Optical fiber mechanical resonance probe sensor and protein folding process monitoring method

Abstract

The invention discloses an optical fiber mechanical resonance probe sensor and a protein folding process monitoring method. In the sensor, a laser sends a laser signal to an optical fiber grating through a circulator; the driving device drives the fiber bragg grating and the vibration part to synchronously do subharmonic vibration, the grating period of the fiber bragg grating changes periodically in the vibration process, the reflection spectrum of the fiber bragg grating shifts periodically, and after the fiber bragg grating receives the laser signal, the vibration part vibrates; the intensity of the reflected light reversely transmitted to the photoelectric detector through the circulator also changes periodically; the characteristic parameters of the to-be-detected substance inserted into the vibration part are different, and the periodic change degrees of the reflected light intensity are different; the photoelectric detector converts the reflected light into an electric signal, and the analysis device determines the characteristic parameters of the to-be-detected substance according to the intensity change degree of the electric signal. According to the method, accurate and high-sensitivity measurement of characteristic parameters can be realized, fluorophore does not need to be introduced in the protein folding process, and accurate and high-sensitivity artifact-free characterization can be realized.

Description

Technical Field

[0001] This invention belongs to the field of acousto-optic modulation, specifically relating to an optical fiber mechanical resonant probe sensor and a method for monitoring the protein folding process. Background Technology

[0002] The mechanical properties of biofluids (such as viscosity and surface tension) can serve as indicators for early disease diagnosis, providing rich information for understanding complex physiological and pathological processes. For example, protein aggregation is a key pathological marker of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Early detection of protein aggregation provides valuable insights into the pathophysiology of various neurodegenerative diseases. Fluorescence methods using thioflavin T (ThT) have traditionally been considered the "gold standard" for detecting protein aggregation. ThT exhibits increased quantum yield when binding to the β-sheet structure of aggregated proteins. However, introducing fluorophores into protein aggregation studies can interfere with the aggregation process by directly interacting with the proteins. Therefore, developing label-free methods is crucial for achieving accurate and artifact-free characterization of protein aggregation processes.

[0003] Viscosity can be used as a label-free indicator of protein aggregation. During aggregation, proteins form higher-order structures through self-association, leading to changes in intermolecular interactions and thus increasing the viscosity of the solution. Traditional viscosity measurement techniques include capillary viscometers, rotational rheometers, microrheology, and microfluidic viscometers. However, their measurement sensitivity, large sample size, and complex manufacturing processes often fail to meet the requirements. Summary of the Invention

[0004] This invention provides an optical fiber mechanical resonant probe sensor and a method for monitoring protein folding processes, which solves the problems of low sensitivity, large sample requirements, and complex manufacturing of current characteristic parameter detection methods. It also solves the problems of low accuracy and inability to achieve artifact-free characterization in current protein aggregation processes, which require the introduction of fluorophores for labeling.

[0005] According to a first aspect of the present invention, a fiber optic mechanical resonant probe sensor is provided, comprising a fiber optic grating, a driving device, a circulator, a laser, a photodetector, an analytical device, and a vibrating part inserted into a substance to be tested. The output end of the laser is connected to a first end of the circulator, the second end of the circulator is connected to a first end of the fiber optic grating, the third end of the circulator is connected to the analytical device through the photodetector, and the second end of the fiber optic grating is connected to the vibrating part. The laser transmits the laser signal to the fiber Bragg grating via the circulator. The driving device is located at the first end of the fiber Bragg grating and drives the fiber Bragg grating and the vibrating part to perform subharmonic vibration synchronously. During the vibration, the grating period of the fiber Bragg grating changes periodically, and correspondingly, the reflection spectrum of the fiber Bragg grating shifts periodically. After receiving the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator also changes periodically. The degree of periodic change in the intensity of the reflected light varies depending on the characteristic parameters of the analyte inserted into the vibrating part. The photodetector converts the reflected light into an electrical signal, and the analysis device determines the characteristic parameters of the analyte based on the degree of intensity change of the electrical signal.

[0006] Optionally, the wavelength of the laser signal is within the linear region of the unshifted reflection spectrum, and the intensity change of the reflected light is determined by the actual left-right shift of the reflection spectrum. The actual left-right shift of the reflection spectrum depends on the driving frequency and driving voltage of the driving device, the characteristic parameters of the substance to be tested around the vibrating part, and its immersion depth. After the wavelength of the laser signal is determined, before the vibrating part is immersed in the substance to be tested, the driving voltage is gradually increased. At the same time, the analysis device is used to observe whether the electrical signal exhibits a 1 / 2 resonant wave. If it does, the driving voltage at this time is taken as the minimum value of the driving voltage. Thereafter, the driving voltage is gradually increased, and the analysis device is used to observe whether the electrical signal exhibits distortion. If it does, the driving voltage at this time is taken as the maximum value of the driving voltage. In the actual measurement process, the driving voltage takes a value between its minimum and maximum values. When the fiber grating vibrates from the middle position to both sides under the drive of the driving device, the reflection spectrum of the fiber grating shifts in different directions and the amount of shift gradually increases on the corresponding side.

[0007] Optionally, the driving device drives the fiber optic grating and the vibrating part to synchronously perform subharmonic vibration at their respective intrinsic frequencies.

