Bragg reflector to form a Fabry-Perot microcavity; Teflon tubes are connected to both ends of the square capillary quartz tube for connecting to a microfluidic system to achieve extraction of biological solutions; polyethylene glycol is mixed and incubated with biological molecules, and the biological solution is introduced into the Fabry-Perot microcavity through the microfluidic system to achieve ultra-low concentration biological detection.

[0008] In the present invention, the high reflectivity film is formed by alternating two dielectric films with different high and low refractive indices, and its reflectivity is 90-100%.

[0009] In the present invention, the reflection band of the high reflectivity film is from near ultraviolet light to mid-infrared light.

[0010] In the present invention, the cross-sectional height and width of the square quartz tube are 100-1000 μm, the wall thickness is 5-200 μm, and the length is 0.5-10 cm.

[0011] In the present invention, the material of the square quartz tube is preferably silicon dioxide.

[0012] In the present invention, the polyethylene glycol solution is used as a polymerization promoter for enhancing biological signals, and the concentration of the polyethylene glycol solution is 10-100 mg / mL.

[0013] The technical principle realized by the present invention is: Bragg reflectors are directly plated on the upper and lower surfaces of a high-precision square capillary quartz tube to form a high-quality factor Fabry-Perot optical microcavity. Based on the mature preparation process of the square capillary tube, the high parallelism requirement of the traditional Fabry-Perot resonant cavity between the plane reflectors is overcome, and the lateral constraint of the quartz tube wall on the light field greatly reduces the mode volume in the cavity, increases the light energy density in the cavity, and enhances the interaction between light and matter. Combined with the polymerization of polyethylene glycol on biological molecules to enhance the antigen-antibody complex reaction, promote the aggregation of immune complexes, thereby increasing the size and concentration of antigen-antibody nanoparticles, and realizing the amplification of optical signals. The high-quality factor Fabry-Perot resonant cavity realizes the specific detection of ultra-low concentration antigen protein molecules by monitoring the transmission intensity and wavelength peak position. The biosensor can not only achieve the characteristics of low cost, simple structure, convenient preparation, high repetition stability, fast response time, free surface modification and chemical labeling, but also achieves extremely high sensitivity and trace biological analyte detection.

[0014] The present invention has the following characteristics:

[0015] (1) The present invention is significantly different from the traditional Fabry-Perot resonant cavity. In the present invention, no additional plane reflector is required for assembly, and the reflective film is directly plated on the surface of the square tube to form a Fabry-Perot micro-cavity. Due to the lateral constraint of the square quartz tube wall on the light field, the diffraction loss and walk-off loss are suppressed, which can not only further improve the light energy density, but also greatly reduce the mode volume;

[0016] (2) The Fabry-Perot microcavity sensor provided by the present invention overcomes the requirement of the traditional Fabry-Perot resonant cavity for high parallelism alignment of two reflective mirrors, greatly improving the quality factor of the sensor;

[0017] (3) The Fabry-Perot microcavity provided by the present invention is itself a microfluidic channel with a hollow structure, which can simultaneously realize the transmission and detection of the biological analyte solution to be tested;

[0018] (4) The Fabry-Perot microcavity provided by the present invention has an extremely high quality factor and an extremely high optical power density, which significantly increases the interaction between light and matter, thereby enabling the detection of tiny refractive index changes;

[0019] (5) The Fabry-Perot microcavity provided by the present invention is based on monitoring the changes in the spectral peak position caused by small changes in the refractive index, achieving a 7.11×10 -8 Ultra-low detection limit of RIU and ultra-high resolution refractive index sensing of 201.0nm / RIU;

[0020] (6) The Fabry-Perot microcavity provided by the present invention enhances the antigen-antibody complex reaction based on the polymerization effect of polyethylene glycol on biomolecules, promotes the aggregation of immune complexes, thereby increasing the size and concentration of antigen-antibody nanoparticles, and achieving an amplification effect on optical signals;