[0008] Optionally, a vertical displacement platform is also included, on which the driving device is fixed. The side where the first end of the fiber grating is located is fixed to the driving device with UV glue. The vertical displacement platform drives the fiber grating and the vibrating part to move vertically up and down perpendicular to the surface of the material to be tested through the driving device, so that the vibrating part is immersed in the material to be tested. During the vertical movement, the driving device is controlled to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at the immersion depth as the target reflected light at that immersion depth, determines the subharmonic wave intensity in the target reflected light at that immersion depth, and thus obtains the functional relationship between different immersion depths and the subharmonic wave intensity. The analytical device determines the slope of the linear fitting region in the functional relationship, and obtains the characteristic parameters of the substance to be tested based on the determined slope and the correspondence between different characteristic parameters and the slope.

[0009] Optionally, the fiber grating and the vibrating part are made of single-mode fiber. A Bragg grating is etched at the corresponding position of the single-mode fiber to form the fiber grating. The corresponding fiber segment of the single-mode fiber is stretched to obtain a tapered fiber segment. Half of the tapered fiber segment is removed to obtain the vibrating part connected to the fiber grating.

[0010] Optionally, for each immersion depth, if multiple target reflected light are obtained at that immersion depth, the analysis device takes the average value of the secondary resonant wave intensity of each target reflected light at that immersion depth as the secondary resonant wave intensity corresponding to that immersion depth; the characteristic parameter is the viscosity.

[0011] Optionally, when the characteristic parameter is viscosity, the relationship between different characteristic parameters and slope is as follows: as viscosity increases, the slope gradually decreases.

[0012] According to a second aspect of the present invention, a method for monitoring protein folding using the above-described fiber optic mechanical resonant probe sensor is provided, comprising: Step S11: Prepare glycerol-water mixtures of different concentrations as standard solutions. For each concentration of glycerol-water mixture, determine the slope of the glycerol-water mixture of that concentration according to the slope determination method. After obtaining the slopes of glycerol-water mixtures of different concentrations, determine the corresponding relationship between different viscosities and slopes based on the relationship between the viscosity and concentration of the glycerol-water mixture. Step S12: Prepare protein solutions of different concentrations as standard solutions. For each concentration of protein solution, determine the slope of the protein solution of that concentration according to the slope determination method. After obtaining the slope of protein solutions of different concentrations, determine the viscosity of protein solutions of different concentrations according to the correspondence between the different viscosities and the slope. Step S13: For each protein solution of a given viscosity, heat the protein solution under set conditions to promote the protein to undergo folding and aggregation reactions. After heating for different corresponding durations, determine the slope of the protein solution according to the slope determination method. Based on the correspondence between different viscosities and slopes, determine the viscosity of the protein solution after heating for the corresponding duration. This yields the viscosity change of protein solutions of different viscosities during the folding and aggregation reaction process, and the viscosity change reflects the folding and aggregation process of protein solutions of different concentrations. Step S14: For the protein solution to be tested, the slope of the protein solution to be tested is determined according to the slope determination method. Based on the correspondence between different viscosities and slopes, the viscosity of the protein solution to be tested and the viscosity change of the protein solution to be tested during the folding and aggregation reaction are obtained, thereby obtaining the folding and aggregation process of the protein solution to be tested. The slope determination method is as follows: the vibrating part of the fiber optic mechanical resonant probe sensor, which serves as the probe, is oriented downwards. The vertical displacement platform is controlled to drive the fiber optic grating and the vibrating part perpendicular to the surface of the glycerol-water mixture or protein solution. The probe is inserted into the glycerol-water mixture or protein solution and gradually moves downwards. During this process, the driving device is controlled to drive the fiber optic grating and the vibrating part to perform subharmonic vibration synchronously. The subharmonic wave in the spectrometer is observed. The downward movement is stopped when the subharmonic wave disappears. The vertical displacement platform is controlled to move the fiber grating and the vibrating part vertically upward until the vibrating part leaves the surface of the protein solution. During this process, the driving device is controlled to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at that immersion depth as the target reflected light at that immersion depth and determines the subharmonic wave intensity in the target reflected light obtained at that immersion depth. After obtaining the subharmonic wave intensity at each immersion depth, that is, after obtaining the functional relationship between different immersion depths and the subharmonic wave intensity, the slope of the linear fitting region in the functional relationship is determined.

[0013] Optionally, in this slope determination method, for each immersion depth, if multiple target reflected lights are obtained at that immersion depth, the analysis device determines the intensity of the secondary resonant wave in each target reflected light obtained at that immersion depth, and takes the average value of the determined secondary resonant wave intensities as the intensity of the secondary resonant wave at that immersion depth.

[0014] Optionally, the protein is insulin, and in step S13, the set conditions are acidic conditions and 52°C conditions.