[0021] (7) The Fabry-Perot microcavity provided by the present invention is based on uniformly oscillating the fully reacted antigen-antibody complex solution and introducing it into the Fabry-Perot microcavity through a microfluidic system to monitor the transmission intensity and wavelength shift changes in real time;

[0022] (8) The signal intensity enhancement effect of the Fabry-Perot microtubule lumen PEG provided by the present invention helps to achieve more sensitive biomolecule detection, especially in the detection of low concentrations and single molecular weight levels;

[0023] (7) The Fabry-Perot microluminal biosensor provided in the present invention has the characteristics of low cost, simple structure, convenient preparation, high repetitive stability, fast response time, high specificity biological detection without surface modification and chemical labeling;

[0024] (8) The sensor detection system provided in the present invention is easy to build, the detection method is simple, and it is convenient for practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the Fabry-Perot micro-lumen sensor of the present invention.

[0026] Figure 2 It is a schematic diagram of a spatial optical path coupling system for detection of a Fabry-Perot micro-cavity sensor of the present invention.

[0027] Figure 3 It is the measured transmission spectrum of the Fabry-Perot micro-cavity sensor of the present invention.

[0028] Figure 4 The transmission resonance spectrum is obtained when solutions with different refractive indices are passed into the liquid core region of the Fabry-Perot micro-cavity sensor of the present invention.

[0029] Figure 5 It is a characterization diagram of the refractive index sensitivity of the Fabry-Perot micro-cavity sensor of the present invention.

[0030] Figure 6 It is a detection limit diagram of the measured refractive index sensing of the Fabry-Perot micro-cavity sensor of the present invention.

[0031] Figure 7 This is a diagram of the polyethylene glycol-enhanced biomolecular optical signal detection of the Fabry-Perot microluminal sensor of the present invention.

[0032] The numbers in the figure are: 1 is a high reflectivity film, 2 is a quartz tube, 3 is a biological molecule combined with polyethylene glycol, and 4 is a microfluidic channel. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to these examples.

[0034] Example 1

[0035] In this embodiment, the Fabry-Perot micro-lumen sensor (see Figure 1 ), specifically comprising: a square quartz capillary with a cross-sectional height and width of 300 μm and a wall thickness of 100 μm, which is a hollow structure; the upper and lower surfaces of the square quartz tube are coated with a high reflectivity film with a reflectivity of 99.8%.

[0036] The preparation process of the Fabry-Perot microcavity includes:

[0037] (1) Removal of coating layer: Use an alcohol lamp to burn the coating layer on the surface of the square capillary quartz tube, and then clean it with lens cleaning paper;

[0038] (2) Cleaning of square capillary quartz tube: ultrasonically clean the square capillary quartz tube with acetone, alcohol and deionized water for 2 to 5 minutes, and then place it in a culture dish to allow it to air dry naturally;

[0039] (3) Preparation of Bragg reflectors: The Bragg reflectors are prepared on the upper and lower surfaces of a square capillary quartz tube using conventional coating processes, wherein the working band and wavelength of the reflectors can be controlled by coating technology;

[0040] (4) Tube wall corrosion: After the coating is completed, Teflon tubes are connected to the two ends of the square capillary to form a Fabry-Perot micro-cavity sensor. In order to seek higher sensitivity, the tube wall is corroded with 12% hydrofluoric acid solution in the experiment. At a flow rate of 15μL / min, about 8μm can be corroded in one hour;

[0041] (5) Tube wall smoothing: After the tube wall is corroded into a thin wall, it is smoothed using 4%, 1% and 0.5% hydrofluoric acid solutions respectively to reduce the scattering loss introduced by the roughness.