[0015] The beneficial effects of this invention are: 1. This invention uses a vibrating part as a probe, which is inserted into the substance to be tested. A driving device drives the connected fiber optic grating and the vibrating part to perform subharmonic vibration synchronously. This vibration process causes the grating period of the fiber optic grating to change periodically, which in turn causes the reflection spectrum of the fiber optic grating to shift periodically. At this time, when a laser signal is transmitted to the fiber optic grating, the intensity of the reflected light transmitted back by the fiber optic grating will change periodically. Since the vibrating part interacts with the substance to be tested after being inserted, the characteristic parameters of the substance to be tested are different. Under the same driving action, the vibration amplitude of the fiber optic grating and the vibrating part in the substance to be tested is different, and the corresponding degree of periodic change of the reflected light intensity will also be different. Therefore, the characteristic parameters of the substance to be tested can be determined based on the degree of periodic change of the reflected light intensity. This invention measures characteristic parameters based on the grating period change of the fiber optic grating, which has high sensitivity, does not require a large number of samples during measurement, and has a simple structure. 2. In this invention, the wavelength of the laser signal is set within the linear range of the un-shifted reflection spectrum. After the wavelength of the laser signal is determined, the minimum and maximum values ​​of the driving voltage are determined before the vibrating part is immersed in the substance to be tested. When the driving voltage is taken between its minimum and maximum values, the intensity of the reflected light will undergo a monotonically increasing or monotonically decreasing periodic change regardless of the characteristic parameters of the substance to be tested during the actual measurement process. Thus, the characteristic parameters of the substance to be tested can be determined based on the degree of change in the intensity of the reflected light. 3. The present invention uses the local oscillator frequency of the fiber optic grating and the vibrating part to drive them to perform subharmonic vibration, which can make the fiber optic grating and the vibrating part resonate, thereby improving the measurement range and measurement sensitivity; 4. This invention uses a vertical displacement platform to drive the fiber optic grating and vibrating unit to move vertically up and down perpendicular to the surface of the material to be tested. During the up and down movement, the fiber optic grating and vibrating unit are driven to synchronously perform subharmonic vibrations. This allows multiple reflected lights to be obtained at each position. Furthermore, by changing the immersion depth of the vibrating unit, a large amount of reflected light related to the immersion depth can be obtained. Although a large amount of data is obtained, the intensity differences between the reflected lights are very small except for abnormal data. Therefore, improving the accuracy of determining the characteristic parameters of the material to be tested based on the intensity of a large amount of reflected light is not very meaningful. Therefore, this invention proposes to determine the characteristic parameters of the material to be tested based on the intensity of the subharmonic wave in the reflected light. Since the subharmonic wave is very sensitive to external stimuli, the intensity of the subharmonic wave can be adjusted by inserting the vibrating unit to different depths. The intensity of the sub-resonant waves obtained at different depths varies considerably. Therefore, the intensity of a large number of sub-resonant waves can improve the characteristic parameters of the analyte. However, this invention does not average the intensity of each sub-resonant wave obtained at the same depth and different depths to characterize the characteristic parameters of the analyte, because there may be outliers in these intensity data, which would lead to low accuracy in determining the characteristic parameters. Based on this, this invention proposes to establish a functional relationship between the intensity of the sub-resonant wave and the immersion depth, and to determine the characteristic parameters based on the slope of the linear fitting region in this functional relationship. The slope represents a trend of change, and even if there are outliers, it can accurately reflect the characteristic parameters. Thus, this invention can ensure the accuracy of characteristic parameter measurement. 5. For each immersion depth of the vibrating part, the present invention takes the average value of the intensity of the secondary resonant wave in the target reflected light obtained at that immersion depth, and uses the average value as the intensity of the secondary resonant wave corresponding to that immersion depth, thereby improving the accuracy of the intensity of the secondary resonant wave at the corresponding immersion depth. 6. In characterizing the protein folding and aggregation process, this invention is based on a fiber optic mechanical resonant probe sensor, which has high accuracy and sensitivity, and does not require the introduction of fluorescent groups for labeling, thus achieving artifact-free characterization. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of an embodiment of the fiber optic mechanical resonant probe sensor of the present invention; Figure 2 This is a schematic diagram showing the connection relationship between the driving device, fiber optic grating, and vibration unit of the present invention; Figure 3 This is a schematic diagram of the fiber optic grating and the vibrating part of the present invention; Figure 4 This is a schematic diagram of the fabrication process of the fiber optic grating and the vibrating part of the present invention; Figure 5 This is a schematic diagram showing the periodic change of the reflection spectrum of the fiber optic grating when the fiber optic grating and the vibrating part are synchronously undergoing subharmonic vibration. Figure 6It is the functional relationship between different relative displacements and reflected light intensity, and the corresponding relationship between the slope of the linear fitting region in this functional relationship and the viscosity; Figure 7 It describes the relationship between protein solution concentration and viscosity, as well as the viscosity changes of protein solutions during the folding and aggregation process. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, and to make the above-mentioned objectives, features and advantages of the embodiments of the present invention more apparent and understandable, the technical solutions in the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] In the description of this invention, unless otherwise specified and limited, it should be noted that the term "connection" should be interpreted broadly. For example, it can be a mechanical connection or an electrical connection, or it can be a connection between two internal components. It can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above term according to the specific circumstances.

[0019] See Figure 1 This is a schematic diagram of an embodiment of the fiber optic mechanical resonant probe sensor of the present invention. Combined with... Figure 2 and Figure 3 As shown, the sensor may include a fiber Bragg grating 3, a driving device, a circulator 7, a laser 6, a photodetector 8, an analytical device, and a vibrating element 11 inserted into the analyte. The output end of the laser 6 is connected to the first end of the circulator 7, the second end of the circulator 7 is connected to the first end of the fiber Bragg grating 3, and the third end is connected to the analytical device through the photodetector 8. The second end of the fiber Bragg grating 3 is connected to the vibrating element. The laser 6 transmits a laser signal to the fiber Bragg grating 3 through the circulator 7. The driving device is located on the side where the first end of the fiber Bragg grating 3 is located, and it drives the fiber Bragg grating 3 and... The vibrating part 11 synchronously performs subharmonic vibration. During the vibration process, the grating period of the fiber Bragg grating 3 changes periodically, and correspondingly, the reflection spectrum of the fiber Bragg grating 3 shifts periodically. After receiving the laser signal, the intensity of the reflected light transmitted back to the photodetector 8 through the circulator 7 also changes periodically. The degree of periodic change in the intensity of the reflected light varies depending on the characteristic parameters of the substance to be tested inserted into the vibrating part 11. The photodetector 8 converts the reflected light into an electrical signal, and the analysis device determines the characteristic parameters of the substance to be tested based on the degree of change in the intensity of the electrical signal.

[0020] In this embodiment, combined with Figure 3As shown, the fiber grating 3 and the vibrating part 11 can be made of single-mode fiber. A Bragg grating is etched (e.g., using ultraviolet lithography) at the corresponding position on the single-mode fiber to form the fiber grating. The corresponding fiber segment of the single-mode fiber is stretched to obtain a tapered fiber segment. Half of the tapered fiber segment is removed to obtain the vibrating part connected to the fiber grating. This vibrating part serves as a probe. Figure 4 As shown, the fabrication process of the fiber grating and the vibrating part may include: fixing a single-mode fiber 1 with a Bragg grating etched on it on a hydrogen-oxygen fusion tapering platform 2, stripping the fiber coating layer of the single-mode fiber to be tapered and wiping it with alcohol, then controlling the hydrogen-oxygen flame to heat the corresponding position on the single-mode fiber, and simultaneously starting a preset program to pull the single-mode fiber to both sides at a uniform speed for a certain distance, thus obtaining a tapered fiber segment, removing half of the tapered fiber segment to obtain the vibrating part connected to the fiber grating.

[0021] The length of the fiber Bragg grating affects the range of its reflected light spectrum. Increasing the fiber Bragg grating length raises the reflection peak height and narrows the reflection bandwidth, while the center wavelength generally remains constant. In this embodiment, the fiber Bragg grating length can be 1 cm. Because the reflection spectrum of the reflected light transmitted back from the fiber Bragg grating during synchronous subharmonic vibration will shift, the wavelength of the laser signal needs to be set within the linear region of the unshifted reflection spectrum in order to observe the periodic changes in the intensity of the reflected light. If the length of the fiber Bragg grating changes, thus altering the linear region of the unshifted reflection spectrum, the wavelength of the laser signal needs to be adjusted. This change in the intensity of the reflected light is determined by the actual left-right shift of the reflection spectrum, which depends on the driving frequency and driving voltage of the driving device, the characteristic parameters of the test material around the vibrating part, and its immersion depth. After the wavelength of the laser signal is determined, and before the vibrating part is immersed in the substance to be tested, the driving voltage is gradually increased. Simultaneously, the analysis device is used to observe whether a half-resonance wave appears in the electrical signal. If it does, the driving voltage at this point is taken as the minimum value. Subsequently, the driving voltage is gradually increased, and the analysis device is used to observe whether the electrical signal is distorted. If so, the driving voltage at this point is taken as the maximum value. During actual measurement, the driving voltage is taken between its minimum and maximum values. This invention sets the wavelength of the laser signal within the linear range of the un-offset reflection spectrum. After the wavelength of the laser signal is determined, and before the vibrating part is immersed in the substance to be tested, the minimum and maximum values ​​of the driving voltage are determined. When the driving voltage is taken between its minimum and maximum values, during actual measurement, regardless of the characteristic parameters of the substance to be tested, the intensity of the reflected light will undergo a monotonically increasing or monotonically decreasing periodic change. Therefore, the characteristic parameters of the substance to be tested can be determined based on the degree of change in the intensity of the reflected light.

[0022] When the fiber grating vibrates from its central position towards both sides under the drive of the driving device, its reflection spectrum shifts in different directions, with the shift gradually increasing on each corresponding side. Combined with... Figure 5 As shown, the intersection points of reflected light with different reflection spectrum curves at corresponding wavelengths are different, and the reflectivity is different at each intersection point. Therefore, the intensity of reflected light will change periodically.

[0023] Furthermore, the longer the vibrating part is, the farther its tip is from the Bragg grating, which reduces the sensitivity and stability of the probe. Therefore, the length and tip diameter of the vibrating part should be appropriately set. In this embodiment, the vibrating part can be frustum-shaped, with a length (i.e., the height of the frustum) of 1.4 cm and a tip diameter of 16 μm. The first end of the fiber grating 3 can be fixed to the driving device using ultraviolet glue (i.e., UV glue). The farther this fixing position is from the vibrating part 11, the weaker the vibration applied to the vibrating part 11 by the driving device. Therefore, the fixing position should be appropriate. In this embodiment, the fixing position can be 1.1 cm away from the grating area of ​​the fiber grating.

[0024] In this embodiment, the driving device can use the eigenfrequency of the fiber optic grating 3 and the vibrating part 11 to drive them to perform subharmonic vibration synchronously. This invention uses the oscillator frequency of the fiber optic grating and the vibrating part to drive them to perform subharmonic vibration, which can cause the fiber optic grating and the vibrating part to resonate, thereby improving the measurement range and measurement sensitivity.