[0042] The Fabry-Perot microcavity sensor in the present invention utilizes the lateral confinement effect of the Fabry-Perot cavity and the square tube wall on the light field and the high parallelism of the upper and lower surfaces, significantly increasing the effective propagation path and light energy density of the light in the cavity. This not only improves the quality factor of the microcavity, but also reduces the mode volume, thereby achieving high-sensitivity detection of ultra-low concentration analytes to be measured, and is suitable for biomolecule sensing.

[0043] Example 2

[0044] In this example, based on the parameters of Example 1, the performance of the Briperot microcavity sensor prepared by the method is characterized. First, a corresponding spatial optical coupling system is designed and constructed, such as Figure 2 As shown. The output light of the tunable laser is used as the signal light to excite the intrinsic mode of the Bridgman-Perot microtubule cavity, and the transmission spectrum is collected in real time by the photodetector. The detection optical path system consists of three parts: imaging optical path (dashed line), signal detection optical path (solid line) and optical microfluidic system.

[0045] Imaging optical path: A reflective illumination system is used. It consists of a lens, a beam splitter, and a CCD imaging device. After the surface information of the sample is imaged by the lens, it passes through the beam splitter in turn to fold the optical path by 90° and finally transmits it to the CCD imaging device. The imaging optical path can observe the position of the incident light spot relative to the square capillary quartz tube in real time, and realize accurate and efficient coupling of the cavity mode of the Fabry-Perot microcavity.

[0046] Signal detection optical path: After the output light of the tunable laser is collimated by the fiber collimator, it passes through the beam splitter in turn and is focused by the lens to achieve the coupling of the incident light and the cavity mode of the Fabry-Perot microtube. In order to collect the transmitted light signal, the transmitted signal is first collimated into parallel light by an objective lens and transmitted to the remote photodetector. Finally, the lens is used to couple the light signal to the photosensitive area of ​​the photodetector. The transmission spectrum is detected by the photodetector and the spectrum is displayed on the computer for processing and analysis.

[0047] Microfluidic system: The microcavity is connected to the syringe and analyte through a Teflon tube. In the actual test, the extraction speed of the syringe is controlled by a microfluidic pump to ensure that the solution in the microcavity moves at a constant flow rate.

[0048] Example 3

[0049] In this embodiment, based on the spatial optical path coupling system constructed in Embodiment 2, the transmission spectrum of the Fabry-Perot microcavity is obtained. Figure 3 is the transmission spectrum of the resonant mode of the Fabry-Perot microcavity near 910nm, and the line width is fitted by the Lorentzian line shape. Its quality factor can be calculated by the following formula:

[0050] Q=λ / w, (1)

[0051] Where λ is the resonant wavelength and w is the half-width of the resonant mode. According to the detected spectrum and the Lorentzian line shape fitting, the half-width of the resonant mode of the Fabry-Perot microcavity near 910 nm is 0.9 pm. Therefore, the quality factor of this mode is calculated by formula (1) to be 1.0×10 6. This result indicates a high quality factor. The Fabry-Perot microcavity means that the light in the cavity has very little energy loss after multiple reflections. The optical signal in the cavity can be maintained for a long time, which is manifested as very low energy loss. Low loss allows the cavity to maintain the optical signal for a longer time, thereby improving the sensitivity of the optical signal. The Fabry-Perot microcavity can more accurately select and enhance light of specific wavelengths, which helps to achieve high-resolution spectral measurements and precise wavelength selection, and realizes the detection of tiny refractive index changes, which can be used for the detection of ultra-low concentration biological molecules.