[0025] While the characteristic parameters of the test substance can be determined based on the intensity variation of reflected light, when the immersion depth of the vibrating part is fixed, determining the characteristic parameters based on the intensity of a single target reflected light (i.e., the reflected light with the highest or lowest intensity obtained within one vibration cycle of the fiber grating) has low accuracy. Furthermore, determining the characteristic parameters by averaging the intensity of multiple target reflected lights may be affected by abnormal data, resulting in still low accuracy. Therefore, this invention needs further improvement in the accuracy of characteristic parameter measurement. The applicant's research has found that the secondary resonant wave in the reflected light is highly sensitive to external stimuli. The intensity of the secondary resonant wave is sensitive not only to the characteristic parameters of different test substances but also to the different immersion depths of the vibrating part in the same test substance. As the immersion depth of the vibrating part increases, the intensity of the secondary resonant wave fluctuates significantly and rapidly.

[0026] Based on this, the applicant proposes that, by changing the immersion depth of the vibrating part and determining the characteristic parameters based on the intensity change of the secondary resonant wave in the reflected light, the present invention may also include a vertical displacement platform ( Figure 1(Not shown in the image) The driving device is fixed on the vertical displacement platform. The driving device drives the fiber grating 3 and the vibrating part 11 to move vertically up and down perpendicular to the surface of the material to be tested, so that the vibrating part is immersed in the material to be tested. During the vertical movement, the driving device controls the fiber grating and the vibrating part to move synchronously in subharmonic vibration. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity at that immersion depth as the target reflected light at that immersion depth, determines the subharmonic wave intensity in the target reflected light at that immersion depth, and thus obtains the functional relationship between different immersion depths and subharmonic wave intensity. The analysis device determines the slope of the linear fitting region in the functional relationship, and obtains the characteristic parameters of the material to be tested based on the determined slope and the correspondence between different characteristic parameters and the slope. Specifically, for each immersion depth, if multiple target reflected lights are obtained at that depth, the analysis device takes the average value of the secondary resonant wave intensity of each target reflected light at that depth as the secondary resonant wave intensity corresponding to that depth. When the characteristic parameter is viscosity, the relationship between different characteristic parameters and the slope is as follows: as viscosity increases, the slope gradually decreases.

[0027] This invention employs a vertical displacement platform to drive a fiber optic grating and a vibrating section to move vertically up and down perpendicular to the surface of the material under test. After each vertical movement, the fiber optic grating and vibrating section are driven to synchronously perform subharmonic vibrations. This allows for the acquisition of multiple reflected light beams at each position. Furthermore, by changing the immersion depth of the vibrating section, a large amount of reflected light related to the immersion depth can be obtained. Although a large amount of data is obtained, the intensity differences between the reflected light beams are very small, except for anomalous data. Therefore, improving the accuracy of determining the characteristic parameters of the material under test based on the intensity of a large amount of reflected light is not very meaningful. To address this, this invention proposes determining the characteristic parameters of the material under test based on the intensity of the subharmonic wave in the reflected light. Since the subharmonic wave is highly sensitive to external stimuli, the intensity of the subharmonic wave can be adjusted by inserting the vibrating section to different depths. The intensity of the secondary resonant waves obtained at different depths varies considerably. Therefore, using the intensity of a large number of secondary resonant waves can improve the characteristic parameters of the analyte. However, this invention does not average the intensities of the secondary resonant waves obtained at the same depth and different depths to characterize the characteristic parameters of the analyte, because these intensity data may contain anomalies, which would lead to low accuracy in determining the characteristic parameters. Based on this, this invention proposes to establish a functional relationship between the intensity of the secondary resonant waves and the immersion depth. The characteristic parameters are determined based on the slope of the linear fitting region in this functional relationship. The slope represents a trend of change, and even with anomalies, it can accurately reflect the characteristic parameters. Thus, this invention can ensure the accuracy of characteristic parameter measurement. Furthermore, for each immersion depth of the vibrating part, this invention averages the intensity of the secondary resonant waves in the target reflected light obtained at that immersion depth, and uses this average value as the secondary resonant wave intensity corresponding to that immersion depth, thereby improving the accuracy of the secondary resonant wave intensity at the corresponding immersion depth.

[0028] In this embodiment, the driving device may include a signal generator 5 and a piezoelectric driver 4 connected together. The signal generator 5 can send a sinusoidal signal to the piezoelectric driver 4. The piezoelectric driver 4 can apply an acoustic signal to the fiber optic grating 3 and the vibrating part 11 according to the sinusoidal signal to drive the fiber optic grating 3 and the vibrating part 11 to synchronously perform subharmonic vibration. The side where the first end of the fiber optic grating 3 is located can be fixed at the center of the piezoelectric driver 4. In addition, the analysis device may include a spectrometer 9 and a mixed-signal oscilloscope 10. The vertical displacement platform can be an electric displacement platform. The wavelength of the laser signal can be 1549.94 nm. The photodetector can be a Thorlabs PDB450C. The mixed-signal oscilloscope can be a Tektronix MSO2024B. The spectrometer can be a Rigol DSA815.

[0029] Based on the aforementioned fiber optic mechanical resonant probe sensor, the detection of the characteristic parameters of the substance to be tested can be achieved by following these steps: Step S1: Configure analytes with different characteristic parameters as standard substances (e.g., configure glycerol-water mixtures of different concentrations as standard solutions). For each standard solution with corresponding characteristic parameters, with the vibrating part 11 of the fiber optic mechanical resonant probe sensor facing downwards, control the vertical displacement platform to drive the fiber optic grating 3 and the vibrating part 11 perpendicular to the surface of the analyte, insert them into the analyte and gradually move them downwards. During this process, control the driving device to drive the fiber optic grating and the vibrating part to perform subharmonic vibration synchronously, and observe the subharmonic wave in the spectrometer. When the subharmonic wave disappears, stop moving downwards. The reason for the disappearance of the subharmonic wave is the result of the strong interaction between the vibrating part and the analyte. The intermolecular forces within the analyte will hinder and suppress the subharmonic vibration of the vibrating part.

[0030] Step S2: Control the vertical displacement platform to move the fiber optic grating 3 and the vibrating part 11 vertically upwards until the vibrating part 11 leaves the surface of the material to be measured (at which point the sub-resonant wave intensity reaches its maximum value). During this process, control the driving device to drive the fiber optic grating 3 and the vibrating part 11 to synchronously perform sub-resonant vibrations. After the fiber optic grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at that immersion depth as the target reflected light for that immersion depth, determines the sub-resonant wave intensity in each target reflected light obtained at that immersion depth, and takes the average value of each determined sub-resonant wave intensity as the sub-resonant wave intensity at that immersion depth. After obtaining the sub-resonant wave intensity at each immersion depth, i.e., obtaining the functional relationship between different immersion depths and sub-resonant wave intensity, determine the slope of the linear fitting region in the functional relationship, and correlate this slope with the characteristic parameter, thereby obtaining the correspondence between different characteristic parameters and the slope, such as... Figure 6 As shown.

[0031] Step S3: For the substance to be tested, control the vertical displacement platform to drive the fiber grating 3 and the vibrating part 11 perpendicular to the surface of the substance to be tested, insert them into the substance to be tested, and gradually move them downward. During this process, control the driving device to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously, and observe the subharmonic wave in the spectrometer. Stop moving downward when the subharmonic wave disappears. Then, control the vertical displacement platform to drive the fiber grating 3 and the vibrating part 11 to move vertically upward until the vibrating part 11 leaves the surface of the substance to be tested. During this process, control the driving device to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. The fiber grating receives... After the laser signal is transmitted back to the photodetector via the circulator, the intensity of the reflected light will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity at that immersion depth as the target reflected light at that immersion depth, determines the intensity of the secondary resonant wave in each target reflected light obtained at that immersion depth, and takes the average value of each determined secondary resonant wave intensity as the secondary resonant wave intensity at that immersion depth. After obtaining the secondary resonant wave intensity at each immersion depth, that is, after obtaining the functional relationship between different immersion depths and secondary resonant wave intensity, the slope of the linear fitting region in the functional relationship is determined.

[0032] Step S4: Determine the characteristic parameters of the substance to be tested based on the slope corresponding to the substance and the correspondence between different characteristic parameters and the slope. The resonant wave can be a 1 / 2 resonant wave.

[0033] As can be seen from the above embodiments, the present invention uses a vibrating part as a probe, which is inserted into the substance to be tested. A driving device is used to drive the connected fiber optic grating and the vibrating part, so that the two perform subharmonic vibration synchronously. This vibration process causes the grating period of the fiber optic grating to change periodically, thereby causing the reflection spectrum of the fiber optic grating to shift periodically. At this time, when the laser signal is transmitted to the fiber optic grating, the intensity of the reflected light transmitted back by the fiber optic grating will change periodically. Since the vibrating part interacts with the substance to be tested after being inserted, the characteristic parameters of the substance to be tested are different. Under the same driving action, the vibration amplitude of the fiber optic grating and the vibrating part in the substance to be tested are different, and the corresponding degree of periodic change of the reflected light intensity will also be different. Therefore, the characteristic parameters of the substance to be tested can be determined according to the degree of periodic change of the reflected light intensity. The present invention measures characteristic parameters based on the grating period change of the fiber optic grating, which has high sensitivity, does not require a large number of samples during measurement, and has a simple structure.