[0052] Example 4

[0053] In order to evaluate the refractive index sensing performance of the Fabry-Perot microcavity, different volume ratios (0%, 0.02%, 0.04%, 0.06%, 0.08% and 0.1%) of dimethyl sulfoxide and deionized water solution were introduced into the microcavity microfluidic channel in this case, and the refractive index values ​​of the corresponding solutions ranged from 1.33 to 1.330142. Figure 4 The resonance spectra of the Fabry-Perot microcavity when solutions with different refractive index values ​​are introduced are shown. Figure 5 The relationship between the change in the refractive index of the solution and the shift in the resonance wavelength was demonstrated. The experimental results showed that with the increase in the concentration of the dimethyl sulfoxide solution, the resonance wavelength showed an obvious red shift phenomenon, and there was a significant linear relationship between the resonance wavelength and the refractive index value. According to the experimental data fitting, the sensitivity was 201.0nm / RIU. It is worth noting that the actual detection limit of the Fabry-Perot microcavity is as low as 7.11×10 -8 RIU (see Figure 6 ). These outstanding performance indicators are attributed to the lateral light field confinement of the microtube sidewalls, which greatly reduces the mode volume. In addition, the precise parallelism of the square capillary quartz tube minimizes diffraction and lateral drift losses, and the ultra-high detection sensitivity lays the foundation for ultra-low concentration biomolecule detection.

[0054] Example 5

[0055] In this case, the antigen-antibody complex reaction is enhanced by the aggregation-promoting effect of polyethylene glycol on protein molecules, which promotes the aggregation of immune complexes, thereby increasing the size and concentration of antigen-antibody complex nanoparticles and achieving signal amplification. This case compares the wavelength shift changes of antigen-antibody complex solutions containing polyethylene glycol and those without polyethylene glycol in the range of human epidermal growth factor receptor 2 protein (HER2) concentration from 100ag / mL to 100pg / mL. In this label-free biomolecule detection, the human epidermal growth factor receptor 2 solution is first mixed and incubated with the polyethylene glycol solution, and then the antibody solution is added for mixed reaction. The fully reacted antigen-antibody complex solution is evenly oscillated and introduced into the Fabry-Perot microtube cavity through a microfluidic system to monitor the wavelength shift changes of the transmission spectrum in real time. As the concentration of human epidermal growth factor receptor 2 protein increases, the wavelength of the antigen-antibody complex solution blue shifts. It is worth noting that under the same human epidermal growth factor receptor 2 protein concentration conditions, the antigen-antibody complex solution containing polyethylene glycol showed a significantly enhanced wavelength shift effect. Therefore, the experimental results show that polyethylene glycol promotes the polymerization reaction of proteins in antigen-antibody binding and enhances the biological monitoring signal, thus helping to achieve more sensitive biological molecule detection, especially in low-concentration detection. It has important advantages.

Claims

1. An enhanced Fabry-Perot microcavity sensor for ultra-low concentration biological detection, characterized in that: Specifically include: The central area of ​​the square capillary quartz tube with a hollow structure serves as a microfluidic channel. The upper and lower surfaces of the square quartz tube are coated with a high-reflectivity film, which serves as a Bragg reflector to form a Fabry-Perot microcavity. Teflon tubes are connected to both ends of the square capillary quartz tube for connecting to a microfluidic system to extract biological solutions. Polyethylene glycol is mixed and incubated with biological molecules, and the biological solution is introduced into the Fabry-Perot microcavity through the microfluidic system to achieve ultra-low concentration biological detection.

2. The enhanced Fabry-Perot microcavity sensor according to claim 1, characterized in that: The high reflectivity film is formed by alternating two dielectric films with different high and low refractive indices, and its reflectivity is 90-100%.

3. The enhanced Fabry-Perot microcavity sensor according to claim 2, characterized in that: The reflection wavelength band of the high reflectivity film is from near ultraviolet light to mid-infrared light.

4. The enhanced Fabry-Perot microcavity sensor according to claim 3, characterized in that: The cross-sectional height and width of the square quartz tube are 100-1000 μm, the wall thickness is 5-200 μm, and the length is 0.5-10 cm.

5. The enhanced Fabry-Perot microcavity sensor according to claim 4, characterized in that: The polyethylene glycol is used as a polymerization promoter for enhancing biological signals, and the concentration of the polyethylene glycol solution is 10-100 mg / mL.

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