[0034] In addition, the present invention also provides a protein folding monitoring method based on the above-mentioned fiber optic mechanical resonant probe sensor, which may include: Step S11: Prepare glycerol-water mixtures of different concentrations as standard solutions. For each concentration of glycerol-water mixture, determine the slope of that concentration using the slope determination method. After obtaining the slopes of glycerol-water mixtures of different concentrations, determine the correspondence between different viscosities and slopes based on the relationship between the viscosity and concentration of the glycerol-water mixture. Figure 6 As shown in Figure b; Step S12: Prepare protein solutions of different concentrations as standard solutions. For each concentration of protein solution, determine the slope of that concentration according to the slope determination method. After obtaining the slopes of different concentrations of protein solutions, determine the viscosity of different concentrations of protein solutions based on the correspondence between the different viscosities and the slopes. Figure 7 As shown in Figure a; Step S13: For each protein solution of a given viscosity, heat the protein solution under set conditions to facilitate the folding and aggregation reaction of the protein (e.g., heating a 500 nM protein solution in an acidic water bath and at 52°C for different times: 0-7 h). After heating for different corresponding durations, determine the slope of the protein solution according to the slope determination method. Based on the correspondence between different viscosities and slopes, determine the viscosity of the protein solution after heating for the corresponding duration. This yields the viscosity change of protein solutions of different viscosities during the folding and aggregation reaction process. This viscosity change reflects the folding and aggregation process of protein solutions of different concentrations. Figure 7 As shown in Figure b; Step S14: For the protein solution to be tested, the slope of the protein solution to be tested is determined according to the slope determination method. Based on the correspondence between different viscosities and slopes, the viscosity of the protein solution to be tested and the viscosity change of the protein solution to be tested during the folding and aggregation reaction are obtained, thereby obtaining the folding and aggregation process of the protein solution to be tested. The slope determination method is as follows: the vibrating part of the fiber optic mechanical resonant probe sensor, which serves as the probe, is oriented downwards. The vertical displacement platform is controlled to drive the fiber optic grating and the vibrating part perpendicular to the surface of the glycerol-water mixture or protein solution. The probe is inserted into the glycerol-water mixture or protein solution and gradually moves downwards. During this process, the driving device is controlled to drive the fiber optic grating and the vibrating part to perform subharmonic vibration synchronously. The subharmonic wave in the spectrometer is observed. The downward movement is stopped when the subharmonic wave disappears. The vertical displacement platform is controlled to move the fiber grating and the vibrating part vertically upward until the vibrating part leaves the surface of the protein solution. During this process, the driving device is controlled to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at that immersion depth as the target reflected light at that immersion depth and determines the subharmonic wave intensity in the target reflected light obtained at that immersion depth. After obtaining the subharmonic wave intensity at each immersion depth, that is, after obtaining the functional relationship between different immersion depths and the subharmonic wave intensity, the slope of the linear fitting region in the functional relationship is determined.

[0035] In this embodiment, in the slope determination method, for each immersion depth, if multiple target reflected lights are obtained at that immersion depth, the analysis device determines the intensity of the secondary resonant wave in each target reflected light obtained at that immersion depth, and takes the average value of the determined secondary resonant wave intensities as the secondary resonant wave intensity at that immersion depth. The protein can be insulin. This invention can fabricate corresponding fiber optic mechanical resonant probe sensors and set appropriate operating points according to specific measurement requirements and the characteristic changes of different proteins during folding and aggregation. This allows for the monitoring of protein folding.

[0036] As can be seen from the above embodiments, the present invention characterizes the protein folding and aggregation process based on a fiber optic mechanical resonant probe sensor, which has high accuracy and sensitivity, and does not require the introduction of fluorophores for labeling, thus achieving artifact-free characterization.

[0037] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims.

[0038] It should be understood that the present invention is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is defined solely by the appended claims.

Claims

1. A fiber optic mechanical resonant probe sensor, characterized in that, It includes a fiber Bragg grating, a driving device, a circulator, a laser, a photodetector, an analytical device, and a vibrating part inserted into the substance to be tested. The output end of the laser is connected to the first end of the circulator, the second end of the circulator is connected to the first end of the fiber Bragg grating, the third end of the circulator is connected to the analytical device through the photodetector, and the second end of the fiber Bragg grating is connected to the vibrating part. The laser transmits the laser signal to the fiber Bragg grating via the circulator. The driving device is located at the first end of the fiber Bragg grating and drives the fiber Bragg grating and the vibrating part to perform subharmonic vibration synchronously. During the vibration, the grating period of the fiber Bragg grating changes periodically, and correspondingly, the reflection spectrum of the fiber Bragg grating shifts periodically. After receiving the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator also changes periodically. The degree of periodic change in the intensity of the reflected light varies depending on the characteristic parameters of the analyte inserted into the vibrating part. The photodetector converts the reflected light into an electrical signal, and the analysis device determines the characteristic parameters of the analyte based on the degree of intensity change of the electrical signal.

2. The fiber optic mechanical resonant probe sensor according to claim 1, characterized in that, The wavelength of the laser signal is within the linear region of the unshifted reflection spectrum, and the intensity change of the reflected light is determined by the actual left-right shift of the reflection spectrum. The actual left-right shift of the reflection spectrum depends on the driving frequency and driving voltage of the driving device, the characteristic parameters of the material to be tested around the vibrating part, and its immersion depth. After the wavelength of the laser signal is determined, before the vibrating part is immersed in the substance to be tested, the driving voltage is gradually increased. At the same time, the analysis device is used to observe whether the electrical signal exhibits a 1 / 2 resonant wave. If it does, the driving voltage at this time is taken as the minimum value of the driving voltage. Thereafter, the driving voltage is gradually increased, and the analysis device is used to observe whether the electrical signal exhibits distortion. If it does, the driving voltage at this time is taken as the maximum value of the driving voltage. In the actual measurement process, the driving voltage takes a value between its minimum and maximum values. When the fiber grating vibrates from the middle position to both sides under the drive of the driving device, the reflection spectrum of the fiber grating shifts in different directions and the amount of shift gradually increases on the corresponding side.

3. The fiber optic mechanical resonant probe sensor according to claim 1 or 2, characterized in that, The driving device drives the fiber grating and the vibrating part to perform subharmonic vibration synchronously at their respective eigenfrequencys.

4. The fiber optic mechanical resonant probe sensor according to claim 3, characterized in that, It also includes a vertical displacement platform on which the driving device is fixed. The first end of the fiber grating is fixed to the driving device with UV glue. The vertical displacement platform drives the fiber grating and the vibrating part to move vertically up and down perpendicular to the surface of the material to be tested through the driving device, so that the vibrating part is immersed in the material to be tested. During the vertical movement, the driving device is controlled to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at the immersion depth as the target reflected light at that immersion depth, determines the subharmonic wave intensity in the target reflected light at that immersion depth, and thus obtains the functional relationship between different immersion depths and the subharmonic wave intensity. The analytical device determines the slope of the linear fitting region in the functional relationship, and obtains the characteristic parameters of the substance to be tested based on the determined slope and the correspondence between different characteristic parameters and the slope.

5. The fiber optic mechanical resonant probe sensor according to claim 4, characterized in that, The fiber grating and the vibrating part are made of single-mode fiber. A Bragg grating is etched at the corresponding position of the single-mode fiber to form the fiber grating. The corresponding fiber segment of the single-mode fiber is stretched to obtain a tapered fiber segment. Half of the tapered fiber segment is removed to obtain the vibrating part connected to the fiber grating.

6. The fiber optic mechanical resonant probe sensor according to claim 4, characterized in that, For each immersion depth, if multiple target reflected light are obtained at that immersion depth, the analysis device takes the average value of the secondary resonant wave intensity of each target reflected light at that immersion depth as the secondary resonant wave intensity corresponding to that immersion depth; the characteristic parameter is the viscosity.

7. The fiber optic mechanical resonant probe sensor according to claim 4, characterized in that, When the characteristic parameter is viscosity, the relationship between different characteristic parameters and slope is as follows: as the viscosity increases, the slope gradually decreases.

8. A method for monitoring protein folding based on the fiber optic mechanical resonant probe sensor according to any one of claims 1 to 7, characterized in that, include: Step S11: Prepare glycerol-water mixtures of different concentrations as standard solutions. For each concentration of glycerol-water mixture, determine the slope of the glycerol-water mixture of that concentration according to the slope determination method. After obtaining the slopes of glycerol-water mixtures of different concentrations, determine the corresponding relationship between different viscosities and slopes based on the relationship between the viscosity and concentration of the glycerol-water mixture. Step S12: Prepare protein solutions of different concentrations as standard solutions. For each concentration of protein solution, determine the slope of the protein solution of that concentration according to the slope determination method. After obtaining the slope of protein solutions of different concentrations, determine the viscosity of protein solutions of different concentrations according to the correspondence between the different viscosities and the slope. Step S13: For each protein solution of a given viscosity, heat the protein solution under set conditions to promote the protein to undergo folding and aggregation reactions. After heating for different corresponding durations, determine the slope of the protein solution according to the slope determination method. Based on the correspondence between different viscosities and slopes, determine the viscosity of the protein solution after heating for the corresponding duration. This yields the viscosity change of protein solutions of different viscosities during the folding and aggregation reaction process, and the viscosity change reflects the folding and aggregation process of protein solutions of different concentrations. Step S14: For the protein solution to be tested, the slope of the protein solution to be tested is determined according to the slope determination method. Based on the correspondence between different viscosities and slopes, the viscosity of the protein solution to be tested and the viscosity change of the protein solution to be tested during the folding and aggregation reaction are obtained, thereby obtaining the folding and aggregation process of the protein solution to be tested. The slope determination method is as follows: the vibrating part of the fiber optic mechanical resonant probe sensor, which serves as the probe, is oriented downwards. The vertical displacement platform is controlled to drive the fiber optic grating and the vibrating part perpendicular to the surface of the glycerol-water mixture or protein solution. The probe is inserted into the glycerol-water mixture or protein solution and gradually moves downwards. During this process, the driving device is controlled to drive the fiber optic grating and the vibrating part to perform subharmonic vibration synchronously. The subharmonic wave in the spectrometer is observed. The downward movement is stopped when the subharmonic wave disappears. The vertical displacement platform is controlled to move the fiber grating and the vibrating part vertically upward until the vibrating part leaves the surface of the protein solution. During this process, the driving device is controlled to drive the fiber grating and the vibrating part to perform subharmonic vibration synchronously. After the fiber grating receives the laser signal, the intensity of the reflected light transmitted back to the photodetector through the circulator will change periodically. For each immersion depth, the analysis device takes the reflected light with the maximum or minimum intensity obtained at that immersion depth as the target reflected light at that immersion depth and determines the subharmonic wave intensity in the target reflected light obtained at that immersion depth. After obtaining the subharmonic wave intensity at each immersion depth, that is, after obtaining the functional relationship between different immersion depths and the subharmonic wave intensity, the slope of the linear fitting region in the functional relationship is determined.

9. The protein folding monitoring method according to claim 8, characterized in that, In this slope determination method, for each immersion depth, if multiple target reflected lights are obtained at that immersion depth, the analysis device determines the intensity of the secondary resonant wave in each target reflected light obtained at that immersion depth, and takes the average value of the determined secondary resonant wave intensities as the intensity of the secondary resonant wave at that immersion depth.

10. The protein folding monitoring method according to claim 8, characterized in that, The protein is insulin, and in step S13, the set conditions are acidic conditions and 52°C conditions.

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